UDC 615.272.4.015.4:616.61-008.64-005.4

V.G. Zeleniuk

Bukovinian state medical university, Chernivtsi

Interconnection of renoprotective and pleiotropic properties of statins under THE ischemia-reperfusion acute renal failure

RESUME

In the experiment on white rats the renoprotective properties of statins (atorvastatin, lovastatin, simvastatin) were found, realized in the increase of the glomerular filtration rate, recovery of tubular reabsorption and reduction of proteinuria by normalization of prooxidant-antioxidant balance and prevention of proximal tubular cells’ mitochondrial damage.

Key words: statins, acute kidney failure, ischemia-reperfusion, prooxidant-antioxidant balance

INTRODUCTION

Ischemia-reperfusion of the kidney is common method among experimental models of acute renal failure (ARF) induction, and includes the mechanisms of renal tubular cell damage due to depletion of oxygen and ATP, leading to the progression of energy metabolism disorders [6], that cause loss of microvillies of brush border, cellular connections, disintegration of the cytoskeleton with Na+-K+-ATPase dysfunction and oxidative stress development [22]. Activation of lipid peroxidation is one of the reasons for the mitochondrial membranes damage, their death and the consequent progressive disturbances of energy metabolism, complicated by subsequent activation of lipid peroxidation caused by oxygen influx in the cells during reperfusion, resulting in the formation of an abundance of free radicals [10, 15].

One of the goals of pharmacotherapy of acute renal failure is the use of drugs with antioxidant properties [13, 20]. Statins, despite their primary hypolipidemic effects, are able to exhibit antioxidant, anti-inflammatory and immunomodulatory properties, normalizing endothelial function [12, 17, 21]. Inhibiting the synthesis of low-density lipoproteins and thus the formation of their oxidized forms, exacerbating ischemia [18], statins are able to significantly improve the course of ARF.

The purpose of research was to establish a relationship between renoprotective and pleiotropic properties of some statins (atorvastatin, lovastatin, simvastatin) in ischemia-reperfusion ARF.

MATERIALS AND METHODS

In vivo studies were carried out on 40 nonlinear mature white laboratory rats weighting 140-180 g, maintained under the vivarium conditions with constant temperature and humidity and free access to food and water.Rats were randomly divided into five groups (n=8): І– control group, which includes sham-operated animals; ІІ –group of animals with ARF; ІІІ, IV, V – groups of animals with ARF, treated with studied drugs (atorvastatin, lovastatin, simvastatin respectively) at a dose 20 mg/kg in 1% starch solution (1 ml suspension of the drug per 100g body weight). Ischemia was simulated in compliance with aseptic technique under general anesthesia (etaminal sodium, 40 mg/kg): median laparotomy was performed, each kidney was isolated followed by clamping a renal stem (the artery, vein and ureter) for a period of 75 min, sealing of the abdominal cavity adhering to thetemperature regime[4, 11]. After the clamp removal abdominal cavity layers were sewn, followed by 24-hour reperfusion and subsequent kidney function assessment under the conditions of induced diuresis (enteral administration of drinking water in a volume of 5% of body weight, urine was collected during 2 h). The animals were euthanized by decapitation under the thiopental anesthesia (80 mg/kg) for blood and kidneys sampling [19].

The concentration of creatinine in plasma was determined by the Popper method in Merzon modification, in urine – by the Folin method [8], the protein content in urine – by the reaction with sulfosalicylic acid [7]. State of prooxidant-antioxidant balance was evaluated by the malondialdehyde (MDA) level [9] and the glutathione peroxidase (GPx) activity in the kidney tissue[1]. The concentration of potassium and sodium ions in urine and blood plasma was assessed by flame photometry on «FPL-1» (Ukraine) [8]. The β-lipoproteins concentration in blood plasma was determined by turbodimetric Burstein-Samay method [2]. State of energy supply of the kidneys was determined by the succinate dehydrogenase (SDH) activity [5]. The extent of tubular membrane pathology [14, 16] was evaluated by the γ-glutamyl transpeptidase (GGTP) activity [3]. Statistical analysis of the data was performed with «Statistica 6.0» software. Multiple comparisons among the groups were made by Student’s t-test (for normal distribution) and Mann-Whitney U-test (in case of non-normal distribution).Correlation analysis of samples was performed using Spearman's coefficient.

