RU2704022C1 - Method of proteomic correction of oxalate nephrolithiasis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns application of tetrapeptide Leu-Ile-Lys-His, leucine-isoleucine-lysine-histidine, as agent for proteome correction of oxalate nephrolithiasis.

EFFECT: invention provides a wider range of methods for pharmacological correction of oxalate nephrolithiasis and high antilithogenic activity.

1 cl, 5 dwg, 4 tbl, 2 ex

Description

The invention relates to medicine, namely pharmacology, urology and can be used for pharmacological correction of oxalate nephrolithiasis.

According to modern data, the epidemiology of the ICD is comparable to that for diabetes mellitus - every 11th resident of developed countries suffers from nephrolithiasis, and the cost of a set of medical, preventive and rehabilitation measures in the United States alone exceeds $ 10 billion annually. At the same time, despite the achievements of modern urology, lithotripsy in its various variants, which is a very traumatic method and is accompanied by a number of serious side effects, remains the main method of kidney stone litholysis. Against this background, the pharmacological support for the treatment of ICD is mainly auxiliary in nature. Means with specific activity for nephrolithiasis, practically does not exist.

One of the most modern approaches to the development of new effective antilithogenic agents is the so-called proteomic approach, according to which the target for pharmacological litholysis of urolite or the prevention of its formation is an organic matrix of kidney stones. Considering the protein nature of the basic structural and functional elements of the matrix, it is believed that specific protein and amino acid molecules can be an external ligand with affinity for this type of “substrate” (or processes that determine its formation). The development of new antilithogenic agents is based on the search for such molecules.

The closest to the achieved result the prototype is a peptide agent with antilithogenic action, obtained by acetic acid extraction from pork kidneys (RF patent 2652339). It is administered orally at a dose of 15 mg. The method provides an antilithogenic effect, expressed in 100% destruction of large and medium kidney stones against the background of oxalate nephrolithiasis.

The disadvantages of this tool are the inability to accurately establish the biologically active substances that make up its composition and determine its therapeutic effect in urolithiasis, as well as the presence of a significant amount of related substances in the composition of the drug.

The authors propose a new method of proteomic correction of oxalate nephrolithiasis, characterized by high therapeutic efficacy.

The technical result of the claimed invention is to expand the range of methods for pharmacological correction of oxalate nephrolithiasis.

The technical result is achieved by the fact that for the proteomic correction of oxalate nephrolithiasis, the Leu-Ile-Lys-His tetrapeptide (leucine - isoleucine - lysine - histidine) is used.

The effectiveness of the method of proteomic correction of oxalate nephrolithiasis was found experimentally.

Experiments to study the effectiveness of the method of proteomic correction of oxalate nephrolithiasis were carried out on 20 male Wistar rats with an average weight of 150 g), divided equally into 2 groups: control (modeling of oxalate nephrolithiasis) and experimental (modeling of oxalate nephrolithiasis + administration of Leu-Ile-Lys-His tetrapeptide) (leucine - isoleucine - lysine - histidine).

Throughout the experiment, the animals were in individual cages adapted for collecting urine, in the conditions of a standard laboratory diet. According to the experimental conditions, rats were divided into 2 groups of 15 animals each. In the first (control group), for the purpose of modeling oxalate nephrolithase for 6 weeks, the rats were provided with a free 1% ethylene glycol solution as a drink. In the second (experimental group) rats, for 6 weeks, a 1% solution of ethylene glycol was also freely available, but from the 4th week until the end of the experiment, they administered Leu-Ile-Lys-His tetrapeptide daily at a dose of 2.8 mg

Every week during the experiment, in each group of rats, a daily urine volume was collected in which the protein concentration, creatinine and the activity of marker enzymes of nephrothelial damage - lactate dehydrogenase (LDH) were determined to assess the degree of damage to renal tissues when modeling oxalate nephrolithiasis using a DIRUI CS-T240 automatic biochemical analyzer. and γ-glutamyl transferase (GGT). Taking into account the daily volume of diuresis, the excretion of protein and creatinine was calculated.

