RU2281505C1 - Method for predicting lead nephropathy in experimental animals in case of chronic intoxication - Google Patents

Abstract

FIELD: medicine, experimental biology, ecology, toxicology.

SUBSTANCE: the present innovation deals with studying pathogenetic mechanisms of toxic actions of heavy metals, lead, in particular, upon renal functional state and refers to the method for predicting lead nephropathy in experimental animals in case of chronic intoxication. In animal erythrocytes and renal tissue it is necessary to detect the concentration of malonic dialdehyde (MDA) simultaneously with the activity of Na⁺, K⁺, -ATP-ase of renal tissue and at values of MDA being 5.28±0.26 nM/ml erythrocytic mass and more, and in the cells of cortical and cerebral substance of renal tissue 2.79±0.27 and 3.69±0.33 nM/mg protein and more, correspondingly and activity values of Na⁺, K⁺, -ATP-ase of cortical and cerebral substances of renal tissue being 2.12±0.29 and 5.49±0.52 mcM Rn/mg protein/h and less, correspondingly, one should diagnose lead nephropathy. The innovation takes into account disorders of water- and electrolyte-excretory renal function in case of lead intoxication and pathogenetic mechanisms of these disorders.

EFFECT: higher accuracy of diagnostics.

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Description

The invention relates to medicine, experimental biology, ecology, toxicology and can be used to study the pathogenetic mechanisms of the toxic effects of heavy metals, in particular lead, on the functional state of the kidneys.

The kidneys are the main organ that secretes toxins absorbed by the body. A large number of nephrons provides an extensive surface of glomerular endothelial cells and tubule epithelium for contact with them (Osmanov I.M., 1996). Impairment of renal function during lead intoxication may be due to the direct effect of lead on the enzyme systems of the renal tubule epithelium, which provide active secretion and reabsorption. Thiol poison - lead blocks sulfhydryl groups of membrane proteins of the renal tubule cells, which affects their functional ability. There is evidence that impaired renal function can serve as an indicator of the harmful effects of abnormal concentrations of heavy metals on the body.

There is literature data indicating that lead, as a metal with variable valency, is able to initiate free radical oxidation processes (Ezhkova, T.S. et al., 1987). Some authors specifically indicate that lead is able to increase the level of active oxygen radicals (Adonaylo V.N., Oteiza P.I., 1999; Stohs S.J., Bagchi D., 1995). Moreover, penetrating red blood cells and disrupting heme synthesis, lead causes lead anemia, followed by hemic hypoxia of tissue cells, which also contributes to the formation of oxygen free radicals and the development of oxidative stress.

Along with the literature data, it should be noted that the question of the pathogenetic role of lipid peroxidation in the development of impaired water and electrolyte excretory function of the kidneys and the activity of membrane enzymes, in particular, Na, K-ATPase, of renal tissue remains insufficiently studied.

A known method for the diagnosis of lead nephropathy, based on a morphological study of the renal tissue of animals, resulting in hypotrophy and vacuolization of medium and small caliber arteries, mucous edema and muscle hypertrophy in arterioles, death of the brush border of cells, intranuclear inclusions in the proximal tubules (Sanchez-Fructuoso AI et al. Experimental lead nephropathy: treatment with calcium disodium ethylenediamine-tetraacetate. // Am. J. kidney Dis. - 2002. - Vol.40 (1) .- P.202-204). This model was taken by us as a prototype.

The disadvantage of the described method is that, firstly, violations of water and electrolyte excretory function of the kidneys during lead intoxication are not reflected, and secondly, a biochemical analysis revealing the pathogenetic mechanisms of these disorders is not performed.

