EFFECTS OF “NEPHROPHYTE” ON CASPASE-3 ACTIVITY AND PRESERVATION OF TISSUE ATP IN RENAL ISCHEMIA/REPERFUSION / Монголын эм зүй, эм судлал

This paper describes the effects of a multicomponent plant preparation developed on the basis of Tibetan prescriptions, including ethanolic extracts of Arctostaphylos uva- ursi (L.) Spreng, Orthosiphon stamineus (L.), Polygonum aviculare (L.), on caspase-3 activity and recovery of tissue ATP content in ischemic renal cells of albino Wistar rats. It has been shown that after “Nephrophyte” administration at a dose of 150 mg/kg body wt. (overnight and 1 hour before ischemia) the activity of caspase-3 significantly decreased and the recovery of tissue ATP was significantly enhanced. These results suggest that “Nephrophyte” may protect the kidney against ischemia/ reperfusion injuries, at least, by ameliorating apoptotic process and preserving tissue ATP content. KEY WORDS: kidney, ischemia/reperfusion, caspase-3, ATP, “Nephrophyte” INTRODUCTION Acute renal failure (ARF) as a result of ischemia/reperfusion (I/R) is of great clinical significance because of its frequent occurrence, high morbidity and mortality. It is important to improve the ability of kidney to tolerate ischemic damages. ATP depletion and apoptosis are widely recognized to be implicated in ischemic renal injury. Hence, research efforts designed to prevent or ameliorate I/R injury have focused on the pharmacological inhibition of apoptotic process, preservation and recovery of intracellular ATP. However, the above approaches remain to be fully explored. It is now widely recognized that several pathological processes of biological tissues are caused by ischemia-reperfusion processes. Acute renal failure (ARF) is a clinical syndrome characterized by an abrupt loss of kidney function resulted from different forms of renal injury including ischemic and toxic stimuli. Tubular cell death through apoptosis is supposed to play a significant role in the genesis of ARF that has been confirmed by studies both in animal models and in clinical kidney diseases [12; 16]. Therefore, the control of apoptosis could be a potential target for therapeutic interventions with the aim to prevent or at least to alleviate the severity of ARF. Among the biochemical events leading to apoptotic changes, activation of caspases is of great importance since they are responsible for almost all the biochemical and morphological features of apoptosis, caspase-3 being a key mediator of apoptotic death in different cell types. Earlier, it has been shown that protease inhibitors, especially peptide-based inhibitors are highly effective in preventing programmed cell death in both in vitro and in vivo models [8; 10]. Recently, natural compounds of plant origin became a focus of interest in regulating cell survival. The induction of apoptosis by plant extracts largely through caspase-3 activation has been reported in a number of studies. For example, solamargine, a steroidal alkaloid glycoside from Solanum incunum triggers apoptosis in human hepatoma cells [9]; triterpenoid saponins from Acacia victoriae induce apoptosis in Jurkat cells [6]; Tylophora alkaloids were shown to induce apoptosis in human erythroleukemic cells involving cytochrome c release and caspase-3 activation [3]. As to the data on the natural inhibitors of apoptosis, there are few reports in the present-day literature. Luo et al. [11] have found that the standardized Ginkgo biloba extract EGb761 significantly attenuates mitochondrion- initiated apoptosis and decreases caspase-3 activity in neuroblastoma cells [19]. The inhibitory effect of caffeic acid on the activity of caspase-3 in cultured cerebellar granule neurons subjected to apoptosis has been shown in a more recent study [18], as well as protective effect of tirofuban on ischemia-induced renal apoptosis [5]. Here, we report the inhibition of caspase-3 activity and significant recovery of tissue ATP by “Nephrophyte”, a multicomponent plant preparation developed on the Tibetan prescriptions, in rat ischemic kidney cells. Our previous studies showed antioxidant, anti- inflammatory and nephroprotective activity of the phytopreparation in question due to its phytoconstituents (flavonoids, phenolic acids and free amino acids) that was the rationale for the present research [13; 15]. MATERIALS AND METHODS ANIMALS Albino Wistar rats, weighing 180-200 g each of either sex were used. The animals were maintained under a standard light cycle (12 h light, 12 h dark) and temperature (20˚C), with free access to food and water. The research was approved by the animal study committee of the local institutional review board and conducted according to the guide for the care and use of laboratory animals.

