RU2696580C1 - Method for preventing renal dysfunction with an arginase ii inhibitor in experiment - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to experimental pharmacology and urology, and concerns preventing ischemia-reperfusion renal injuries. For this purpose, Wistar rats 120 minutes before simulating renal ischemia by applying non-traumatic clamps on renal pedicles for 40 minutes followed by reperfusion of blood flow, arginase II KUD975 inhibitor 3 mg/kg is administered intragastrically through a probe.EFFECT: method provides effective prevention of renal ischemia-reperfusion injury, including due to endothelium-protective properties of the preparation administered at the experimentally established time.1 cl, 2 tbl, 1 ex

Description

The invention relates to medicine, in particular to experimental pharmacology and urology.

Acute renal damage remains a serious public health problem, especially in hospitalized and critical patients, and is a major risk factor for chronic kidney disease [Belayev L.Y., Palevsky P.M. The link between acute kidney injury and chronic kidney disease. // Curr Opin Nephrol Hypertens - 2014 .-- 23 .-- 149-154; Coca S.G., Singanamala S., Parikh C.R. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. // Kidney Int - 2012. - 81. - 442-448]. Despite supportive care using renal replacement therapy, acute renal failure is still associated with increased short-term and long-term mortality, not only due to renal failure, but also due to concomitant dysfunction of other organs and systems [Kao CC, Yang WS, Fang JT, et al. Remote organ failure in acute kidney injury. // Journal of the Formosan Medical Association. - 2018]. The leading cause of acute renal damage is insufficient perfusion of the kidney tissue, exacerbated by further reperfusion [Martin, J.L., Gruszczyk, A.V., Beach, T.E. et al. Mitochondrial mechanisms and therapeutics in ischaemia reperfusion injury // Pediatr Nephrol - 2018. https://doi.org/10.1007/s00467-018-3984-5].

A known method for the prevention of ischemic-reperfusion injury of kidneys in an experiment (Arginase-2 mediates renal ischemia-reperfusion injury / Raup-Konsavage WM, Gao T., Cooper TK, et al. // American Journal of Physiology-Renal Physiology. - 2017. - 313: 2. - F522-F534), including modeling of pathology by applying atraumatic clamps to the renal legs of laboratory mice for 28 minutes. For the prevention of ischemic reperfusion injury, a single intraperitoneal administration of the non-selective arginase inhibitor S- (2-boronoethyl) -L-cysteine 18 hours before the ischemic stimulus was used. The method provides a reduction in the severity of histopathological changes, processes of oxidative stress and apoptosis, the synthesis of pro-inflammatory cytokines; increased the formation of nitric oxide and phosphorylation of eNOS, the expression of coactivator 1 alpha γ-receptor-activator of proliferation of peroxisomes and contributed to the preservation of mitochondrial ultrastructure.

The main disadvantage of this method is that for prophylaxis a non-selective inhibitor of the arginase enzyme is used as a pharmacological agnet, which reduces the activity of both arginase II and arginase I. As a result of the suppression of the activity of arginase I, side effects such as hyperammonemia, manifested in insufficiency, can be observed. of the urea enzyme cycle and leading to ammonia poisoning of the body, which can aggravate the symptoms of accumulation of other nitrogen metabolism products in acute renal respiration - creatinine and urea. On the other hand, the functional state of the kidneys was not evaluated as the main prognostic criterion used in clinical practice.

Closest to the claimed is a method of preventing ischemic reperfusion injury of the kidneys in the experiment (Lempiäinen J, Finckenberg P, Levijoki J, Mervaala E. AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney. British Journal of Pharmacology. 2012; 166 (6) : 1905-1915. Doi: 10.1111 / j.1476-5381.2012.01895.x.), Including reproduction of a pathology model and administration of 5-amino-4-imidazolecarboxamide riboside-1-β-D-ribofuranoside to a laboratory animal at a dose of 500 mg / kg once, moreover, 5-amino-4-imidazolecarboxamide riboside-1-β-D-ribofuranoside is administered intravenously 30 minutes before the modeling of ischemia and the severity of froprotektivnyh properties produce once after 24 hours of reperfusion without calculating glomerular filtration rate and fractional sodium excretion.

The main disadvantage of this method is that the prevention of renal dysfunction is evaluated only by measuring serum concentrations of creatinine and urea without calculating the glomerular filtration rate and fractional sodium excretion, as well as the lack of data on the dynamics on the third day of the experiment, when pathological changes reach their maximum, which It does not allow us to fully talk about the effective prevention of renal impairment in the experiment.

On the other hand, 5-amino-4-imidazole carboxamide riboside-1-β-D-ribofuranoside in clinical trials of the third phase has not proved its effectiveness in ischemic and reperfusion heart injuries that have a similar pathogenesis with the studied model [Pokrywka A, Cholbinski P, Kaliszewski P, Kowalczyk K, Konczak D, Zembron-Lacny A. Metabolic modulators of the exercise response: Doping control analysis of an agonist of the peroxisome proliferator-activated receptor δ (GW16) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). J Physiol Pharmacol. 2014; 65 (4): 469–76.]. In addition, isoflurane, which according to the literature has nephroprotective activity, was used as an anesthetic, which could distort the results of the study [Carraretto, A.R. et al. Does propofol and isoflurane protect the kidney against ischemia / reperfusion injury during transient hyperglycemia ?. Acta Cir. Bras. [online]. 2013, vol. 28, n.3 [cited 2018-05-27], pp. 161-166.].

