CC BY
55
UDC 616.613-003.7:616.151-07
BIOCHEMICAL MARKERS OF DAMAGE TO THE KIDNEY TISSUES BY EXPERIMENTAL OXALATE NEPHROLITHIASIS
1 Altai State Medical University, Barnaul
2 Novosibirsk State Medical University, Novosibirsk
N.N. Yakushev 1, P.G. Madonov 2, O.Sh. Atabayeva 1
The aim of the research was to study the dynamics of y-glutamyltransferase activity and cyclooxygenase-1 concentration in urine and the dynamics of malonic dialdehyde concentration in rat kidney tissue by experimental oxalate nephrolithiasis.
Experiments were performed on 15 male Wistar rats aged 2-3 months and weighing 180-220g. Oxalate nephrolithiasis was modeled for 6 weeks in accordance with the generally accepted ethylene glycol model. Prior to the start of the experiment, and after 3 and 6 weeks, urine collection was performed, in which y-glutamyl transferase (GGT) activity and cyclooxygenase-1 (COX-1) concentration were determined. At the end of 6 weeks, in the homogenate of renal tissue, the concentration of thiobarbituractivative products (TBRP) was determined. The results of the experiments showed that the activity of GGT in the urine after 6 weeks of nephrolithiasis simulation increased by 6.6 times, the concentration of TBRP in the kidney homogenate relative to the level of healthy rats increased by 1.3 times, and there was a tendency to the increase of COX-1 concentration in the urine by 1.7 times. Experimental oxalate nephrolithiasis shows an increase in activity of biochemical markers of lithogenesis - GGT and TPBP. This indicates the development of a damaging factor in the kidneys, which is based on the activation of lipid peroxidation.
Key words: oxalate nephrolithiasis, y-glutamyltransferase, malonic dialdehyde, cyclooxygenase-1.
Kidney stone disease is still one of the most common and serious diseases of the urinary system. The epidemiology of nephrolithiasis covers every 10th inhabitant of developed countries [1]. Modern ideas about the pathogenesis of nephroli-thiasis indicate the initiating role in the stone formation process of the so-called "damaging factor" - destructive changes in the structure and function of the tubular epithelium of the kidney, resulting in formation of crystalline material from urine at a certain site, and the primary focus of lithogen-esis is formed [2]. In this regard, it is considered that certain signaling molecules, indicating the development of a damaging factor, may be biochemical markers of lithogenesis. This may be important not only in the study of the pathogenesis of neph-rolithiasis, but also in assessing the effectiveness of the developed methods of drug treatment of urolithiasis.
The results of a number of studies show that the cascade of reactions provoking the development of a damaging factor begins with the destruction of nephrocyte cell membranes under the influence of crystallization driving forces [3]. Therefore, we were interested in studying the dynamics of activity and/or content of y-glutamyltransferase, malonic dialdehyde and type 1 cyclooxygenase in the kidneys by experimental nephrolithiasis. As is known, y-glutamyltransferase is a membrane-bound enzyme that is present in large amounts in kidneys [4]. Malonic dialdehyde is the main product of perox-idation of membrane phospholipids [5]. Cyclo-ox-ygenase-1 is an enzyme involved in the catabolism of membrane phospholipids during the conversion
of arachidonic acid to prostaglandins [6]. These signaling molecules are found in the kidneys in significant amounts, and therefore, they can potentially be biochemical markers of the development of oxa-late nephrolithiasis.
Thus, the research objective is to study the dynamics of y-glutamyltransferase activity and cyclo-oxygenase-1 concentrations in the urine and the dynamics of the concentration of malonic dialdehyde in the renal tissue of rats by experimental oxalate nephrolithiasis.
Materials and methods
The experiments were carried out on 15 male Wiatar rats, 2-3 months old and weighing 180-220g. The studies were carried out in accordance with the requirements of the "Rules of work with the use of experimental animals." Throughout the study, the animals were located in individual metabolic cages adapted for urine collection. Oxalate nephrolithiasis was modeled according to the generally accepted ethylene glycol model, according to which the rats were provided with free-access 1% ethylene glycol (EG) for 6 weeks [7].