RESULTS AND DISCUSSION

Renal ischemia followed by reperfusion led to oliguria. Thus, in the group of untreated animals urine output decreased by 31% as compared with the control group, and the use of statins provided the recovery of urination to the index of sham-operated group (tabl. 1).Under the conditions of simulated pathology a significant, 3-fold, fall in GFR was observed. The administration of atorvastatin contributed to the increase of GFR by 2.7 times, lovastatin – by 2.5 times, simvastatin – by 2.8 times. In untreated animals with ARF sodium excretion increasedby 70%, associated with a profound disturbance of tubular reabsorption. It is confirmed by an increase of the fractional excretion of sodium to 2.4%, indicating the development of acute tubular necrosis. Statins contributed to normalization of electrolyte-excretory kidney function, reducing the sodium excretion, as well as its fractional excretion below 1%, which corresponds to parameters of normalcy. In untreated animals significantly worsened proximal transport of sodium ions – by 3.2-fold, and distal transport to a lesser extent – by 1.3-fold. Studied drugs restored the reabsorption of sodium ions, particularly atorvastatin improved proximal transport relatively to the control group by 21%.The administration of statins helped to reduce hyperkalemia that developed during ischemia-reperfusion ARF by the increase of potassium excretion by an average 64% to all drugs.A similar trend was observed for the restoration of water reabsorption under the influence of drugs to the level of the control group. The protein concentration in the urine of untreated animals with ARF exceeded the indicator of control animals by 2.5 times, and the use of statins contributed to the a significant reduction of proteinuria, with the best results noted in the simvastatin group – 2.4-fold against 1.9-fold and 1.8-fold in the atorvastatin and lovastatin groups, respectively.

The severity of ARF is evidenced by the increased activity of GGTP by 49 times as compared with the control group (table 1), correlating with an increase in fractional excretion of sodium (r=0,71), decrease in potassium excretion (r=0,81) and proteinuria (r=0,69). A significant decrease in GGTP activity under the influence of statins indicates their ability to facilitate the protection of tubules’ nephrocytes in ischemic ARF, limiting the development of tubular necrosis and loss of brush border.

Development of ischemia-reperfusion ARF was accompanied by violation of prooxidant-antioxidant balance in the kidney tissue: MDA content increased by 65%, GPx activity decreased by 2.6 times (tabl. 2). When applying statins decrease in the MDA level by 34% and increase GPx activity by 92% (average for all drugs) were observed, with better results noted in the simvastatin group – by 55% and by 2.3 times, respectively. Damage of mitochondrial membranes by free radicals due to development of renal ischemia resulted in energy metabolism disturbances and reduced SDH activity in kidney tissue by 2.6 times (fig. 1), confirmed by the inverse correlation between the MDA content in kidney tissue and SDH activity (r=-0.88), as well as a direct correlation between GPx and SDH activity (r=0.72). Through the inhibition of free radical oxidation processes a positive trend was managed to achieve the SDH activity recovery under the influence of statins: by 2.2-fold in the atorvastatin group, by 1.7-fold – in the lovastatin group and 2.3-fold - in the simvastatin group. Such influence of drugs can be associated with a decrease of the β-LP content in blood plasma of rats by an average 53% (fig. 2), since in addition to their atherogenic influence, β-LP are able to be modified in the oxidized forms and aggravate ARF, as evidenced by an increase of their contents in the pathology by 50% and connections with MDA level (r=0.46), GPx activity (r=-0.86), GFR (r=-0.56) and protein concentration in the urine (r=0.35). Among the applied drugs the most expressive hypolipidemic effect was demonstrated by simvastatin, as indicated by the reduction on 25% plasma β-LP level as compared with atorvastatin and lovastatin.

CONCLUSION

Under the conditions of ischemia-reperfusion ARF statins (atorvastatin, lovastatin, simvastatin) show renoprotective effects at a dose 20 mg/kg through the restoration of renal excretory function, normalization of prooxidant-antioxidant balance and energy metabolism in nephrocytes, and among the studied drugs simvastatin showed the best effectiveness that can be explained by its higher lipophilicity and stronger antiatherogenic effect under the current conditions.

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УДК615.272.4.015.4:616.61-008.64-005.4

В.Г. Зеленюк

Буковинский государственный медицинский университет, г. Черновцы

СВЯЗЬ НЕФРОПРОТЕКТОРНЫХ И ПЛЕЙОТРОПНЫХ СВОЙСТВ СТАТИНОВ ПРИ ИШЕМИЧНО-РЕПЕРФУЗИОННОЙ ОСТРОЙ ПОЧЕЧНОЙ НЕДОСТАТОЧНОСТИ

АННОТАЦИЯ

В эксперименте на белых крысахустановлены нефропротекторныесвойства статинов (аторвастатин, ловастатин, симвастатин), реализовавшиесяв повышениискорости клубочковой фильтрации, восстановлении канальцевой реабсорбции иуменьшении протеинурииблагодаря нормализации прооксидантно-антиоксидантного балансаипредупреждению повреждения мембран митохондрий клеток проксимальных канальцев.

