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UDC 616.62-003.7-092.4
PHARMACOLOGICAL MODULATION OF THE EXPRESSION OF INTRARENAL INHIBITORS OF CRYSTALLIZATION AND OXIDATIVE STRESS MARKERS OF NEPHROCYTES BY EXPERIMENTAL KIDNEY STONE DISEASE
Altai State Medical University, Barnaul A.Yu. Zharikov, N.N. Yakushev
The present research is aimed at the study of sodium citrate and a-tocopherol acetate effect on the level of expression of Tamm-Horsfall protein, osteopontin, bikunin, malondialdehyde and superoxide dismutase in the kidney tissue of rats by experimental kidney stone disease.
The experiments were performed in 60 male Wistal rats at the age of 2-3 months and 250-300 g of weight. The animals were equally divided into 4 groups: the group of intact rats - Gint (42 days in standard laboratory conditions without KSD modelling and without injection of the studied drugs), control group - Gc (42 days of KSD modelling), group of treatment by sodium citrate - Gnc (42 days of KSD modelling + daily injection of sodium citrate from 22nd to 42nd day of experiment), group of treatment by a-tocopherol acetate - Ga (42 days of KSD modelling + daily injection of a-tocopherol acetate from 22nd to 42nd day of experiment). The experimental kidney stone disease was modelled in accordance with the generally accepted ethyleneglycol model. The expression of Tamm-Horsfall protein, osteopontin, bikunin, superoxide dismutase and malondialdehyde was determined by means of indirect two-stage streptavidin-biotin method with the control of reaction specificity.
It was determined, that the expression of Tamm-Horsfall protein, osteopontin, bikunin, superoxide dismutase and malondialdehyde in conditions of KSD modelling was exposed to considerable alterations in relation to the healthy rats. On this background, long-term continuous injection of sodium citrate and a-tocopherol acetate was accompanied by the normalization of expression of intrarenal inhibitors of crystallization and oxidative stress markers of nephrocytes.
Key words: kidney stone disease, intrarenal inhibitors of crystallization, oxidative stress markers, sodium citrate, a-tocopherol acetate.
Introduction
According to modern concepts, the qualitative and quantitative changes in the expression of intrarenal crystallization inhibitors, such as the Tamm-Horsfall protein (THP), osteopontin (OPN) and bikunin (BIK), play a crucial role in the process of stone formation by kidney stone disease (KSD), as a result of which they lose their inhibitory properties or even become stimulants of lithogenesis [1-4]. In addition, the destructive role of the process of free radical oxidation (FRO) by KSD, which leads to the formation of a primary source of lithogenesis with subsequent precipitation of the crystalline material from urine [5-7], is not subject to doubt. Histochemical markers of the FRO process can be the level of expression of malonic dialdehyde (MDA) and superoxide dis-mutase (SOD) in kidneys, whose role in activation and inhibition of FRO is well known [5-7].
In this connection, changes in the parameters of the intraluminal expression of mentioned substances can become an important indicator of the effectiveness of a method of pharmacological correction of urolithiasis. Given this circumstance, we were interested in the possibility of evaluating the effect of some known antilithogenic agents on the level of expression of intrarenal crystallization inhibitors and markers of oxidative stress in the kidney tissue.
Research objective
To study the effect of sodium citrate and a-tocopherol acetate on the level of expression of the Tamm-Horsfall protein, osteopontin, bikun-in, malonic dialdehyde and superoxide dismutase in rat kidney tissue by experimental kidney stone disease.
Materials and methods
The experiments were carried out in 60 male Wistar rats aged 2-3 months and weighing 250300 grams grown in the nursery of the Research Institute of Cytology and Genetics of the SB RAS (Novosibirsk). The studies were carried out in accordance with the principles of humanity set out in the directives of the European Community (86/609 / EEC) and the Helsinki Declaration, in accordance with the "Rules for Working with Experimental Animals". The animals were divided equally into 4 groups: a group of intact rats - Gint (42 days of maintenance in standard laboratory conditions without KSD modelling and without the administration of studied drugs), control group Gc (42 days of KSD modeling), group of sodium citrate treatment - Gs c (42 days of KSD modeling + daily administration of sodium citrate from 22nd to 42nd days of the experiment), group of a-tocopherol acetate treatment - Gt a (42 days of KSD modeling +
daily administration of a-tocopherol acetate from the 22nd to 42nd days of the experiment).
Experimental kidney stone disease was modeled in accordance with the generally accepted ethylene glycol model, according to which animals were given a 1% solution of ethylene glycol on a daily basis as a drink [8]. a-tocopherol acetate is a classic antioxidant used in the treatment of KSD [7]. The drug was injected through the probe in the form of an oily solution at a dose of 300 mg/kg. Sodium citrate is a drug that has the ability to chelate lithogenic ions and/or their insoluble salts in the urine [9]. Was injected through the probe in a dose of 200 mg / kg.