To determine the concentration of the protein, a biuret method was used, which is based on the formation of a blue-violet complex with copper ions, the optical density of which is directly proportional to the protein concentration. Creatinine concentration was determined by the kinetic method without deproteinization, based on the Jaffe reaction of the formation of a red-orange colored complex. The principle of the method for determining the activity of LDH is based on the reaction of reduction of pyruvate to lactic acid, the reaction rate is proportional to the activity of LDH in the sample. The determination of GGT activity is based on the transfer reaction of L-γ-glutamyl-3-carboxy-p-nitroanilide to glycylglycine, which is catalyzed by this enzyme to form colored 5-amino-2-nitrobenzoate.

After 6 weeks of the experiment, the rats were euthanized under ether anesthesia and both kidneys were removed, one of which was used to determine the activity of free radical oxidation in the renal tissue, and the other to conduct morphological studies.

The activity of the process of free radical oxidation in the kidney tissue was evaluated by the combination of prooxidant and antioxidant parameters. The former included the content of thiobarbitrate reactive products (TBRP) and total prooxidant activity (OPA), and the latter included total antioxidant activity (OAA) and the activity of antioxidant enzymes: glutathione peroxidase (GPO), superoxide dismutase (SOD), and catalase (CAT).

Thiobarbiturate-reactive products (TBPD) were determined by the colorimetric method, measuring the color intensity of the solution during the chemical reaction of TBPP with thiobarbituric acid. Total prooxidant activity (OPA) was evaluated by the accumulation of peroxidation products of TWIN-80, reacting with thiobarbituric acid.

Total antioxidant activity (OAA) was evaluated as an integrative indicator of the activity of all enzymatic and non-enzymatic neutralization factors of free radicals by the degree of inhibition of Fe ²⁺ / ascorbate-dependent oxidation of TWIN-80 tissue homogenet. To determine the activity of glutathione peroxidase (GPO), the concentration of reduced glutathione was measured in a color reaction with Ellman's reagent. Superoxide dismutase (SOD) activity was determined by suppressing the formation of nitroformazan, a colored oxidation product of nitrotetrazolium by superoxide radicals formed by the interaction of phenazine metasulfate and nicotine amididine nucleotide (NADH). Catalase activity (CAT) was evaluated by suppressing the oxidation enzyme of sodium molybdate with hydrogen peroxide.

To conduct morphological studies, the kidneys were fixed in a 10% formalin solution, processed according to the standard method, and a cross section of 6 μm thick was prepared through the renal papilla. The obtained sections were stained with hematoxylin and eosin, methenamine-silver. Calcium deposits were identified by the Koss histochemistry method and using a computer program, the number of calcium deposits in the field of view was calculated on the images and their size was determined. Morphometric studies were performed using ImageJ 1.43 and AxioVision 3.1 software packages.

Statistical processing of the results was carried out using the computer program Statistica 12.0. The results of biochemical studies are presented by the median (M) and interquartile range (25%, 75%). The results of morphometric studies are presented by the mean (M) and standard error of the mean (m). Statistical comparisons between groups were carried out using the non-parametric Kruskal-Walless criteria and the Mann-Whitney U-test, comparisons within the group relative to the initial level were carried out using the non-parametric Wilcoxon test. The results were recognized reliable with a value of the confidence indicator p <0.05.

The method is illustrated by the figures:

Figure 1 - along the horizontal axis - the name of the indicator and the unit of measurement. The vertical axis is the value of the indicator. Values are presented as the median and minimum-maximum, excluding emissions (non-outlier range);

Figure 2 - along the horizontal axis - the name of the indicator and the unit of measurement. The vertical axis is the value of the indicator. Values are presented as the median and minimum-maximum, excluding emissions (non-outlier range);

Figure 3 - coloring according to Koss. Magnification × 400;

- контрольная группа,

подопытная группа. Значения представлены в виде медианы и минимума-максимума без учета выбросов (non-outlier range). р_к - уровень статистической значимости изменений значения соответствующего показателя относительно контрольной группы;Figure 4 - along the horizontal axis - the name of the indicator and the unit of measurement. The vertical axis is the value of the indicator.

