RU2716509C1 - Method for preventing experimental toxic nephropathy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental biology and pharmacology, and can be used for preventing rhabdomyolysis-induced renal injury. An experimental animal is intramuscularly injected with 50 % glycerol in dose of 0.8 ml/100 g of body weight. From the following day after experimental renal injury is reproduced for 30 days, acizole in dose of 30 mg/kg is administered intragastrically daily to animals.

EFFECT: method provides studying the preventive effect of acizole on restoring renal function on the background of toxic nephropathy caused by rhabdomyolysis, due to the fact that from the following day after experimental renal damage within 30 days, acizole is administered.

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Description

The invention relates to medicine, experimental biology, pharmacology and can be used to study the mechanisms of prevention of toxic nephropathy.

Kidney disease is becoming increasingly important for modern health care. So, according to Kaprin A.D., Apolikhina O.I. and others. For the period from 2002 to 2014 in Russia, the increase in the number of patients with glomerular and tubulointerstitial kidney diseases (nephropathies) amounted to + 3.2%. There is also a large increase in the number of patients with renal failure - 131.7%. (Kaprin A.D., Apolikhin O.I., Sivkov A.V., Solntseva T.V., Komarova V.A. Analysis of Uronephrological Morbidity and Mortality in the Russian Federation for the period 2002-2014 according to official statistics. Experimental and clinical urology. No. 3, 2016).

Nephropathy is a group of kidney diseases characterized by damage to the glomeruli and tubules of the nephron. At the same time, despite the polyetiological nature, they have common pathogenetic mechanisms. So, non-specific factors determining the progression of nephropathy are such as a change in the state of aggregation of the blood, a violation of protein transport, a change in the expression of mediators of cell damage. A consequence of the complex action of these processes is the fibrosis of kidney tissue and the loss of the functioning mass of nephrons (Kalyuzhin V.V., Urazova O.I., Kalyuzhina E.V., Sibireva O.F., Tkalich L.M., Zibnitskaya L.I. , Terentyeva N. N. Nonspecific mechanisms of progression of chronic kidney disease // Bulletin of Siberian medicine. 2015. No. 4. P. 87-98).

An important role in the pathogenesis of kidney diseases is played by lipid peroxidation (LPO) of cell membranes. It was established that oxidative stress is one of the key links in the development of nephropathy (Zolotukhin P.V., Aleksandrova A.A., Dovzhik A.D., Lebedeva Yu.A., Kuzminova O.N., Gutnikova L.V. Interactomics - an analytical tool for studying the molecular basis of nephropathy // Nephrology. 2013. No. 5. P. 9-15). A large number of free radicals formed in this process cause alteration and destruction of cells. Destructive processes in lipid peroxidation are caused by the destruction of cell membranes or damage to cell DNA, which causes PARP1-dependent repair. PARP1, PARP1 - poly (ADP-ribose) polymerase 1, an enzyme that, when DNA breaks occur, binds to the break points due to the so-called "zinc fingers" and is simultaneously activated. The result is chromatin decondensation at the rupture site, which facilitates the access of repair enzymes. This leads to the depletion of the pool of NAD + and ATP, which leads to an irreversible failure of the energy systems of the cell and its death. These processes are aggravated by ischemia and hypoxia, since in this case the activity of the GSK3B enzyme, which is a factor of apoptosis and necrosis, increases in the kidney tissue. In addition, it inhibits the work of the antioxidant system. (Zolotukhin P.V., Belanova A.A., Lebedeva Yu.A., Batyushin M.M., Chistyakov V.A. Cellular Physiology of damage and restoration of the kidneys // Nephrology. 2015. No. 5. P. 17-22 )

An oxidative explosion and the consequent peroxidation of lipids in cell membranes is especially evident in toxic nephropathies. In addition, a strong inflammatory reaction develops in the kidney tissue, which exacerbates the processes of lipid peroxidation and causes circulatory disorders in the target organ. With the complex action of all pathogenetic links, the glomeruli and proximal tubules are affected, which causes a violation of the filtration and reabsorption processes, due to which diuresis and excretory function as a whole are disturbed.

The described effects are observed in experimental toxic nephropathies, including rhabdomyolysis-induced, which makes it relevant to search for ways to prevent these pathological conditions.

There is no specific treatment for toxic nephropathy. For the treatment of these diseases, drugs of various groups are prescribed to restore kidney function (diuretics, infusion preparations, amino acids and others). However, in the generally accepted tactics of treating toxic damage to the kidneys, no place is given to drugs that correct oxidative status. In this regard, we selected the drug acizole.

Acyzole (bis- (1-vinylimidazole) zinc diacetate) was created at the Irkutsk Institute of Chemistry named after A.E. Tabor SB RAS and is a derivative of 1-alkenylimidazole. This drug is a powerful antihypoxant that is effective for carbon monoxide poisoning. In conditions of low oxygenation, this drug is able to reduce the need for organs in oxygen and increase their resistance to oxygen deficiency. Acizole is able to compensate for the lack of zinc in the body and thereby normalizes the work of Zn-dependent enzymes. In addition, it is able to act as a membrane protector and has antioxidant properties. You can also note its vasodilating effect.