At the end of 6 weeks of the experiment, the rats were decapitated under complete ether anesthesia, after which kidneys were removed to conduct an immunohistochemical study.
Determination of the expression of the Tamm-Horsfall protein, osteopontin and bi-kunin, as well as superoxide dismutase and malo-nic dialdehyde, was carried out with the help of an indirect two-step streptavidin-biotin method with control of the specificity of the reaction [10]. After the standard procedure of dewaxing and rehydra-tion, endogenous peroxidase was blocked according to the recommendations of the antibody manufacturer (Santa Cruz, USA).
Morphometric studies were carried out using the software packages ImageJ 1.43 and AxioVi-sion 3.1. The degree of expression was evaluated by a semi-quantitative method using the ct2 criterion for the intensity of DAB staining using ImageJ 1.43 image analysis software. For the convenience of interpreting the results, the obtained data were calculated by the formula:
100xD
E% = 100--
256
Where: E% - percent of expression;
256 - maximum color intensity of the cutoff field.
Results and discussion
As a result of the conducted experiments, it was found that in the Gint rats, the expression of the Tamm-Horsfall protein was noted mainly in the cytoplasm and on the apical membranes of epithelial cells of the distal tubules of the cortex and the thick ascending section of the Henle loop of the outer zone of the medulla. Expression of os-teopontin was recorded mainly in the cytoplasm of the epitheliocytes of the nephron tubules, collecting tubes, transitional epithelium of the cup-and-pelvis system. In addition, moderate bikunin expression of cytoplasmic localization in the epithelium of the nephron tubules and collecting tubes and the absence of such in the interstitium were observed. The quantitative indices of the expression
of THP, OPN, BIK and also MDA and SOD are presented in Table 1.
Against this background, under the conditions of KSD modeling, significant changes in the qualitative and quantitative indices of the expression of THP, OPN, BIK, MDA and SOD were observed in the control group. It turned out that the expression of THP in the interstitium of the renal papilla, which was most pronounced in the region of the base and middle third, was also noted, i.e. in places where deposits of calcium predominate. After 6 weeks of ethylene glycol implementation, the degree of expression of THP in relation to intact rats was significantly increased by 4.6% (Table 1). There was no topological difference in the expression of arterial hypertension with respect to intact animals in the control of the disease. However, by the long-term use of ethylene glycol, the expression of OPN was weakened by 3.2% compared to that of intact animals (Table 1). In the kidneys of the rats in the control group with similar overall localization, the BIK expression was also detected in the interstitium of the renal papilla, i.e. in the place of preferential deposition of calcium deposits. At the same time, the level of BIK expression in quantitative terms slightly exceeded the indices of the intact group (Table 1).
The level of MDA expression in points in the Gc group increased from 2+ in norm to 3+ (Table 1). In percent, the recorded increase constituted 3.5%. Against this background, the level of SOD expression, on the contrary, declined. So, if in intact rats this indicator was estimated as 3+, then after six weeks of modeling the disease, it was reduced to 2+. In quantitative terms, the regression of the degree of SOD expression constituted 5.8%.
The experiments conducted in the Gsc group showed that as a result of the course of sodium citrate implementation, the expression of inhibitors of crystallization was normalized. Thus, the topology of the expression of THP, the signs of which were detected in the interstitium of the renal papilla in the control of the disease, i.e. in the place of preferential deposition of calcium deposits, in the rats treat with sodium citrate completely corresponded to the intact group: cytoplasm and apical membranes of the epithelial cells of the distal tubules of the cortex and the thick ascending section of the Henle loop of the outer zone of the medulla without signs of interstitial localization. At the same time, there was no topological difference in the expression of OPN and BIK between the groups.
Despite this, the quantitative characteristics of the expression of all the proteins described by the results of the course of treatment underwent significant changes. As follows from Table 1, the level of THP expression decreased relative to control from 3+ points to 2+ points, which in arithmetic interpretation constituted 3.2%. Expression of acute renal failure, on the contrary, increased from 2+
points to 3+ points, i.e. by 2.5%, and the expression of BIK - weakened by 3.4%.
Thus, the pattern of expression of intrarenal crystallization inhibitors as a result of a three-week administration of sodium citrate was generally consistent with that of healthy animals.