[M31R and R335C polymorphic variants of the IL8RA gene in Russian and Buryat patients with atopic bronchial asthma].

To test the M31R and R335C polymorphisms of the Il8RA gene for association with atopic bronchial asthma (BA), the allele and genotype frequency distributions of the polymorphisms were studied in Russian patients from Moscow and Buryat patients from Ulan-Ude. The study involved two Russian groups, one including 291 DNA samples of patients with atopic BA, and the other, 266 DNA samples of healthy people. The two Buryat groups included 124 and 152 DNA samples from patients with atopic BA and healthy people, respectively. The M31R polymorphism proved to be associated with atopic BA in Russians. Allele Arg and genotype Met/Arg suggested a higher risk of BA (OR= 4.45, P = 0.003 and OR = 4.58, P = 0.003, respectively), while allele Met and genotype Met/Metwere associated with a lower risk (OR = 0.22, P = 0.003 and OR = 0.22, P = 0.003, respectively). The R335C polymorphism was not associated with atopic BA in Russians and was in Buryats. Allele Arg and homozygous genotype Arg/Arg suggested a higher risk of the disease (OR = 3.06, P = 0.030 and OR = 3.20, P = 0.027, respectively), while allele Cys and genotype Arg/Cys suggested a lower risk (OR = 0.33, P = 0.030 and OR = 0.31, P = 0.027, respectively). The results support the role of the IL8RA gene in atopic BA.

[Effect of nephrophyte on the course of the hepatorenal syndrome caused by a complex of antituberculous drugs].

A complex of antituberculous drugs causes the hepatorenal syndrome in albino rats. The course administration of the plant agent nephrophyte in an experimental therapeutic dose during tuberculostatic therapy has a positive effect on the course of the hepatorenal syndrome.

[Interrelationship between structural variants of the apolipoprotein B and ischemic heart disease and plasma lipid levels]. / Vzaimosviaz' strukturnykh variantov gena apolipoproteina B s ishemicheskoi bolezn'iu serdtsa i urovnem lipidov plazmy krovi.

Xba I and EcoR I polymorphism of the apolipoprotein B (APOB) gene was studied by PCR. A significant increase in the frequency of allele X+ and haplotype H+E+ was demonstrated in patients with coronarographically documented coronary heart disease (CHD) over that of the general population. Association of allele E- with increased levels of serum triglycerides was found. The results provide evidence about the contribution of structural variants of the APOB gene to determining CHD.

PvuII restriction fragment length polymorphism of lipoprotein lipase in Russians.

The PvuII restriction fragment length polymorphism at the lipoprotein lipase gene was studied by the polymerase chain reaction. The distribution of the genotypes and allele frequencies in Russians from West Siberia were determined.

[PvuII restriction fragment length polymorphism at the lipoprotein lipase gene]. / PvuII restriktsionnyi polimorfizm gena lipoproteinlipazy.

Using the polymerase chain reaction a PvuII polymorphism at the lipoprotein lipase gene was studied. The distribution of the genotypes and alleles frequency in 90 unrelated Russians were determined. Comparative data on pVUII polymorphism in various populations are presented.

Influence of genetic and demographic factors on etiology and pathogenesis of chronic disease in north Siberian aborigines.

The relationship between genetico-demographic parameters (endogamy, inbreeding, mating structure, gene diversity) and regularities of the prevalence of hereditary and common diseases (ischemic heart disease, arterial hypertension) and their risk factors in the North Khanty, a small, semi-isolated Siberian ethnic group of Finno-Ugric linguistic affinities, were studied. It has been shown that among hereditary diseases autosomal-dominant and polygenic ones are most frequent. Special features of the genetic liability to common diseases were described. The contributions of hereditary factors to the total variability of the 20 studied morphophysiological traits were shown to vary significantly.

The role of hereditary factors in phenotypic variability of hormone levels in the population genetically adapted to circumpolar environment.

The variability of the plasma cortisol, T3, T4, TSH and estradiol levels were studied in the North Khanty population genetically adapted to the extreme conditions of the North. The relationships between genetic markers and hormone concentrations were also studied. Negative linear correlations of T3 and T4 levels with age were obtained. Cortisol, estradiol and T3 levels showed nonlinear age dependency. T4 concentrations were significantly lower in August than in February and November. The highest heritability estimate was shown for estradiol levels (44%).

