Abstract: Background: Renal tubular cells are exposed to high toxin concentrations more than other tissues because of active tubular secretion, reabsorption and urine concentration mechanisms in kidneys. Due to this reason, renal tubules are direct targets of nephrotoxicity. Alpha-linolenic acid is a carboxylic acid, whose anti-inflammatory and anti-oxidant effects are shown in various studies. Because of this, a study to examine the protective effects of alpha-linolenic acid on nephrotoxicity is planned. Materials and Methods: Gentamicin is administered to mice for 9 days to form nephrotoxicity and alpha-linolenic acid is administered to mice for 9 days to evaluate protective effects. Cyclooxygenase-2, phospholipase A2 and inducible nitric oxide synthase enzymes in their kidneys are analyzed by using ELISA method to compare the nephrotoxicity levels. Results: Gentamicin administration increased the expression of cyclooxygenase-2, phospholipase A2 and inducible nitric oxide synthase enzymes. Alpha-linolenic acid administration to mice that are administered gentamicin previously decreased the rate of increase of the cyclooxygenase-2 and inducible nitric oxide synthase caused by gentamicin while, it didn't have any effect on phospholipase A2 increase. Conclusion: Gentamicin caused an increase in cyclooxygenase-2, phospholipase A2, inducible nitric oxide synthase enzymes in kidneys. Application of alpha-linolenic acid decreased the increase of cyclooxygenase-2 and nitric oxide synthase enzymes significantly while, having no effect on the increase of phospholipase A2. In conclusion, this study shows that the gentamicin administration causes nephrotoxicity and the use of alpha-linolenic acid can be helpful against this toxic effect.
INTRODUCTION
Saturated fatty acids can accumulate in the body since they are in solid state at room temperature. However, poly unsaturated fatty acids are in liquid state in room temperature and they are also vital for human survival. Due to this reason they are named as fundamental fatty acids and they are separated in to two groups which are n-6 and n-3. Main sources for n-6 fatty acids are corn and soybean oils and main sources for n-3 fatty acids are flaxseed oil, nuts and fish oils. Flaxseed oil and nuts mostly contain alpha-linolenic acid while, fish oil mostly contain eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The EPA and DHA should be ingested because they can not be synthesized and therefore called essential fatty acids1.
Alpha-linolenic acid is protective for heart and cardiovascular system2,3. It is a precursor for EPA and DHA which joins arachidonate structure4. Blood pressure of spontaneous hypertensive rats is shown to decrease which are on a alpha-linolenic acid diet5. Alpha-linolenic acid also have antioxidant properties. It reduces oxidative stress which helps to prevent the inflammation6. It also reduces the inflammation caused by lipopolysaccharides (LPS). In addition, alpha-linolenic acid inhibits the translocation of nuclear factor kappa-B (NF-κB) and the phosphorylation of mitogen-activated protein kinase (MAPK) which decreases the expression of inflammation factors such as inducible Nitric Oxide Synthase (iNOS), cyclooxygenase (COX)-2 and TNF-alpha7.
Various studies shown that one of the main reasons for acute intrinsic renal insufficiency is aminoglycoside nephrotoxicity. Nephrotoxicity can be defined as nephrotoxic renal insufficiency. Kidneys are prone to be affected by toxic effects of the drugs and other endogenic and exogenic toxins due to their function in blood perfusion, metabolic activity and excretion. Renal tubular cells are exposed to high toxin concentrations more than other tissues because of active tubular secretion, reabsorption and urine concentration mechanisms in kidneys. Due to this reason, renal tubules are direct targets of nephrotoxicity. Tubular necrosis is the significant feature of the nephrotoxicity. Due to this, it is also called acute tubular necrosis8.
Aminoglycoside antibiotics are widely used in control and treatment of the Gram (-) aerobic infections. Although, gentamicins have the most widespread application areas among these antibiotics, its usage is limited because of its nephrotoxic effects. Aminoglycoside antibiotics are responsible for nearly 10% of the acute renal insufficiency incidences. Due to this, aminoglycoside antibiotics are used as a model in acute renal insufficiency incidences8.