Closest to the claimed is a method for the prevention of toxic nephropathy using watermelon seeds, taken as a prototype (Patent No. 2144829 C1, publ. 01/27/2000, Bull. No. 3, Application: 98110824/14, 06/04/1998, A61K 35/78 (2000.01 ), authors: Dmitrienko N.V., Emtsov V.I.), which consists in the fact that 0.1 ml of watermelon seed oil was administered intragastrically per 100 g of animal weight, starting from the next day from the moment of a single inoculation of carbon tetrachloride ( CHU).

The disadvantages of the prototype include the difficulty of maintaining and controlling the optimal composition of substances in oil from watermelon seeds, which has a nephroprotective effect. There is also no reliable data on the preventive effect of this therapeutic composition on the pathogenetic links of nephropathy, including damage to cell membranes by free radicals. Limited areas for growing watermelon do not allow the widespread use of oil from their seeds as a means of preventing kidney disease.

The aim of the study was to study the prophylactic effect of the drug acizole on the level of lipid peroxidation and renal function in the presence of toxic nephropathy caused by rhabdomyolysis.

The invention is aimed at solving the problem of developing a method for the prevention of glycerol nephropathy in experimental animals.

The solution to this problem provides the restoration of renal function in case of toxic damage by improving tissue respiration, reducing the level of lipid peroxidation due to the cytoprotective effect, and improving organ blood supply due to the vasodilating effect of acyzole.

To achieve this technical result, the claimed invention of a method for the prevention of toxic nephropathy in experimental animals with rhabdomyolysis of the kidney has the following distinctive features: animals daily, once a day, injected with acyzole at a dose of 30 mg / kg through a tube into the stomach the next day after a single intramuscular injection 50% glycerol solution at a dose of 0.8 ml / 100 g weight.

Acyzole, being a powerful antihypoxant and zinc donor, improves tissue respiration processes, stabilizes cell membranes and reduces the destructive effect of free radical oxidation products. Also, in the laboratory of the Department of Normal Physiology, SOGMA, it was found that acizole accelerates glomerular filtration.

Daily administration of acizole for a month, once a day, through a tube into the stomach at a dose of 30 mg / kg after the initiation of rhabdomyolysis and toxic damage to the kidneys with myoglobin leads to a decrease in pathological processes in experimental animals. The claimed method is effective, cost-effective and easily reproducible.

According to the information available to the authors, the set of essential features characterizing the essence of the claimed invention is not known, which allows us to conclude that the invention meets the criterion of "novelty."

According to the authors, the essence of the claimed invention should not be obvious to specialists from a known level of medicine, since it does not reveal the above-mentioned possibility of obtaining a method for the prevention of toxic nephropathy in experimental animals with glycerol damage to the kidneys, including administering acyzole every day through an atraumatic tube into the stomach in within 30 days, which allows us to conclude that the criterion of "inventive step".

The set of essential features characterizing the essence of the invention, in principle, can be repeatedly used in medicine with obtaining a result consisting in an effective and easily reproducible method for the prevention of toxic nephropathy by restoring kidney function by improving tissue respiration, reducing the level of lipid peroxidation due to cytoprotective action, improvement of organ blood supply due to the vasodilating effect of acizole, which allows us to conclude that zobreteniya criterion of "industrial applicability".

This method is as follows.

To simulate toxic glycerol nephropathy, a 50% glycerol solution is introduced into the femoral muscle of experimental rats at a dose of 0.8 ml / 100 g of weight, which causes the release of myoglobin from the destroyed myocytes (has a nephrotoxic effect) and reactive oxygen species into the systemic circulation. Dissolving 3 g of acyzole in 100 ml of distilled water, we obtain a solution of acyzole in the ratio of 30 mg of substance in 1 ml. Every day, once a day for a month, we inject into the stomach through an atraumatic tube with a syringe 0.1 ml of acyzole solution for every 100 g of weight of experimental animals.

After 14 and 30 days, studies were carried out on the level of peroxidation products, the activity of antioxidant enzymes and the study of the functional parameters of the kidneys - volume of urine output, glomerular filtration rate, relative reabsorption of water, excretion of sodium, potassium and protein in urine. Animals were withdrawn from the experiment by intraperitoneal administration of sodium thiopental.

Example. The experiment was conducted on 65 male Wistar rats with an average weight of 267 ± 13 g, divided into 5 groups: 1-intact group; 2 animals with toxic nephropathy, which were withdrawn from the experiment after 14 days (2 weeks); 3- animals with toxic nephropathy, which were removed from the experiment after 30 days (4 weeks); 4 animals with prophylactic intragastric administration of acyzole against toxic nephropathy for 14 days; 5- animals with prophylactic intragastric administration of acyzole against toxic nephropathy for 30 days.

