The effect of derivatives of sulfur-containing amino acids (ademetionine, taurine and glutathione) on survival of animals with acute kidney injury of various genesis

Aim. To study the nephroprotective effect of sulfur-containing amino acids (ademetionine, taurine and glutathione) on different models of acute kidney injury (AKI) by the integral index of the survival rate of animals. Materials and methods. The experiments were performed on 105 non-linear mature white laboratory rats and 35 mice. The drugs studied – ademetionine (Geptral, Abbott SpA, Italy) in the dose of 20 mg/kg, glutathione (TAD 600, Biomedica Foscama, Italy) in the dose of 30 mg/kg and taurine (Sigma-Aldrich, USA) in the dose of dose 100 mg/kg – were injected intraperitoneally 3-7 days before AKI modeling. Rhabdomyolysis-induced AKI was modeled by the intramuscular injection of 50 % glycerol solution in the dose of 10 ml/kg, toxic AKI – by the injection of gentamicin solution in the dose of 80 mg/kg and the subcutaneous injection of ethylene glycol in the dose of 10 ml/kg. Simulation of renal ischemia was performed under general anesthesia (ethaminal sodium, 40 mg/kg) by clamping of both renal pedicles for a period of 60 min, followed by reperfusion for 24 h. Results and discusion. The experimental results show a significant improvement in the survival of animals under the effect of the drugs of derivatives of sulfur-containing amino acids studied in conditions of various models of AKI. Ademetionine, taurine and glutathione reduced the lethality of animals on models of rhabdomyolytic, gentamicin, ischemia-reperfusion AKI. In conditions of ethylene glycol intoxication the highest nephroprotective activity by the survival criterion was shown by glutathione (57.1 % survival at 48 h after administration of ethylene glycol against 100 % of mortality in the control group (p < 0.05). Conclusions. All derivatives of sulfur-containing amino acids studied (ademetionine, taurine and glutathione) exhibit the nephroprotective activity by the criterion of survival of animals on various models of AKI. Glutathione demonstrates the best survival of animals with acute kidney injury of various genesis, while ademetionine has the lowest survival.

Ключевые слова: нефропротекция; острое повреждение почек; выживаемость; адеметионин; таурин; глутатион Acute kidney injury (AKI) is a concept that replaced the well-known term "acute kidney failure", but did not change the dynamics of progression of this pathology. The incidence of AKI varies from 1 to 31 %, and the mortality rate reaches from 19 to 83 % [1,2]. Toxic kidney injury (toxic nephropathy) is usually the result of an accidental domestic food, biological, or industrial professional poisoning. In recent years chemicals that are used in everyday life and industry are of particular importance, therefore, the frequency of toxic nephropathy increases. Despite the widespread introduction of renal replacement therapy, mortality in severe forms of AKI is not characterized by a tendency to decrease [3]. In all cases development of AKI involves some common mechanisms: disturbances of renal (especially cortical) circulation, reduction of glomerular filtration, reduction of reabsorption with total diffusion of the glomerular filtrate through the wall of damaged tubules, compression of the tubules with swollen interstitium, disturbances of the oxidative-reduction processes, in particular activation of free radical oxidation processes against the background of the antioxidant system dysfunction [4].
One of the common types of toxic AKI is ethylene glycol poisoning. Ethylene glycol is a nephrotoxin that quickly causes fatal kidney injury, and its damaging effect on kidneys is due to the toxic effects of its metabolites (oxalic acid). In this case, oxalates of calcium monohydrate are crystallized in the tubular lumen, causing destruction of membrane phospholipids and deve-lopment of acidosis [5][6][7][8][9]. The model of ethylene glycol intoxication is one of the methods of screening research, in which the integral criterion of the nephroprotective effect is survival of animals, which allows reliable verification of the protective effect of nephroprotective compounds.
In order to verify the nephroprotective effect of the derivatives of sulfur-containing amino acids studied (ademetionine, taurine and glutathione) the integral index of animal survival on different models of AKI have been selected. The potential nephroprotective activity of the drugs selected is due to the biological presence of their active components in the form of sulfur-containing amino acids in the body and their biochemical interconversions. Ademethionine is an active metabolite of methionine, has the antioxidant, membranoprotective, cytoprotective, anti-inflammatory and analgesic activity [10,11]. Taurine as a sulfur-containing amino acid acts as an osmoregulator of cells, a membranoprotector, a detoxifier, an antioxidant, it provides resynthesis of ATP, exhibits the anti-inflammatory action [12,13]. Glutathione is an endogenous tripeptid, an antioxidant, a detoxicant, a membranoprotector, is involved in maintenance of the thiol/ disulphide homeostasis in tissues, and participates in the protein and hydrocarbon metabolism [14,15].
The aim of the study was to compare the nephroprotective effect of sulfur-containing amino acids (ademetionine, taurine and glutathione) on different models of AKI by the integral index of the survival rate of animals.