Various studies showed that the COX pathway, which functions in inflammation, accompanies gentamicin nephrotoxicity. In a study, selective COX-2 inhibitor is reported to be decreasing the nephrotoxicity caused by gentamicin9. Furthermore, gentamicin application is determined to be causing an increase in the activity of phospholipase A210. In another study conducted, nephorotoxicity formed by gentamicin application caused an increase in iNOS enzyme11.
The aim of this study was to evaluate the effects alpha-linolenic acid, which is shown to have antiinflammatory effects on iNOS, phospholipase A2 and COX-2 enzymes whose levels are increased in nephrons by the gentamicin.
MATERIALS AND METHODS
Male mice (8 weeks old, balb/c, albino) that are obtained from the Experimental Animal Center in Çukurova University, in Adana are used in study. This study was approved by the Animal Care Committee and Ethics Committee of Cukurova University.
Mice are divided into three groups which are: Control group, gentamicin group and alpha-linolenic acid group. To gentamicin group, 100 mg kg–1 gentamicin is applied intraperitoneally once a day for 9 days. To alpha-linolenic acid group, 100 mg kg–1 gentamicin is applied intraperitoneally together with 70 mg kg–1 alpha-linolenic acid which is applied by using a gavage for 9 days. Physiological serum is applied intraperitoneally to control group under same experimental conditions for 9 days. At the end of the protocol described above, cervical dislocation is applied to the mice. Kidneys of the mice are stored in eppendorf tubes at -80°C for later use in quantitative analysis.
Quantitative analysis
Tissue homogenization: Frozen tissue samples that are stored in eppendorf tubes are treated with 3 mL g–1 RIPA (Radio-immunoprecipitation assay) buffer, 30 μL PMSF (phenylmethanesulfonyl fluoride), 30 μL sodyum vanadate and 30 μL protease inhibitor. Then, homogenates are obtained by using ultrosonication on those tubes on ice. Homogenates are then centrifuged at 10,000 RPM for 10 min and supernatants are taken and pellets are discarded.
Protein quantification: Bradford method is used to quantify the protein in homogenized tissues. By using Bovine serum albumin (1 μg mL–1), 1, 2, 3, 5, 7, 8, 10 μg mL–1 standards are prepared. Then, 10 μL is taken from every sample and completed to 100 μL by adding distilled water. Lastly, 1 mL Bradford solution is added to standards and samples, vortexed and absorbances at 595 nanometer are measured manually. Protein quantification (μg μL–1) is done according to the standart curve drawn in Prism software.
ELISA (Enzyme linked immunosorbent assay) test: The ELISA test is used to examine the expression and activity of COX-2, phospholipase A2 and iNOS enzymes.
Statistic analysis: Results were expressed as Mean±SEM and n refers to the number of animals used for each experiments. Differences in results between tissues were tested by analysis of variance (ANOVA) corrected for multiple comparisons (Bonferroni corrections). The p-values less than 0.05 were considered to be significant.
RESULTS
ELISA COX-2 enzyme quantification: While, gentamicin application caused an increase in the COX-2 enzyme in kidneys, application of alpha-linolenic acid decreased this increase significantly (Fig. 1). Mean values of COX-2 concentrations for control, gentamicin and gentamicin+ alpha-linolenic acid groups are found to be 9499 pg mL–1 (SEM 241.2), 11422 pg mL–1 (SEM 461.1), 9617 pg mL–1 (SEM 107.8), respectively.
| Fig. 1: | Effect of alpha-linolenic acid on cyclooxygenase-2 enzyme of the gentamicin applied mice (n = 6). Statistical analysis: ANOVA, post hoc: Bonferroni, *Control at p<0.05, #Gentamicin at p<0.05 |
ELISA phospholipase A2 enzyme quantification: While, gentamicin application caused an increase in the phospholipase A2 enzyme in kidneys, application of alpha-linolenic acid had no effect on this increase (Fig. 2). Mean values of phospholipase A2 concentrations for control, gentamicin and gentamicin+alpha-linolenic acid groups are found to be 1766 pg mL–1 (SEM 72.9), 2448 pg mL–1 (SEM 139.5), 2603 pg mL–1 (SEM 112.3), respectively.