УДК615.272.4.015.4:616.61-008.64-005.4

В.Г. Зеленюк

Буковинський державний медичний університет, м. Чернівці

ЗВ'ЯЗОК НЕФРОПРОТЕКТОРНИХ ТА ПЛЕЙОТРОПНИХ ВЛАСТИВОСТЕЙ СТАТИНІВ ПРИ ІШЕМІЧНО-РЕПЕРФУЗИВНІЙ ГОСТРІЙ НИРКОВІЙ НЕДОСТАТНОСТІ

АНОТАЦІЯ

В експерименті на білих щурах встановлено нефропротекторні властивості статинів (аторвастатин, ловастатин, симвастатин), які реалізувались у підвищенні швидкості клубочкової фільтрації (ШКФ), відновленні канальцевої реабсорбції та зменшенні протеїнурії завдяки нормалізації прооксидантно-антиоксидантного балансу та попередженню ушкодження мембран мітохондрій клітин проксимальних канальців.

Table 1

EFFECT OF STATINS ON RENAL FUNCTION IN RATS UNDER THE CONDITIONS OF ISCHEMAI-REPERFUSION ACUTE RENAL FAILURE(M±m,N=8)

Indices / Control / Pathology
(ARF) / ARF +
atorvastatin / ARF +
lovastatin / ARF +
simvastatin
Diuresis, ml/2 h / 2,93±0,15 / 2,24±0,09** / 2,97±0,11## / 2,79±0,26## / 2,99±0,16##
Glomerular filtration rate,μl/min / 669,3±97,1 / 223,5±36,9** / 601,1±68,2## / 569,9±47,8## / 636,3±62,4##
Water reabsorption, % / 95,99±0,45 / 89,94±1,87** / 95,64±0,39## / 95,73±0,49## / 95,91±0,36##
Urine protein concentration, g/l / 0,022±0,008 / 0,056±0,004* / 0,029±0,003## / 0,031±0,004## / 0,023±0,003##
Na+excretion, μmol/2 h / 2,06±0,38 / 3,51±0,34* / 1,72±0,25## / 2,10±0,39# / 1,68±0,23##
Na+proximal transport, mmol/2 h / 15,61±2,42 / 4,85±0,83** / 18,86±0,75## / 10,88±1,45## / 15,51±1,60##
Na+distal transport, mmol/2 h / 0,59±,03 / 0,44±0,02* / 0,58±0,03## / 0,43±0,04 / 0,63±0,04##
Fractional excretion of Na+, % / 0,51±0,09 / 2,38±0,46** / 0,15±0,02## / 0,27±0,07## / 0,13±0,02##
K+ excretion, μmol/2 h / 25,50±2,60 / 15,13±2,18* / 26,23±1,24# / 23,73±2,39 / 24,79±2,33#
Urine GGTP activity, mmol/(h*l) / 0,09±0,01 / 4,43±0,42** / 0,70±0,08## / 0,81±0,08## / 0,50±0,11##

Notes(here and in tabl. 2):

1. Ascompared with the intact control group – * (р≤0,05), ** (р≤0,01);

2. As compared with the untreated pathology (ARF) group – #(р≤0,05), ##(р≤0,01);

GGTP – γ-glutamyl transpeptidase.

Table2

EFFECT OF STATINS ON PROOXIDANT-ANTIOXIDANT BALANCE (M±m,N=8)

Indices / Control / Pathology
(ARF) / ARF +
atorvastatin / ARF +
lovastatin / ARF +
simvastatin
MDA content in kidney tissue, μmol/g / 33,19±1,52 / 54,87±2,35** / 40,07±2,04## / 47,21±4,46 / 35,31±2,88##
GPx activity in kidney tissue, μmol/mg of protein / 214,86±9,46 / 82,67±7,07** / 157,84±11,46# / 129,87±15,13# / 189,35±12,9##

Notes: MDA – malone dialdehyde, GPx – glutathione peroxidase.

Fig. 1. Succinate dehydrogenase activity in kidney tissue of rats.

Notes(here and in fig. 2):

1. Ascompared with the intact control group – * (р≤0,05), ** (р≤0,01);

2. As compared with the untreated pathology (ARF) group – #(р≤0,05), ##(р≤0,01);

ARF– acute renal failure.

Fig. 2. β-lipoprotein content in blood plasma of rats.

1