The objective of the invention is to provide a more effective method for the prevention of impaired renal function using an arginase II KUD975 inhibitor.

This object is achieved in that a method for the prevention of renal impairment using an arginase II KUD975 inhibitor is proposed, which includes drug prophylaxis by administering a single solution of it to laboratory animals, followed by modeling of pathology by applying atraumatic clamps to the kidney legs for 40 minutes, followed by reperfusion of blood flow in the kidneys moreover, Wistar white rats are used as laboratory animals, an argi inhibitor is used as a medicine Naza II KUD975, administered at a dose of 3 mg / kg intragastrically via a probe 120 minutes before ischemia simulation, and the severity of changes in functional parameters is assessed after 24 and 72 hours of reperfusion.

The technical result of the invention is an effective method for the prevention of impaired renal function using an arginase II KUD975 inhibitor in an experiment, confirmed by the results of biochemical studies and calculating the glomerular filtration rate and fractional sodium excretion. The substance KUD975 under study is a synthesized compound of a phenolic nature - methyl ester (2 - ((1-hydroxynaphthalen-2-yl) thio) acetyl) -D-proline.

An important role in the pathogenesis of ischemic reperfusion damage to the kidneys is played by an enzyme that regulates the exchange of L-arginine and nitric oxide - arginase, which exists in two isoforms: arginase I and arginase II [Arginase-2 mediates renal ischemia-reperfusion injury / Raup-Konsavage WM, Gao T., Cooper TK, et al. // American Journal of Physiology-Renal Physiology. - 2017 .-- 313: 2. - F522-F534].

The main advantage of the proposed method is that the introduction of an arginase II KUD975 inhibitor at a dose of 3 mg / kg once 120 minutes before the induction of ischemia leads to a pronounced prevention of renal dysfunction in the experiment, which is confirmed by the results of biochemical studies and the calculation of glomerular filtration rate and fractional sodium excretion .

The method is carried out as follows.

The experiments were performed on 80 male Wistar rats weighing 180-220 g. Quarantine-free rats were taken for the study.

The choice of male rats in the experiment is associated with the presence of cyclic hormonal changes in females and the renoprotective effects in estrogens, which can affect the purity of the experiment.

Each group included 10 rats. The first group is a group of false-operated animals (24 hours), the second group is a group of false-operated animals (72 hours), the third group is with modeling of ischemic reperfusion damage to the kidneys (control, 24 hours), the fourth group is with modeling of ischemic reperfusion damage of the kidneys ( control, 72 hours), the fifth group - with pathology correction with an arginase II KUD975 inhibitor (24 hours of reperfusion), the sixth group - with pathology correction with an arginase II KUD975 inhibitor (72 hours of reperfusion), the seventh group - with pathology correction L-norvalino (Comparison formulation, 24 hours of reperfusion), the eighth group - correction pathology L-norvaline (comparing drug 72 hours of reperfusion).

Simulation of ischemic reperfusion damage to the kidneys was performed as follows: under general anesthesia (chloral hydrate, 300 mg / kg intraperitoneally), median laparotomy was performed, renal legs were isolated, and atraumatic vascular clamps were sequentially applied to both legs with an ischemic period of 40 minutes under the control of microcirculation. Then clamps were removed, the abdominal cavity was washed with 0.9% sodium chloride solution and the wound was sutured in layers. To obtain urine samples, animals were placed in metabolic cells with free access to water for 12 or 24 hours. Next, diuresis was measured and samples were taken for further research. After 24 or 72 hours of reperfusion, the laboratory animal was anesthetized by intraperitoneal injection of chloral hydrate at a dose of 300 mg / kg of animal body weight, relaparotomy was performed and blood was taken from the right ventricle for biochemical studies.

In the fifth and sixth experimental groups, animals were injected with an arginase inhibitor II KUD975 at a dose of 3 mg / kg once intragastrically through a probe 120 minutes before the application of vascular clamps. In the seventh and eighth experimental groups, animals were injected with L-norvaline at a dose of 100 mg / kg once intraperitoneally 30 minutes before the application of vascular clamps.

The severity of renoprotective properties was judged by the results of biochemical studies and the calculation of glomerular filtration rate and fractional sodium excretion after 24 and 72 hours of the reperfusion period.

Serum creatinine and urea levels were determined using the photocolorimetric method using standard reagent kits. The concentration of potassium and sodium ions in the blood serum was determined by the standard method attached to the sets on an automatic analyzer.

The clearance of endogenous creatinine (glomerular filtration rate (GFR) was calculated as follows:

Fractional sodium excretion (FEna) was calculated using the following formula:

The reliability of changes in the absolute parameters was determined by the difference method of variation statistics with finding the average values of the shifts, the arithmetic mean and the probability of a possible error (p) according to Student tables. Differences were evaluated as significant at p <0.05. For calculations, we used the statistical analysis program Microsoft Excel.