The claimed invention has the following essential features: in experimental animals, against the background of lead intoxication in chronic poisoning, the activity of lipid peroxidation (LPO) is studied by the concentration of the final LPO product - malondialdehyde (MDA) in the membranes of red blood cells and renal tissue and at the same time the activity of Na ⁺ , K ⁺ ATP-basics of renal tissue, and with MDA values of 5.28 ± 0.26 nmol / ml of erythrocyte mass or more, and in the cells of the cortical and brain substances of the renal tissue, 2.79 ± 0.27 and 3.69 ± 0, respectively , 33 nmol / mg bel and more and activity values of Na ^+, K ⁺ -ATPase cortex and medulla of kidney tissue 2.12 ± 0.29 and 5.49 ± 0.52 pmol _P and / mg protein / hour or less, respectively diagnosed lead nephropathy . Red blood cells are universal representatives of tissue cells, therefore, a change in the content of MDA in red blood cells reflects its content in other cells, in particular in the cells of renal tissue. Those. changes in the concentration of MDA in red blood cells and cells of the kidney tissue are unidirectional, therefore, this indicator can be used to diagnose lead nephropathy not only in experimental animals, but also in humans.

The claimed invention is aimed at solving the problem of developing a method for the diagnosis of lead nephropathy in experimental animals with chronic poisoning.

The solution to this problem allows us to expand our understanding of the pathogenetic mechanisms of the development of nephropathy against the background of chronic lead intoxication in experimental animals.

To achieve this technical result, the claimed invention "A method for the diagnosis of lead nephropathy in experimental animals with chronic poisoning" includes the following essential features: in animals, the concentration of malondialdehyde (MDA) and the activity of Na ⁺ , K ⁺ -ATPase are determined in animals in red blood cells and renal tissue renal tissue, and with MDA values of 5.28 ± 0.26 nmol / ml of erythrocyte mass or more, and in the cells of the cortical and brain substances of the renal tissue, 2.79 ± 0.27 and 3.69 ± 0.33 nmol / mg, respectively protein and more and s Achen activity of Na ^+, K ⁺ -ATPase cortex and medulla of kidney tissue 2.12 ± 0.29 and 5.49 ± 0.52 pmol _P and / mg protein / hour or less, respectively diagnosed lead nephropathy.

Between the features of the claimed invention and the technical result, there is the following causal relationship: at the above MDA concentrations in the membranes of erythrocytes and homogenates of the renal tissue and the activity of Na ⁺ , K ⁺ -ATPase of the cortical and brain substances of the renal tissue, a violation of the water and electrolyte excretory function of the kidneys , i.e. lead nephropathy develops.

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 the above method for diagnosing lead nephropathy is not detected from it, which allows us to conclude that the criterion of "inventive step" is met.

The set of essential features characterizing the essence of the invention, in principle, can be used in medicine with obtaining a result consisting in a more accurate method for the diagnosis of lead nephropathy. Given that the red blood cell is the most accessible model for research in animals and humans, and the lipid peroxidation processes in it proceed similarly to tissue, the MDA content in red blood cells can be used to diagnose lead nephropathy not only in experimental animals, but also in humans. All this allows us to conclude that the invention meets the criterion of "industrial applicability".

The method is as follows. Lead acetate is dissolved in distilled water in such a way that 0.2 mg of lead accounts for 1.0 mg of lead for administration through a probe of a dose of 10 mg / kg weight (10.0 mg / kg weight = 1.0 / 100 g / 0, 2), and 0.1 mg of solution accounts for 1.0 mg for subcutaneous administration of the same dosage (10.0 mg / kg of weight = 1.0 / 100 g / 0.1 ml). For every 100 g of rat weight, 0.2 ml of lead acetate solution is injected through a probe and 0.1 ml of solution subcutaneously, which is not an excessive water load on the body of the experimental animal. The resulting solution of lead acetate is administered daily 1 time per day for 16-18 days.