The study of the effect of long-term administration of a-tocopherol acetate on the expression of intrarenal crystallization inhibitors and markers of oxidative damage to nephrocytes made it possible to establish that the studied drug leads to a characteristic change in the expression of THP, OPN, and BIK. It turned out that expression of THP in the kidneys of Gt a rats was detected in the cytoplasm and on the apical membranes of epithelial cells of the distal tubules and the thick ascending section of the Henle loop of the outer zone of the medulla, which fully corresponded to the topology of intact rats. It is important that there was no interstitial localization of the THP expression as a result of the treatment, although it was fixed in the control of the disease, which was a characteristic immunohistochemical sign of the pathology. There was no visual topological difference in the expression of OPN between the groups. With respect to BIK expression, it was found that in the kidneys of Gt a rats it is present in the epithelium of the nephron tubules and collecting tubes, and there was no interstitial localization in the region of the renal papilla that took place in the control of the disease.
A quantitative assessment of the changes in the expression of the above proteins, as shown in Table 1, revealed a decrease in the expression of the THP from 3+ points in the control to 2+ points in the rat kidneys of the Gta group, which in the percentage measurement was 3.3%. In parallel with the 1+ points in the control, up to 2+ points after the course of a-tocopherol acetate (by 4.4%), the expression of OPN increased, as well as from 2+ points in sick animals to 1+ points in rats with long-term a-tocopherol acetate administration, the degree of expression of BIK decreased. In percent, this decline was 4.1%. The recorded changes led to the fact that the level of expression of THP and OPN after the course of antioxidant therapy corresponded to that of healthy animals, and the same indicator for BIK was even inferior to intact figures of 2.7%.
Moreover, in Gt a rats, the level of MDA expression relative to disease control decreased from 3+ points to 2+ points, which in quantitative terms was 4.1% (Table 1). The same indicator for SOD increased from 2+ points in the control to 3+ points in the treatment group, which was 5.7%. Such changes were realized in the fact that the degree of MDA expression corresponded exactly to the value of healthy rats, and the expression of SOD even exceeded that of 2.2% (Table 1).
Thus, continuous administration of a-tocoph-erol acetate was accompanied by normalization of expression of intrarenal crystallization inhibitors and markers of oxidative damage to nephrocytes.
Summarizing the foregoing, we note that the qualitative and quantitative characteristics of the expression of THP, OPN, BIK, MDA and SOD under the conditions of KSD modeling were subject to significant changes with respect to healthy rats. These changes could be both pathological and compensatory. Nevertheless, in both cases, they can be recognized as markers for the development of the kidney stone disease. In this case, the three-week administration of sodium citrate and a-tocopherol acetate resulted in normalization of the expression indices of the above substances. As is known, sodium citrate is a chelating agent, and a-tocopherol acetate is a direct non-enzyme antioxidant [7,9]. These substances have now been proven to possess antilithogenic properties. Both drugs affect the main driving forces of crystallization: sodium citrate - to supersaturation of urine with calcium ions, a-tocopherol acetate - to oxida-tive damage to nephrothelia [7,9]. It is not excluded that these substances, targeting the proper links of the pathogenesis of nephrolithiasis, significantly weakened the severity of the pathology, which neutralized the conditions for changes in the pattern of expression of THP, OPN, BIK, MDA and SOD, bringing it back to normal. Of course, the features of the modulation of the expression of nephrolithi-asis markers by the KSD and in the conditions of its treatment require a more careful study. However, based on the results of this study, with some confidence, it can be assumed that qualitative and quantitative changes in the expression of the mentioned proteins can be a very informative indicator for evaluating the effectiveness of pharmacological treatment of urolithiasis.
Conclusion
The three-week administration of sodium citrate and a-tocopherol acetate by experimental kidney stone disease leads to normalization of the qualitative and quantitative parameters of the expression of THP, OPN, BIK, MDA and SOD significantly altered under the conditions of the disease.
References
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lauie i Indices of expression of intrarenal crystallization inhibitors and markers of oxidative stress in renal tissue
Groups of animals
KSD markers Gint Gc G s.c. Gt.a
Rate of expression
Points % Points % Points % Points %
Tamm-Horsfall protein 2+ 49,8 3+ 54,4 2+ 51,2 2+ 51,1
Osteopontin 2+ 50,0 1+ 46,8 2+ 49,3 2+ 51,2
Bikunin 2+ 50,6 2+ 52,0 2+ 48,6 1+ 47,9
Malondialdehyde 2+ 48,0 3+ 51,5 2+ 47,6 2+ 47,4
Superoxide dismutase 3+ 52,5 2+ 46,8 3+ 51,0 3+ 54,7
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Contacts
Corresponding author: Zharikov Aleksandr Yur-yevich, Doctor of Biological Sciences, Associate Professor, Head of the Department of Pharmacology of ASMU, Barnaul. 656056, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 24-18-59, 56-68-91. E-mail: zharikov@agmu.ru