- control group,

experimental group. Values are presented as the median and minimum-maximum, excluding emissions (non-outlier range). p _to - the level of statistical significance of changes in the value of the corresponding indicator relative to the control group;

контрольная группа, -

подопытная группа. Значения представлены в виде медианы и минимума-максимума без учета выбросов (non-outlier range), р_к - уровень статистической значимости изменений значения соответствующего показателя относительно контрольной группы.Figure 5 - along the horizontal axis - the name of the indicator and the unit of measurement. The vertical axis is the value of the indicator. -

control group, -

experimental group. The values are presented in the form of the median and minimum-maximum without taking into account emissions (non-outlier range), p _k - the level of statistical significance of changes in the value of the corresponding indicator relative to the control group.

The research results are presented in the examples.

Example 1

The results of the experiments showed that in rats of the control group characteristic biochemical signs of the development of oxalate nephrolithiasis were observed (Table 1).

So, during the experiment, there was a decrease in urine output, creatinine and protein excretion. The level of urination at weeks 4 and 5 was inferior to baseline values by 1.6 times and 2.1 times, respectively (p = 0.047 and p = 0.025). At the same time, creatinine excretion also reached a minimum at the 5th week, when it was 2.7 times less than at the initial level (p = 0.022). Protein excretion significantly decreased already from the 2nd week, reaching a minimum on the 42nd day of the observation period, when it was 13.5 times lower than the initial value (p = 0.017).

In addition, the activity of marker enzymes of damage to the renal epithelium increased (Table 2).

LDH activity, expressed in U / L, consistently increased throughout the observation period. As a result, by the end of the 6th week it was 3.3 times higher than the initial level (p = 0.022). In addition, LDH activity, expressed in units / mg of creatinine per day, also increased starting from the 5th week of the experiment.

As a result, on day 42, it exceeded the initial level by 4.9 times (p = 0.017). In parallel, a tendency toward an increase in GGT activity, expressed in units / L, was recorded. Over the 6 weeks of the observation period, the value of this indicator increased 1.7 times relative to the initial level.

The study of the activity of the process of free radical oxidation in the kidneys of rats of the control group made it possible to determine the following parameters of oxidative damage to the kidneys when modeling nephrolithiasis.

Indicators of prooxidant status — the concentration of TBRP and OPA — had values of 1.9 (1.3; 10.9) μmol and 46.0 (38.6; 54.0)%, respectively (Fig. 1).

Indicators of antioxidant status - OAA, GPO activity, CAT activity and SOD activity - were 58.1 (52.5; 63.5)%, 49.7 (37.4; 53.9)%, 48.2 (43 , 7; 50.9)% and 13.2 (11.7; 20.0)%, respectively (Fig. 2).

A morphological study of the kidneys of rats in the control group showed that when stained with hematoxylin and eosin in the areas of nephrolithiasis, the tubular epithelium was in a state of hyaline droplet dystrophy. The apical edge of the cells was destroyed in places, the brush border is not visible in some places. At the same time, the tubules looked cystic-distended. Nephrothelial cells of the lining tubules were destroyed, either flattened or atrophic. Around the tubules, mild or moderate inflammatory lympho-plasmacytic infiltration was determined. The phenomena of nephrosclerosis were noted. The vessels were in a state of pronounced plethora.

When histochemical staining for calcium was performed according to the Koss method, in the tubules of the kidneys of rats of the control group, deposits of brownish-black stone deposits of various shapes and sizes, arranged individually or in groups, were noted. The number of deposits in the gaps of the tubules ranged from 6 to 19 and averaged 7.9 ± 1.6 in the field of view with an increase of × 400, with a modal value of 6 (Fig. 3).