At the end of the experiment, to study the activity of LPO and Na ⁺ , K ⁺ -ATPase, rats were killed under thiopental anesthesia; blood was taken from the heart using a 2.8% EDTA solution as an anticoagulant. An abdominal cavity was opened, clamps were placed on the renal arteries, and kidneys were removed, which, to study the activity of Na ⁺ , K ⁺ -ATPase, were placed in mortars with ice (t = + 4 ° С). The activity of lipid peroxidation was studied by the concentration of MDA (the end product of lipid peroxidation) according to the method of Osacawa T., the activity of Na ⁺ , K ⁺ -ATPase as the growth of inorganic phosphorus in the incubation medium.

Example 1. Male Wistar rats were injected with a solution of lead acetate through a tube into the stomach and subcutaneously at a dose of 10 mg / kg of animal weight every 16 days for 16-18 days. At the end of the experiment, the functional state of the kidneys was studied: diuresis, glomerular filtration, tubular reabsorption of water, excretion of sodium and potassium, their filtration charges and relative tubular reabsorption of sodium; the MDA concentration in erythrocytes and cells of the renal tissue was determined, the activity of Na ⁺ , K ⁺ -ATPase of the renal tissue was studied. Comparison of the data obtained during the experiment was carried out with a group of intact animals (control group).

The data obtained showed that there is a violation of the functional ability of the kidneys, which is manifested by an increase in spontaneous diuresis (p <0.001) (Table 1), due to a decrease in tubular reabsorption of water (p <0.05 for oral administration, p <0.001 for subcutaneous). With subcutaneous administration of this dosage, a significant decrease in glomerular filtration also contributes to an increase in urine output (p <0.05).

Table 1.
Indicators of water excretory function of the kidneys in rats with the introduction of lead acetate per os at a dose of 10 mg / kg body weight under conditions of spontaneous diuresis (M ± m). Groups of animals Diuresis Glomerular filtration Tubular reabsorption of water ml / h / 100 g ml / h / 100 g % Control group 0.085 ± 0.003 12.18 ± 0.42 99.41 ± 0.038 Pb - 10 mg / kg body weight, h-probe 0.11 ± 0.005 *) 12.1 ± 0.24 99.2 ± 0.088 *) Pb - 10 mg / kg body weight, s / c 0.12 ± 0.009 *) 10.94 ± 0.47 *) 98.83 ± 0.083 *) *) - the reliability of the results of the experimental group regarding control.

Exposure to lead at a dose of 10 mg / kg of the animal’s weight is accompanied by an increase in sodium excretion (p <0.001) as a result of a decrease in tubular reabsorption of this cation (p <0.02 with oral administration of lead acetate, p <0.02 with subcutaneous) (Table 2 ) Potassium excretion is increased with subcutaneous administration of a given dosage (p <0.05) due to an increase in its filtration charge.

Table 2.
Changes in the electrolyte excretory function of the kidneys in rats with the introduction of lead acetate per os at a dose of 10 mg / kg body weight under conditions of spontaneous diuresis (M ± m). Groups of animals Excretion Na Na filtration charge Na reabsorption Excretion K Filtration charge K μmol / h / 100 g % μmol / h / 100 g Control group 9.38 ± 0.26 1658.16 ± 52.93 99.49 ± 0.092 6.32 ± 0.36 53.85 ± 1.47 Pb - 10 mg / kg body weight, h-probe 12.14 ± 0.66 *) 1677.94 ± 71.96 99.16 ± 0.1 *) 6.7 ± 0.34 48.81 ± 3.67 Pb - 10 mg / kg body weight, s / c 12.82 ± 0.57 *) 1500.28 ± 48.94 *) 99.14 ± 0.094 *) 7.64 ± 0.33 *) 64.87 ± 2.65 *) *) - the reliability of the results of the experimental group regarding control.

Responsible for the processes of sodium reabsorption is a biomechanism that functions in the thin ascending knee of the Henle loop and distal tubules and is represented by Na ⁺ , K ⁺ -ATPase. Therefore, we studied the activity of Na ⁺ , K ⁺ -ATPase in the homogenates of the cortical and brain matter of the renal tissue. The data showed that the activity of Na ⁺ , K ⁺ -ATPase is reduced both in the cortical (p <0.05 for oral seeding, p <0.01 for subcutaneous) and in the medulla of the renal tissue (p <0.001 for both routes of administration of lead acetate) (Table 3), moreover, with subcutaneous administration of lead acetate, more pronounced changes are noted.