EXAMPLE OF SPECIFIC PERFORMANCE

After 24 hours of reperfusion in the group with pathology modeling, the serum creatinine level did not differ significantly from the group of false-operated animals and amounted to 55.7 ± 0.8 μmol / L and 57.9 ± 2.38 μmol / L, respectively. However, due to a decrease in urine output, a glomerular filtration rate decreased from 0.51 ± 0.03 ml / min in the group of false-operated animals to 0.17 ± 0.02 ml / min in the ischemia-reperfusion group. After 72 hours, there was a progressive decrease in the filtration capacity of the kidneys, expressed in an increase in serum creatinine from 56 ± 1.52 μmol / L to 120 ± 3.45 μmol / L and a decrease in glomerular filtration rate by 8.2 times from 0.49 ± 0 , 03 ml / min in the group of false-operated animals to 0.06 ± 0.01 ml / min in the group with modeling of pathology.

A study of serum urea concentration showed that modeling a 40-minute bilateral model of renal ischemia-reperfusion led to an increase in this indicator after 24 hours of reperfusion from 5.35 ± 0.21 mmol / L to 9.7 ± 0.68 mmol / L, which somewhat leveled after 72 hours, reaching the level of 8.33 ± 0.23 mmol / l.

In the group of false-operated animals, the level of fractional sodium excretion after 24 and 72 hours was 0.38 ± 0.02% and 0.5 ± 0.02%. Modeling of acute renal damage of ischemic reperfusion genesis led to an increase in FeNa to 2.24 ± 0.12% to 7.4 ± 0.78% after 24 and 72 hours of reperfusion, respectively.

Prevention of renal impairment by an arginase II KUD975 inhibitor led to a decrease in serum creatinine concentration after 72 hours of reperfusion to 73.7 ± 2.22 μmol / L and an increase in glomerular filtration rate to 0.31 ± 0.02 ml / min and 0.26 ± 0.02 ml / min on the first and third days of the experiment, respectively, significantly differing from the parameters of the control group and the comparison drug group (p <0.05). The level of serum urea concentration also decreased to 6.47 ± 0.37 mmol / L and 6 , 9 ± 0.23 mmol / L after 24 and 72 hours of reperfusion, respectively. Fractional sodium excretion against the background of prophylactic use of KUD975 at a dose of 3 mg / kg, reflecting tubular dysfunction, also decreased, amounting to 1.14 ± 0.11% and 1.8 ± 0.09% at different time points of the experiment, exceeding the effectiveness of the comparison drug .

Prevention of renal impairment by L-norvaline led to a decrease in serum creatinine concentration after 72 hours of reperfusion to 93.4 ± 3.39 μmol / L and an increase in glomerular filtration rate to 0.21 ± 0.02 ml / min and 0.15 ± 0 , 01 ml / min on the first and third days of the experiment, respectively, differing significantly from the parameters of the control group only on the third day of the experiment (p <0.05). The level of serum urea concentration decreased to 7.8 ± 0.29 mmol / L and 7, 8 ± 0.48 mmol / L after 24 and 72 hours of reperfusion, respectively. Fractional sodium excretion against the background of the prophylactic use of L-norvaline was 1.42 ± 0.13% and 2.76 ± 0.21% at various time points in the experiment. The dynamics of biochemical markers of acute renal damage, glomerular filtration rate and fractional sodium excretion are presented in tables 1 and 2.

Table 1

Dynamics of biochemical markers of acute renal damage in the experimental groups (M ± m; n = 10)

Note: ^x - p <0.05 in comparison with the group of false-operated animals; ^y - p <0.05 compared with the ischemia-reperfusion group.

table 2

Dynamics of glomerular filtration rate and fractional sodium excretion in experimental groups (M ± m; n = 10)

Note: ^x - p <0.05 in comparison with the group of false-operated animals; ^y - p <0.05 compared with the ischemia-reperfusion group.

Thus, in the proposed method, intragastric administration of an arginase II inhibitor KUD975 at a dose of 3 mg / kg once 120 minutes before the induction of ischemia leads to a more pronounced prevention of impaired renal function than administration of L-norvaline at a dose of 100 mg / kg, which is confirmed by biochemical results research and calculation of glomerular filtration rate and fractional sodium excretion after 24 and 72 hours of the reperfusion period.

Claims (1)

A method for the prevention of renal impairment using an arginase II KUD975 inhibitor, which includes drug prophylaxis by administering a single solution of it to laboratory animals, followed by modeling of pathology by applying atraumatic clamps to the kidney legs for 40 minutes, followed by reperfusion of blood flow in the kidneys, characterized in that as laboratory animals use white Wistar rats; as a medicine, an arginase II KUD975 inhibitor administered at a dose of 3 mg / kg int trizheludochno gavage for 120 minutes to simulate ischemia, and evaluation of expression changes of functional parameters carried in dynamics after 24 and 72 hours of reperfusion while avoiding the side effects typical of small and non-selective inhibitors of arginase.