Prior to the experiment, as well as after 3 and 6 weeks, urine was collected, in which the activity of y-glutamyltransferase (GGT) and the concentration of cyclooxygenase-1 (COX-1) were determined. GGT activity (U/mg creatinine per day) was determined by an optimized kinetic method on a Vitalon 400 semi-automatic analyzer. Under the action of y-glutamyltransferase in the transfer reaction of L-y-glutamyl-3-carboxy-p-nitroanilide to glycylglycine, a colored 5-amino-2-nitrobenzo-
ate is formed. The rate of increase in the optical density of the sample at a wavelength of 405 nm is proportional to the activity of GGT in the analyzed sample. For the quantitative determination of cyclooxygenase-1 in the urine using enzyme immunoassay, a kit for the determination of pros-taglandin-endoperoxide synthetase 1 (PTGS 1) by Cloud-Clone Corp was used.
By the end of 6 weeks of nephrolithiasis modeling, the animals were euthanized under ether anesthesia, both kidneys were removed, one of which served to determine the concentration of thiobar-biturate reactive products, the main representative of which is malondialdehyde, as well as to conduct morphological studies aimed at the confirmation of stone formation processes. The concentration of thiobarbiturate-reactive products (TBRP) in the homogenate of the renal tissue was determined by the colorimetric method, measuring the color intensity of the solution during the chemical reaction of the TBRP with thiobarbituric acid. For morphological studies, the kidneys were fixed in a 10% formalin solution, processed by a standard technique, and a 6 ^m thick cross section was made through the renal papilla. The obtained sections were stained with hematoxylin and eosin. Calcium deposits were identified using the Koss histochem-ical method, and the number of calcium deposits in the field of view was calculated using a comput-
of TBRP in the homogenate of the renal tissue of rats by a six-week experimental nephrolithiasis constituted 7.6 (7.23; 7.65) |jmol, which is 1.3 times higher than in healthy rats - 6.1 ( 5.37; 6.91) ^mol (p = 0.00428).
By histochemical staining on calcium by the Koss method, single crystals or groups of deposits of brownish-black stones of various shapes and sizes were observed in the tubules of the rat kidneys (Figure 2). The number of deposits in the gaps of the tubules ranged from 3 to 7 and averaged 4.6 ± 0.2 in the field of view with the zoom of x 400, with a modal value of 4. When performing computer morphometry, the area of stone deposits
er program, and their size was determined. Mor-phometric studies were performed using the Im-ageJ 1.43 and AxioVision 3.1 software packages.
The statistical processing of the results was carried out using the computer program "Statistica 12.0". The results are represented by the median (M) and interquartile range (25%, 75%) for GGT, COX-1, and TBRP, as well as the mean and standard error of the mean (M ± m) for morphometric parameters. Statistical comparisons of dependent samples were carried out using the Wilcoxon non-parametric test. The results were considered reliable by the value of the significance indicator p <0.05.
Results and discussion
As a result of the experiments, it was established that the activity of GGT in the urine by the end of the 3rd week of the experiment increased from the initial level by 5.0 times. Subsequently, the growth of the value of the described indicator continued, as a result of which, at the end of 6 weeks, it already exceeded the initial figures by 6.6 times. Against this background, the concentration of COX-1 in the urine by the end of the 6th week of the observation period increased relative to the initial level by 1.7 times. This change was not statistically significant and only manifested itself in the form of a trend (Table 1).
171.9 ± 27.6 |jm2.
Discussing the results obtained, we note that under the conditions of a modeled oxalate nephrolithiasis, GGT activity underwent the most pronounced growth. As is known, GGT is a membrane enzyme that is localized on the outer side of the membrane of many cells of the body [4]. Therefore, on the one hand, an increase in its activity in the urine by nephrolithiasis may indicate the destruction of the cell membranes of nephro-cytes and the release of the enzyme into the lumen of the tubules. On the other hand, it is known, that GGT activity is a marker of oxidative stress in body tissues [8]. It has been established, that the most
Indicators of GGT activity and COX-1 concentration in the urine by six Table 1 -week experimental oxalate nephrolithiasis
Week GGT activity (U/mg creatinine per day) COX-1 Concentration (ng / ml)
Initial level 0,25 (0,05 ; 0,85) n=14 3,9 (2,18 ; 5,69) n=15
3rd week 1,24 (0,36 ; 2,10) n=14 p=0,0231 Not determined
6th week 1,64 (0,87 ; 4,63) n=11 p=0,0166 6,6 (6,0 ; 7,4) n=10 p=0,114
Note: n - number of urine samples for analysis; p - indicator of the significance of changes relative to the initial level.