[Restriction polymorphism of mitochondrial DNA among Russian residents of Western Siberia]. / Restriktsionnyi polimorfizm mitokhondrial'noi DNK sredi russkogo naseleniia zapadnoi sibiri.

Hereditary polymorphism of mitochondrial DNA is described for 5 restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI in 60 Russian individuals living in Tomsk. 13 morphotypes of mitochondrial DNA were revealed as a whole for the enzymes analysed. Comparative analysis of mitochondrial DNA morphs and morphotypes with the literature data was conducted.

Mitochondrial DNA polymorphism in Russians from west Siberia.

The mitochondrial DNA (mtDNA) of 60 Russians from West Siberia was analyzed with the following restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI that recognize 6 bp. The observed restriction fragment length polymorphisms (morphs) were classified into 13 types of distinct cleavage patterns (mitotypes). The distributions of the mtDNA morphs were compared with those characteristic of some other human populations.

[Y chromosome heterochromatin and human morphophysiological variability].

Eighty morphophysiological traits were studied in 55 males with a morphologically identical heterochromatin subtotal deletion of Y chromosome and in 55 males with a normal Y chromosome. No significant differences were found between these groups in mean values of most traits except some hematologic parameters. Using the pattern recognition algorithm a combination of 20 traits which differentiated the groups under comparison with the recognition errors 4.6% was extracted. The recognition system consisted the informative traits including electrocardiographic parameters (25% of the traits), some anthropological and hematologic traits and subjects' age. The results suggest that the Y chromosome heterochromatin appears to have a modifying effect on the phenotypic relationship between morphophysiological traits during human ontogenesis.

The role of hereditary factors in phenotypic variability of hormone levels in a population genetically adapted to the circumpolar environment.

The variability of plasma cortisol, T3, T4, TSH, and estradiol levels was studied in the North Khanty population genetically adapted to the extreme conditions of the North. Negative linear correlations of T3 and T4 levels with age were obtained. Cortisol, estradiol, and T3 levels showed nonlinear age dependency. T4 concentrations were significantly lower in August than February and November. The highest heritability estimate was shown for estradiol levels (44%). The relationships between genetic markers and hormone concentrations were studied.

Structure of ischemic heart disease risk factors in the North Khanty population of western Siberia.

The relationships between serum lipids, blood pressures, anthropometric traits, age, and ischemic changes of the electrocardiogram (ECG) in the North Khanty population were studied. To this end, eight morphophysiological variables--presumably risk factors for ischemic heart disease--have been subjected to a principal components analysis. Two principal components were extracted. The first revealed age dependency of the traits not associated with ischemic changes of myocardium in both sexes. A comparative analysis of the second component has shown that the chosen traits correlated with ischemic heart disease in males but not in females. Interpretation is given of the components obtained.

[Complex medico-genetic study of the populations of western Siberia. IV. A multivariate statistical analysis of the interrelation of quantitative and polymorphic monogenic traits]. / Kompleksnoe mediko-geneticheskoe izuchenie populiatsii Zapadnoi Sibiri. Soobshchenie IV. Mnogomernyi statisticheskii analiz vzaimosviazei mezhdu kolichestvennymi i polimorfnymi monogennymi priznakami.

Multidimensional statistical analysis for a set of morpho- and physiological traits (anthropological, hemodynamic and biochemical) was done by the principal components method with interpretation of the latter. Special features of phenotype relationships were established for some traits of pathogenetic values for human chronic pathology. Heterogeneity of distributions of genotype frequencies for polymorphic traits and chromosome Q segments in histogramms of principal components was shown. The distributions are considered to be of nonrandom patterns and reflect common population genetics processes affecting both classes of traits under study.

[Genetic and epidemiologic study of blood pressure in an autochthonous population of the Northern Ob basin. Evaluation of heredity of blood pressure parameters and correlations of arterial hypertension and genetic markers]. / Genetiko-épidemiologicheskie issledovanie AD u korennogo naseleniia obskogo severa: otsenka nasleduemosti parametrov AD i vzaimosviazi arterial'noi gipertonii s geneticheskimi markerami.