Acyzole was administered using an atraumatic intragastric tube daily at a dose of 30 mg / kg to the fourth and fifth group of rats.

After a month, the concentration in the blood of such lipid peroxidation products of cell membranes as hydroperoxides (GP) in plasma was determined according to the method of Gavrilova VB, Gavrilova AR and Mazhul L.M. and malondialdehyde (MDA) in red blood cells according to a method based on its interaction with thiobarbituric acid. Catalase activity was also determined by the method of E. Beutler and superoxide dismutase (SOD) in red blood cells. In blood and urine, the content of endogenous creatinine and protein was determined on a UNICO 2800 UV / VIS spectrophotometer using Olvex diagnostic kits. The concentration of Na and K in the blood was measured using an AEK-01 electrolyte analyzer, and in urine, using a PFA-378 flame photometer. Based on the data obtained, the functional parameters of the kidneys were calculated: the volume of urine output (ml / h / 100 g), glomerular filtration rate (GFR, ml / h / 100 g), relative tubular reabsorption of water (R _H2O ,%), filtration charge (FZ ) sodium, potassium and calcium (µmol / h / 100 g), their excretion (E _x , µmol / h / 100 g) and relative reabsorption of sodium and calcium (R _x ,%). Statistical processing of the results using Student t-test was carried out using GraphPad Prism 6.01 software.

The data obtained indicated that the content of lipid peroxidation products was significantly higher than the control in animals with toxic nephropathy and with the introduction of acyzole (Table 1). However, the content of malondialdehyde against the background of the use of acyzole at all periods was significantly lower than against the background of glycerol nephropathy. At the same time, there were no differences in the content of MDA on the 14th and 30th days of drug administration (Table 1). A similar picture was observed in the study of the concentration of hydroperoxides (table. 1). Catalase activity increases only at 4 weeks of rhabdomyolysis-induced damage to the kidneys, whereas against the background of the use of acizole, it increased already from the second week (Table 1). The introduction of acyzole contributed to an increase in enzyme activity, and no significant differences were found at different times of the studies with the use of the drug (Table 1). The activity of another enzyme of the antioxidant system - superoxide dismutase - increased only on the 14th day of toxic nephropathy, while on the 30th day it was even significantly lower than intact indices. In animals with intragastric administration of acyzole, the activity of SOD corresponded to the control (Table 1).

When studying the water-excreting pattern of the kidneys, the following was noted. Oliguria was reliably detected at week 2 of glycerol nephropathy, and at week 4, diuresis was 2 times higher than background values (Table 2). This was due to a significant decrease in glomerular filtration rate on the 14th day, which was replaced by a certain restoration of glomerular filtration rate on the 30th day with tubular reabsorption remaining at a low level (Table 2). In animals with the introduction of acizole, the volume of urine excreted was significantly higher than on the 14th and 30th days of experimental renal damage, and there were no significant differences between the rates of urine output at the 2nd and 4th week of acizole administration (Table 2). The increase in diuresis in animals of groups 4 and 5 was explained by an increase in glomerular filtration rate, while tubular reabsorption corresponded to control indicators (Table 2).

Analysis of the electrolyte-excreting function of the kidneys showed that in the 2nd group, the excretion of Na decreased, and in the 3rd group it was even higher than in intact animals (Table 2). Such changes at the 2nd week of the experiment were promoted by a significant decrease in the filtration charge of the cation, and at 4 weeks there was a decrease in sodium reabsorption, despite some restoration of its filtration charge in comparison with the two-week period (Table 2). In animals of groups 4 and 5, a significant increase in Na excretion was observed at all periods, which was accompanied by both an increase in cation reabsorption and a multiple increase in its filtration charge (Table 2). Potassium excretion during experimental glycerol nephropathy increased at all times against the background of a reduced filtration charge of the cation (Table 2). The use of acizole contributed to a slight increase in K excretion on the 14th day, and on the 30th day the indicator corresponded to the background (Table 2).

An important marker of impaired renal function is the protein content in the urine. Glycerol damage to the kidney tissue led to an increase in the protein content in the urine (Table 2). Against the background of iv administration of acizole, the protein concentration in the urine decreased, and after a month this indicator did not differ from the control figures (Table 2).

Thus, the use of acizole in toxic glycerol nephropathy reduced the severity of lipid peroxidation of cell membranes upon activation of antioxidant enzymes, and also improved renal function.

From the above it follows that the use of acyzole is an effective way to prevent experimental glycerin nephropathy.

Claims (1)

A method for the prevention of rhabdomyolysis-induced damage to the kidneys, including the formation of rhabdomyolysis-induced damage to the kidneys by intramuscular injection of 50% glycerol solution at a dose of 0.8 ml / 100 g weight, characterized in that from the next day after reproducing the experimental kidney damage for 30 days to animals daily injected acizole intragastrically at a dose of 30 mg / kg