Materials and methods
The series of experiments were performed on 105 nonlinear mature white laboratory rats weighing 130-180 g and 35 mice of both sexes kept in the vivarium at a constant temperature and humidity, with free access to water and food. The rats of each series were divided into 5 groups (n = 7): group I -model pathology, group IIanimals treated with taurine (Sigma-Aldrich, USA) in the dose of 100 mg/kg, group III -animals treated with ademetionine ("Geptral", Abbott SpA, Italy) in the dose of 20 mg/kg, group IV -animals treated with glutathione (TAD 600, Biomedica Foscama, Italy) in the dose of 30 mg/kg, group V -animals treated with the reference drug. As a reference drug in rhabdomyolytic AKI lipoflavone (Biolik, Ukraine) in the dose of 8 mg/kg was chosen; in gentamicin-induced AKI it was canephron (Bionorica CE, Germany) in the dose of 27 mg/kg; in ischemic-reperfusion and toxic ethylene glycol AKI it was mexidol (Pharmasoft, RF) in the dose of 100 mg/kg. All drugs studied (except for canephron, which was used intragastrally) were administered intraperitoneally 3-7 days prior to AKI simulation. The choice of reference drugs in different models of AKI is determined by the evidence base of their nephroprotective potential [16][17][18].
Rhabdomyolytic AKI in rats was induced by intramuscular administration of 50 % glycerol solution in the dose of 8 ml/kg in the muscles of the hind legs. In 40 min after administration of glycerol the animals were given the drugs under study. Gentamicin nephropathy was reproduced by intramuscular administration of 4 % gentamicin sulfate solution (Galychpharm, Ukraine) in the dose of 80 mg/kg once a day for 6 days; the drugs studied were administered 40 min after the gentamicin injection. Ischemic-reperfusion AKI was modeled by clamping of both kidney pedicles for 60 min after the middle laparotomy under general anesthesia (ethaminal sodium, 40 mg/kg) followed by reperfusion for 24 h. Toxic ethylene glycol AKI was caused by a single subcutaneous injection of ethylene glycol in the dose of 10 ml/kg [19]. The statistical evaluation of the effect was carried out using the Fisher's angle transformation criterion. The critical level of significance was accepted at p < 0.05. The studies were conducted in accordance with the provisions of the "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes" [20].

Results and discussion
The research results of the animal survival in different models of AKI with administration of derivatives of sulfur-containing amino acids and the reference drugs are given in Table. In the group of animals with ethylene glycol AKI the highest mortality was observed: survival of mice was 14.3 % in 12 h, and in 24 h it was 0 %. The weakest protective effect in conditions of ethylene glycol AKI was shown by ademetionine: the survival rate in 12 h was 28.6 % (p > 0.05), in 2 days all the animals died.
When taurine was used, an improvement in animal survival compared to the model pathology group was observed only during the first day -42.8 %; however, on day 5 all animals died. Glutathione showed the highest efficacy in ethylene glycol AKI: under its effect a significant increase in the survival rate was seen throughout the observation period, which exceeded the protective effect of the reference drug mexidol.
In rhabdomyolytic kidney injury the mortality of animals was 28.6 %. The use of the drugs studied showed a tendency to decrease the mortality rate; moreover, there was 100 % survival of the animals in taurine and glutathione groups, it corresponded to the effect of the reference drug lipoflavone; when using ademetionine the survival rate was 85.7 % of animals (p > 0.05).
The predictable high mortality rate was observed among animals with gentamicin nephropathy reaching 42.9 %, while the use of drugs reduced the percentage of dead animals with gentamicin nephropathy. The results of the experiment confirm that the effectiveness of ademetionine and the reference drug canephron was lower than the effect of taurine and glutathione, which provided the 85.7% survival of the experimental animals.
A decrease in the survival of rats to 71.4 % occurred in ischemic-reperfusion AKI. Among the drugs studied only glutathione and mexidol completely prevented the death of animals although the use of taurine and ademetionine also showed a tendency to reduce the mortality of rats since the survival rate after their administration was 87.5 % and 71.4 %, respectively. Therefore, the reduction in mortality of the experimental animals under conditions of AKI of different genesis proves the nephroprotective activity of derivatives of sulfur-containing amino acids, which according to this criterion can be placed in the following order from the maximum to the minimal effect: glutathionetaurine -ademetionine.
Thus, according to the results of the study all the simulated forms of AKI were accompanied by the death of a part of the animals, moreover, the expected highest mortality rate in animals was observed in ethylene glycol intoxication. The best protective effect was observed when taking glutathione, as evidenced by 57.1 % survival of mice in 2 days of ethylene glycol AKI development, 100 % survival of rats in rhabdomyolytic and ischemia-reperfusion AKI, and 85.7 % of the animal survival in gentamicin nephropathy. Under the effect of taurine there was a tendency towards improved survival of rats in all AKI models and prolonged life expectancy in ethylene glycol intoxication. The use of ademetionine was the least effective as demonstrated by the highest mortality of mice during the first 12 h after the simulation of ethylene glycol AKI (71.4%) and the lowest survival rate in other models of AKI. CONCLUSIONS 1. All derivatives of sulfur-containing amino acids studied (ademetionine, taurine and glutathione) exhibit the nephroprotective activity by the criterion of survival of animals on various models of AKI.
2. Glutathione demonstrates the best survival of animals with acute kidney injury of various genesis, while ademetionine has the lowest survival.
Conflict of Interests: authors have no conflict of interests to declare.