الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Gentamicin is an aminoglycoside antibiotic widely used to combat Gram-negative bacteria. Gentamicin is described to be a nephrotoxic drug. Moreover, gentamicin-induced nephrotoxicity represents 10-25% of therapeutic courses. Its toxicity is characterized by kidney tubular injury, resulting in reduced tubular reabsorption and thereby increasing excretion of the urinary proteins. Over the last decade, there has been increasing research interest in the proteins containedin urinary exosomes, which are nano-vesicles originating from the multivesicular body, asubcellular component, and their relevance to kidney diseases. Although severalbiomarkers have been reported to be diagnostic of gentamicin-induced nephrotoxicity, none of them are able to detect gentamicin-induced impairment of the renalcollecting ducts. In the present study, we focused on urinary exosomal aquaporin-2, awater channel protein, and TSG101, a frequently used exosome marker protein, andanalyzed their excretory patterns in rats receiving gentamicin. The experiment was conducted with 56 male Sprague-Dawley rats aged 9 weeks (~350 gm/body weight). Rats were randomly divided into control and gentamicin group receiving saline and gentamicin sulfate (160 mg/ kg) daily, respectively, for 8 consecutive days. Urine, blood, and kidney tissue samples were collected for further analyses. The biochemical analyses of blood revealed that gentamicin had significantly increased the plasma creatinine and urea nitrogen concentrationat day 5 and day 3 or later, respectively. The gentamicin group also showed a significant decrease in urinary osmolality and an increase in urine volume at day 7.In addition, there was significant decrease in the urinary pH along with the gentamicin-treatment and a significant loss in body weight was also observed at days 7 and 8 which it might be due increased loss of body fluid as a result of gentamicin- induced renal insufficiency. These data characterize the tubular damage induced by gentamicin. Moreover, in a histopathological examination with PAS-staining revealed a clear kidney tubular injury in gentamicin-treated rats represented by localized vacuolization in the cortex was observed in the early stage of the insult. While, in the late phase, severe tubular damage was demonstrated including loss of brush border, tubular necrosis, cast formation, tubular dilatation, and lymphocytic infiltration, which might be occurred in response to gentamicin-induced oxidative damage and inflammatory pathway. Next, we examined whether gentamicin affected the urinary excretion of exosomal AQP2 protein by western blot analysis. The data indicated that urinary exosomal AQP2 protein excretion was significantly increased and decreasedin the early phase and late phase of the gentamicin insult, respectively. Next, we examined the abundance of renal AQP2 protein on days 2 and 8. Although the levels of AQP2 protein in the cortex and medulla were slightly reduced on day 2, gentamicin had significantly reduced the abundance of renal cortical and medullary AQP2 proteins by day 8. Thereafter, these data were confirmed by immunohistochemistry examination indicating a marked reduction in the level of AQP2 expressionin the outer and inner medulla in the late stage. These immunohistochemistry data supported the immunoblot data described above. Interestingly, there was increased apical expression of AQP2 in the inner medulla in the gentamicin group at day 8. Exosomes are known to be released into the extracellular space by fusion of the outer membrane of multivesicular bodies (MVBs) with the cell surface. Importantly,TSG101 is the core structure of ESCRT-I which is essential for cargo sorting and MVB formation. TSG101 has been commonly used as a marker protein of exosomes in urine. Therefore, we examined the abundance of urinary exosomal TSG101.Interestingly, the urinary exosomal TSG101 excretion was significantly increased in days 1 and 2, but not on day 8. Next, we examined the renal protein level of TSG101 by immunoblotting in days 2 and 8 in a whole-cell lysate fraction in the gentamicin group and the results revealed a significant decreased and tended to be decreased in the cortex and medulla on day 2, respectively. Then, we also examined the renal protein level of TSG101 in a membrane-rich fraction by immunoblotting. The reduced level of renal TSG101 in the fraction in the gentamicin group was less marked in comparison with the results in the whole-cell lysate fraction in the gentamicin group. On day 1 the urinary excretion of both exosomal AQP2 and TSG101 was increased, and therefore we also examined other urinary exosomal proteins including AQP1 and ALIX. The data demonstrated significant increase in both proteins. As mentioned above, our present data indicated that increased excretion of exosomal AQP2 and TSG101 was observed following one day’s treatment with gentamicin, accompanied by a slight decrease in the level of their renal expression. Similarly, at the same time point, excretion of urinary exosomal AQP1 and ALIX was increased. As urinary excretion of all exosomal proteins examined here was increased, it was strongly suggested that treatment with gentamicin increased a greater number of exosomes released from all renal epithelial cells. As a result, slight decreases in renal AQP2 and TSG101 protein abundance might be observed. These data suggest that the increased excretion of exosomal proteins after one day’s treatment with gentamicin might be mediated by the gentamicin-induced increase in the intracellular concentration of Ca2+. In addition, It has been reported that lower pH increases excretion of urinary exosomal AQP2 but our data revealed a reduction in AQP2 in the late phase and the urinary pH was acidic. Therefore, it was unlikely that the urinary acidification with gentamicin was involved in the increased excretion of exosomal proteins. Moreover, it has been found that p53 enhances the secretion of exosomes in the cultured cells and is known that gentamicin increases the expression of p53. Therefore, it is conceivable that increased excretion of urinary exosomes might be due to increased expression of p53. According to the protein analyses data, gentamicin altered the renal expression level of AQP2 and TSG101 proteins. Therefore, we determined the expression level of renal AQP2 and TSG101 mRNA using real-time PCR.The PCR data supported the action of gentamicin in alteration of protein level obtained by immunoblot and indicated that gentamicin nephrotoxicity has induced a compensatory mechanisms for regeneration of the injured tubules. In this study, the urinary exosomal level of AQP2 and TSG101 significantly increased too earlier than traditional markers including plasma creatinine and urea nitrogen concentrations and the histological analysis. In conclusion, our findings suggested thaturinary exosomal AQP2 would be useful for detection of gentamicin-induced collectingduct injury. Furthermore, the excretion levels of urinary exosomal AQP2 and TSG101 incombination also appeared to facilitate early to late detection of gentamicin-inducednephrotoxicity.