ELISA iNOS enzyme quantification: While, gentamicin application caused an increase in the iNOS enzyme in kidneys, application of alpha-linolenic acid decreased this increase significantly (Fig. 3).
| Fig. 2: | Effect of alpha-linolenic acid on phospholipase A2 enzyme of the gentamicin applied mice (n = 6). Statistical analysis: ANOVA, post hoc: Bonferroni, *Control at p<0.05, #Gentamicin at p<0.05 |
| Fig. 3: | Effect of alpha-linolenic acid on iNOS enzyme of the gentamicin applied mice (n = 6). Statistical analysis: ANOVA, post hoc: Bonferroni, *Control at p<0.05, #Gentamicin at p<0.05 |
Mean values of iNOS concentrations for control, gentamicin and gentamicin+alpha-linolenic acid groups are found to be 2251 IU mL–1 (SEM 191.1), 3964 IU mL–1 (SEM 475.5), 2473 IU mL–1 (SEM 232.1), respectively.
DISCUSSION
In this study, the enzymes iNOS, phospholipase A2 and COX-2 which functions in inflammatory response as biological mediators is examined. Various studies confirmed the importance of inflammation in nephrotoxicity caused by gentamicin9-11. The NO produced by one of these mediators, iNOS have a pathogenic role in acute and chronic inflammatory diseases12-14. Studies showed that nephron damage caused by gentamicin is accompanied by iNOS enzyme11. The iNOS enzyme is shown to be decreased by the alpha-linolenic acid in previous studies7. In addition, increased oxidative stress increases superoxide and peroxynitrite production whose formations are also shown to be reduced by alpha-linolenic acid supplement15,16. According to the results of this study, application of alpha-linolenic acid also decreased the increment of iNOS enzyme caused by gentamicin. Alpha-linolenic acid supplements will help to prevent the nephrotoxicity by inhibiting iNOS.
The COX-2 enzyme, which is an inflammatory mediator and prostaglandins synthesized by this enzyme also plays a role in gentamicin nephrotoxicity and there are studies showing that this enzyme increases in nephrons on gentamicin application4. Studies showed that selective COX-2 inhibitors reduce the risk of nephrotoxicity caused by gentamicin. At the same time, alpha-linolenic acid is shown to decrease the LPS induced COX-2 enzyme. Results of this study also show similarities to previous studies and alpha-linolenic acid decreased the increment caused by gentamicin in COX-2 enzyme. However, previous studies determined that the increased iNOS activity reduces the COX-2 expression17. This also suggests that the reduction in COX-2 may have been caused by the increased iNOS.
Another enzyme examined in this study, phospholipase A2 is also shown to be increased by gentamicin application in various studies18. According to the results of this study, application of alpha-linolenic acid couldn’t decrease this increment. Studies showed that, gentamicin application induces the oxidative stress which causes an increase in intracellular calcium levels and this increase causes an increase in the activity of phospholipase A2 enzyme as a result19-21. Alpha-linolenic acid have antioxidative properties because of double bonds in its structure6. While, authors expected to see a decrease in increment of phospholipase A2 caused by gentamicin due to application of antioxidant alpha-linolenic acid, it had no effect according to the results. This can be due to the insufficient effect of antioxidative properties of alpha-linolenic acid in nephrons or gentamicin causing this increment via other mechanisms.
Oxidative stress causes the activation and nuclear translocation of NF-κB which are both key factors in renal inflammation by regulating the gene expression of cytokines and adhesion molecules22. Thereby, reduction of oxidative stress by using alpha-linolenic acid can prevent NF-κB translocation at the early stages of inflammation and thus, can prevent the nephron damage by inhibiting the migration of monocytes and macrophages through decreasing the expression of inflammation mediators such as iNOS and COX-2.
CONCLUSION
This study shows that, application of alpha-linolenic acid reduces the inflammatory response due to increased iNOS and COX-2 enzymes and renal damage caused by gentamicin by inhibiting these enzymes. Thus, application of alpha-linolenic acid reduces the nephrotoxicity risk caused by gentamicin.
ACKNOWLEDGMENT
This study was funded by Cukurova University (TSA-2014-2656).