A study of intrafamilial correlations between systolic and diastolic arterial blood pressure, and the heritability of these parameters as well as relationships between some genetic markers and arterial hypertension in a Northern Khant population is reported. There were no correlations in the BP parameters mentioned between spouses, while the sibs correlations prevailed over the parents-children ones. Heritability was higher for systolic BP, as compared to diastolic BP. A relationship was demonstrated between MN blood group and arterial BP. Males with phenotype O were shown to be less adaptable to diastolic BP rise.

[Complex medico-genetic study of the population of western Siberia. III. Distribution of phenotypes and gene frequencies in the northern Khanty population]. / Kompleksnoe mediko-geneticheskoe izuchenie naseleniia Zapadnoi Sibiri. Soobshchenie III. Raspredelenie fenotipov i gennykh chastot v populiatsii severnykh khantov.

Distribution of phenotypes and gene frequencies for 5 polymorphic loci as well as frequencies of incidence of some anthroposcopic parameters in five subpopulations of the North Khanty population is presented. A comparative analysis was performed for the traits studied among the people of Finno-Ugric group and of North Asia. Wahlund's variance values point to significant subdivision of the population in question. Disruption of the Hardy-Weinberg equilibrium for haptoglobin locus was observed in two subpopulations. Estimates of inbreeding coefficients obtained by different ways (from gene frequencies, genealogy and isonymy) are compared.

[Complex medico-genetic study of the Western Siberian population. II. Population-genetic study of fluorescent chromosome polymorphism in Northern Khanty]. / Kompleksnoe mediko-geneticheskoe izuchenie naseleniia Zapadnoi Sibiri. Soobshchenie II. Populiatsionno-geneticheskoe issledovanie severnykh khantov po fliuorestsentnomu polimorfizmu khromosom.

Polymorphisms of fluorescent variants of 469 human karyotypes from five subpopulations of the Northern Khanty were studied. Significant frequency variability of Q-polymorphic variants of chromosomes was shown. Wahlund's variance for 12 chromosome polymorphic segments was 0.0121, while the total inbreeding coefficient was 0.0069 with a negative nonrandom component. Heterozygosity estimated for chromosome fluorescent polymorphism (0.189) agrees well with that for blood group systems and less for enzymes. The frequencies of Q-polymorphic variants corresponded to Hardy-Weinberg equilibrium, except for chromosome 13 which gave the significant excess of heterozygotes. Possible explanations of this phenomenon are discussed.

[A complex medico-genetic study of West Siberian populations. I. Formulation of a problem. Goals and objectives of the study. Population genetic characteristics of Northern Khanty]. / Kompleksnoe mediko-geneticheskoe izuchenie naseleniia Zapadnoi Sibiri. Soobshchenie I. Postanovka problemy. Tseli i zadachi issledovaniia. Populiatsionno-geneticheskaia kharakteristika severnykh khantov.

Purposes and tasks of the complex medical and genetic study of West-Siberian inhabitants were formulated. Demographic parameters for the North Khanty inhabitants, such as size, dynamics of the tertiary sex ratio, marriage structure and migration processes were presented. Mating and intrapopulation migration patterns are determined by spatial subdivision, because of the vast territory and the traditional way of life, and by isolation by distance. Index of the isolocal endogamy equals to 0.364. The portion of mixed marriages is 22.7% and that of the gametic contribution of immigrants - 7.3%. Inbreeding coefficient by isonymy is 0.00097. Effective population size of the five subpopulations studied as a whole is 25% of their total number.

[Population-genetic study of the blood lipids of the aboriginal population of the Obian North]. / Populiatsionno-geneticheskoe issledovanie lipidov u korennogo naseleniia Obskogo Severa.

Lipid metabolism (total cholesterol, triglycerides, and high-density lipoprotein cholesterol) was shown to be dependent on age, while the atherogenic coefficient was unrelated to age both in males and females. Heritability coefficients have been calculated for the above-mentioned parameters on the basis of intrafamilial correlations analysis. Interclass coefficients of the parent-parent correlations suggest the absence of common environmental effects. Significant associations of all three with phenylthiocarbamide, and of hypertriglyceridemia with phenotype MN (p less than 0.05) were demonstrated in females. The determination coefficient has shown the PTC and MN to make the greatest contribution to blood lipid variability.