Table No. 3.
Indicators of Na ⁺ K ⁺ -ATPase activity in the homogenates of the cortical and brain layers of the renal tissue during lead intoxication (M ± m). Groups of animals Cortical substance Brain substance μmol R _n / mg protein / hour μmol R _n / mg protein / hour Control group 3.0 ± 0.33 6.72 ± 0.35 Pb - 10 mg / kg body weight, h-probe 2.12 ± 0.29 *) 4.82 ± 0.32 *) Pb - 10 mg / kg body weight, s / c 1.93 ± 0.17 *) 4.42 ± 0.26 *) *) - the reliability of the results of the experimental group regarding control.

One of the factors on which the activity of this enzyme depends is the state of the lipid microenvironment. To evaluate the role of lipid peroxidation in reducing the activity of Na ⁺ , K ⁺ -ATPase in our experiments, we studied the free radical oxidation system in erythrocyte membranes, as analogues of tissue cells, and in the homogenates of the cortical and brain layers of renal tissue. It was found that oxidative stress develops in rats, LPO processes are activated, and a statistically significant increase in the concentration of MDA in erythrocytes is observed (p <0.01 for subcutaneous administration of lead acetate, p <0.001 for oral), as well as in cortical cells (p <0.001 ) and brain (p <0.001) layers of renal tissue (Table 4).

Table 4.
LPO in erythrocytes and renal tissue (according to the concentration of MDA) (M ± m). Groups of animals The concentration of MDA in red blood cells The concentration of MDA in cortical cells. renal tissue The concentration of MDA in the brain cells. renal tissue nmol / ml nmol / mg protein nmol / mg protein Control group 4.54 ± 0.16 1.26 ± 0.06 2.73 ± 0.1 Pb - 10 mg / kg body weight, h-probe 5.32 ± 0.18 *) 2.79 ± 0.27 *) 4.08 ± 0.2 *) Pb - 10 mg / kg body weight, s / c 6.09 ± 0.23 *) 2.82 ± 0.046 *) 4.21 ± 0.1 *) *) - the reliability of the results of the experimental group regarding control.

Changes in the processes of SRO in red blood cells and renal tissue are unidirectional, which allows the use of LPO activity indicator in red blood cells for the diagnosis of lead nephropathy in humans.

Thus, LPO activation in erythrocytes and homogenates of the cortical and brain layers of the renal tissue is noted, as evidenced by an increase in the concentration of MDA. As a result of LPO activation, the lipid microenvironment of the Na ⁺ , K ⁺ -ATPase enzyme changes. This leads to a decrease in its activity, as a result of which there is a violation of the electrolyte excretory function of the kidneys. Those. the use of MDA concentration as a diagnostic criterion for lead nephropathy in erythrocytes and in renal tissue and the activity of Na ⁺ , K ⁺ -ATPase are quite justified.

Claims (1)

A method for the diagnosis of lead nephropathy in experimental animals with chronic poisoning, including laboratory tests, characterized in that in animals the concentration of malondialdehyde (MDA) and the activity of Na ⁺ , K ⁺ -ATPase of the renal tissue are determined in animals in erythrocytes and kidney tissue the values of MDA (5.28 ± 0.26) nmol / ml of erythrocyte mass and more, and in the cells of the cortical and medulla of the renal tissue, respectively (2.79 ± 0.27) and (3.69 ± 0.33) nmol / mg of protein or more and activity values of Na ⁺ , K ⁺ -ATPase basics of cortex new and brain matter of the kidney tissue (2.12 ± 0.29) and (5.49 ± 0.52) μmol R _n / mg protein / h or less, respectively, lead nephropathy is diagnosed.