When conducting computer morphometry, the area of stone deposits ranged from 48.8 μm ² to 789.9 μm ² , an average of 298.8 ± 34.2 μm ² . A more detailed analysis of the distribution of stones depending on their area revealed that the number of deposits of stones with an area of 20 μm ² to 100 μm ² was 16.7%, the number of deposits with an area of 100 μm ² to 300 μm ² was 47.2% and the content deposits with an area of more than 300 microns ² and more amounted to 36.1%. The histogram of the distribution of stone deposits had a shift to the right and the distribution peak was in the area of stones from 100 μm ² to 300 μm ² .

Example 2

The experiments showed that with prolonged use of the Leu-Ile-Lys-His peptide for the correction of simulated nephrolithiasis, significant differences were observed in the pathology pattern compared with the control group.

A study of the parameters of the excretory function of the kidneys of rats of the experimental group showed that the dynamics of creatinine excretion was characterized by a significant increase in the value of this indicator in the 5th and 6th weeks of the observation period (Table 3).

As a result, it exceeded the initial level by 85% and 184%, respectively (p = 0.038 and p = 0.021). Recall that in the control group, creatinine excretion, in contrast, decreased in the second half of the experiment. As a result, the creatinine excretion rate at week 5 was statistically significantly higher than that in the control group by 199% (p = 0.030). In addition, protein excretion was weaker than in the control. Therefore, a significant difference compared with the initial level was recorded only at the end of the 6th week. The dynamics of diuresis was also characterized by a slight downward trend.

Very significant changes also occurred in the dynamics of the activity of marker enzymes of damage to the renal epithelium in rats of the experimental group (Table 4).

It turned out that LDH activity, expressed in U / L, throughout the experiment as a whole remained stable without an upward trend, while in the disease control it statistically significantly increased at the 5th and 6th weeks.

In addition, LDH activity, expressed in units / mg of creatinine per day, in rats of the experimental group, starting from the 5th week of the experiment, decreased and differed statistically significantly with the same indicator in the control group: in the 5th week - by 69% (p = 0.013), and in the 6th week - by 94% (p = 0.003).

The decrease in the described indicator relative to the initial level in the experimental group by the end of the experiment was 90% (p = 0.008). Similar overall dynamics characterized changes in GGT activity in the experimental group. The activity of this enzyme, expressed in U / L, remained stable throughout the observation period with no upward trend. GGT activity, expressed in units / mg of creatinine per day, in the 5th and 6th weeks significantly decreased relative to the initial level: by 66% and 86%, respectively (p = 0.008 and p = 0.008). Moreover, it was statistically significantly less than in the control group in the same period of time: in the 5th week - by 73% (p = 0.004), and in the 6th week - by 81% (p = 0.0007).

A study of the activity of the process of free radical oxidation in the kidneys of rats of the experimental group revealed that after prolonged use of the Leu-Ile-Lys-His peptide, the OPA decreased 6.0 times relative to the control group (Fig. 4), amounting to 7.7 (5, 0; 13.0) (p = 0.007). The concentration of TBRP was 7.5 (6.6; 10.7) μmol, which was greater than in the control group, but this difference was not statistically significant.

Against this background, the activity of the antioxidant enzyme KAT after the treatment was weakened 3.5 times relative to the control of the disease and amounted to 13.8 (10.5; 24.7)% (Fig. 5, p = 0.002). The total antioxidant activity, the activity of GPO and SOD amounted to 56.1 (53.9; 58.9)%, 59.8 (58.3; 66.4)% and 6.5 (3.5; 10.7)% respectively, and did not statistically differ from the indicators of the control group.

A morphological study of the kidneys of rats in the experimental group showed that no nephrolithiasis was detected in this group. The kidney tissues were in a normal state, neither inlaying of the tubules with calcium salts, nor the formation of large calculi was observed.

Claims (1)

The use of the tetrapeptide Leu-Ile-Lys-His, leucine - isoleucine - lysine - histidine, as a means for proteomic correction of oxalate nephrolithiasis.

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