As follows from Figure 1, the concentration
ranged from 31.5 ^m2 to 567, 9 ^m2 and averaged
important function of GGT in the body is to maintain the physiological concentration of glutathione in the cytoplasm of cells, which is the main thiol antioxidant in the body [8]. Therefore, it is possible, that such a pronounced increase in GGT activity could be a compensatory response to the developing oxidative stress in the renal tissue by oxalate
nephrolithiasis. This can be confirmed by the increase in the concentration of TBRP recorded in our experiments in the homogenate of the renal tissue. As is known, malonic dialdehyde, the main product of membrane phospholipid peroxidation, is the main representative of TBRP [5].
Figure 1 - The concentration of TBRP by experimental oxalate nephrolithiasis in relation to the group of intact rats. Note: int - indicator of healthy rats; contr - indicator of rats with nephrolithiasis.
Figure 2 - Deposits in the tubules of the rat kidneys by experimental oxalate nephrolithiasis. Coloring according to Koss.
Zoom x 400. Note: brown calcium deposits are in sight.
In addition, we should note the concentration of COX-1 in the urine recorded in our experiments. This enzyme is involved in the metabolism of arachidonic acid to prostaglandin H2 - an intermediate product in the synthesis of a number of prostagladins [6]. The role of the cyclooxygen-ase metabolic pathway of membrane phospho-lipids in the pathogenesis of renal pathologies
is quite complex and diverse. There is an opinion that COX-1 is involved in cytoprotection, the most striking example of which is the regulation of production of protective mucus in the stomach [6]. It is possible that COX-1 may have a similar function in the kidneys. However, of course, this issue is subject to further in-depth study.
Summarizing the above, we note that under conditions of a six-week simulation of experimental oxalate nephrolithiasis, characteristic changes in the dynamics of biochemical markers of the development of pathology were recorded. This was mainly evidenced by a nearly 7-fold increase in GGT activity during the experiment and a significant increase in the concentration of TBRP in the ho-mogenate of the renal tissue by 30%. Confirmation of the development of nephrolithiasis were the results of morphological studies, which showed the formation of a significant amount of calcium deposits in the tubules of the kidneys of experimental rats.
Conclusion
By experimental oxalate nephrolithiasis, an increase in the activity of biochemical markers of lithogenesis — GGT and TBRP — is observed. This indicates the development of a damaging factor in the kidneys, which is based on the activation of lipid peroxidation.
References
1. Scales C.D. Jr., Tasian G.E., Schwaderer A.L. et al. Urinary Stone Disease: Advancing Knowledge, Patient Care, and Population Health. Clin J Am Soc Nephrol. 2016. 11(7): 1305-1312.
2. Zharikov A.Yu., Zverev Ya.F., Bryukhan-ov V.M., Lampatov V.V. Mechanism of formation of crystals in oxalate nephrolithiasis. Nephrology. 2009; 13(4): 37-50.
3. Khan S.R. Histological aspects of the «fixed-particle» model of stone formation: animal studies. Urolithiasis. 2017;45(1):75-87.
4. Castellano I., Merlino A. y-Glutamyltrans-peptidases: sequence, structure, biochemical properties, and biotechnological applications. Cell Mol Life Sci. 2012; 69: 3381-94.
5. Ayala A., Muñoz M.F., Argüelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev. 2014; 2014:360438.
6. Li Y., Xia W., Zhao F., Wen Z., Zhang A., Huang S., Jia Z., Zhang Y. Prostaglandins in the pathogenesis of kidney diseases. Oncotarget. 2018; 9(41):26586-26602.
7. Zharikov A.Yu., Bryukhanov V.M., Zverev Ya.F., Lampatov V.V. Current methods of modeling of oxalate nephrolithiasis. Nephrology. 2008; 12(4): 28-35.
8. Ndrepepa G., Kastrati A. Gamma-glu-tamyl transferase and cardiovascular disease. Ann Transl Med. 2016; 4(24): 481.
Contacts
Corresponding author: Yakushev Nikolai Niko-
layevich, Associate Professor of the Department
of Pharmacology of ASMU, Barnaul.
656031, Barnaul, ul. Papanintsev, 126.
Tel.: (3852) 566891.
E-mail: yakushevnn@mail.ru
Author information
Madonov Pavel Gennadyevich, Doctor of Medical Sciences, Professor, Head of the Department of Pharmacology, Clinical Pharmacology and Evidence-Based Medicine, Novosibirsk State Medical University, Novosibirsk. 630075, Novosibirsk, ul. Zalesskogo, 4. Tel.: (3832) 360902. E-mail: kaffarm@yandex.ru
Atabayeva Olga Shukurullovna, Candidate of Medical Sciences, Associate Professor of the Department of Pharmacology of ASMU, Barnaul. 656031, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566891. E-mail: science@agmu.ru
