Elemental Composition and Morphological Characteristics of Kidney under the Influence of Toxicants

Morphological changes and element status in laboratory animals under the influence of ethanol and cadmium were studied. Male Wistar rats were under investigation. Experimental animals received standard diet and in addition 15% ethanol solution and cadmium sulfate orally. Histologic and morphometric studies were done. Routine Mayer's hematoxylin and eosin stain for renal histological sections was used. Ocular micrometer МОВ-1-15х1500 and software “Test Morpho 4.0” were used for investigation of histological and cytological structures of kidneys. Digital images were produced by microscope “Micros” (Austria). The methods of inductively coupled plasma spectroscopy and atomic emission spectroscopy (Optima 2000 DV and ELAN 9000, PerkinElmer, USA) were used for the elemental study of samples. The combined effect of ethanol and cadmium sulfate caused renal damage which was confirmed morphologically. It was found that 65% nephrons were exposed to destruction with microinfarctions. Volume of 80% renal corpuscles was reduced, but volume of nephrocytes was extended at all levels of nephron. Cavities of the renal tubule were extended in the area of Henle's loop. In addition to kidney damage the concentrations of Mg, P, As, Fe, Cu, Si, Zn were decreased and Cd concentration was increased in the blood of laboratory animals, who were exposed to ethanol and cadmium. The concentration of cadmium was 86 times (p ≤ 0,001) higher and the concentration of lead was 25 times (p ≤ 0.05) higher in the hair of animals from the experimental group than in the hair of animals from the control group.

The purpose of the research is investigation of influence of ethanol and cadmium on the laboratory animals' renal tissue and element status.

Animals
The experimental model of simultaneous influence of ethanol and cadmium was created.The investigation was performed with the usage of male Wistar rats (n=20, weight=180±10.2 g).The animals were kept in clinical and biological laboratory "Vivarium" (Orenburg State University).They were maintained in controlled environment (12:12 h light/dark cycle).The experiment was conducted by observing humane principles as presented by European Community Directive 86/609/EC.

Treatment Design
The duration of the experiment was equal to 45 days.Experimental animals received nutritionally balanced diet and extra 15% (w/v) ethanol solution (42.3-48.9g/kg/day), cadmium sulfate 23.55 mg/kg/day was added to food (the dose was selected according to (Izmerov, 1984), the rats were allowed free access to food anad water.In average the rat received 3.2 -3.4 mg cadmium sulfate/kg body weight per day.The animals of control group received standard diet and tap water ad libitum.At the end of experimental period anesthetized rats were decapitated.

Trace Element Analysis
25 chemical elements were detected in the samples of the rat blood and hair.Analysis were performed in laboratory "Center for Biotic Medicine" (registration number state register RU 0001.513118; May 29 2003; Registration Certificate of ISO 9001: 2000, Number 4017-5.04.06) using the methods of inductively coupled plasma spectroscopy and atomic emission spectroscopy (ICP-AES Optima 2000 DV and ELAN 9000 (Perkin Elmer, USA)).

Histological Analysis
The extraction of histological material from different renal parts was performed during the 30 minutes interval after animals' death.The material was fixed in a 10% neutral buffered formalin, then passed through a battery of alcohols with growing strengths and then covered in paraffin.Routine Mayer's hematoxylin and eosin stain for renal histological sections (thickness -5-6 mkm) was used.Ocular micrometer МОВ-1-15х1500 and software "Test Morpho 4.0"(Russia) were used for investigation of kidney histological and cytological structures, parameters of cell structure and cell nucleus.Each sample of tissue was studied at least from 16 views.Digital images were produced by the microscope "Micros" (Austria).The interconnection between histological indexes was expressed via pair correlation coefficients.

Statistical Analysis
Statistical analysis (calculation of the arithmetical mean, the standard error of the mean and the Student criteria) was performed using standard statistical software ("Excel 2010" «Office XP» and «Statistica 6.0»).The Shapiro-Wilk test was used for testing normality.

Results and Discussion
The renal capsules of rats from the control group were distinct.Glomerular capillaries were identified clearly in renal corpuscles of renal cortex (Figure 1) Cavity of the capsule of the nephron enlarged moderately.Afferent artery and peritubular capillaries were dilated.Nephrocytes were represented at all levels of renal tubule as prismatic, tubular, cubic or flat shape cells.Tubular epithelial cells had light oxyphilic cytoplasm and basophilic nucleus.Experimental rats had a distended and peeled off in some areas renal capsule (Figure 2).There was edema of parenchymatous and stromal elements of renal cortex.The vessels of microvasculature were sludged with erythrocytes.Approximately 65% of nephrons had destruction and certain microinfarctions.There were polymorphocellular infiltrations around Bowman's capsule, leading to aggravation of destruction in all parts of nephrons.80% of glomeruli had reduced volume, but the volume of nephrocytes was extended at all levels of the nephrons.Cavities of renal tubule were extended in the area of Henle's loop.The plasma transudation from extended capillaries to intertubular connective tissue was observed.After aggregating the morphometrical parameters of the kidneys of rats from the control and experimental groups on the basis of interconnected histological elements we performed the statistical analysis (Table 1).Note: statistically significant difference * (p ≤ 0.05).
The relationship between the histological structure of all parts of the kidney of rats in a state of physiological norm ranged from -0.89 to + 0.96.It is mostly positive (78.6%) and insignificant (64.3%).The relative correlation coefficient was equal to 1.07, which means that the system was in the state of dynamic equilibrium.
The correlation was significant between the volume of the nucleus of epithelial cells of the proximal part of nephron and: the volume of the proximal part of nephron epithelial cells (r = 0.96); the diameter of microvessels of glomerulus (r = 0.95); the diameter of the proximal part of the nephron (r = 0.77).
The correlation was significant between the volume of the proximal part of nephron epithelial cells and: the diameter of the proximal part of the nephron (r = 0.92); the diameter of microvessels of glomerulus (r = 0.88); the volume of glomerulus (r = 0.78).The correlation was significant between renal corpuscles volume and: the diameter of the nephron in the Henle's loop area (r = 0.91); the volume of the cavity of the Bowman's capsule (r = 0.82); the diameter of microvessels of glomerulus (r = 0.79).
Thus, the leading structures in the kidney of rats from the control group are: the volume of the nuclei of epithelial cells and of the epithelial cells of the proximal part of nephron themselves, and the volume of renal cells that exhibit a high degree of correlation with the diameters of microvessels of glomerulus, channel of the nephron and volume of the cavity of the Bowman's capsule.
In the kidney of rats from the experimental group the correlation between the histological structure of all parts of а b the kidney ranged from -0.79 to + 0.96.It is positive (64.3%) and mostly insignificant (82.1%).
The relative correlation coefficient was 1.2, which means that the system was implementing a set of adaptive reactions to maintain its dynamic equilibrium.In comparison with the state of the kidney of rats from the control group the complex implemented adaptive reactions expressed in the change of leading structures.The correlation was significant between the volume of glomerulus and: the volume of the cavity of the Bowman's capsule (r = 0.86); the diameters of microvessels of glomerulus (r = -0.79); the diameter of the proximal part of the nephron (r = 0.77).The correlation was significant between renal corpuscle volume and the diameter of the proximal part of the nephron (r = 0.96).
The correlation was significant between the epithelial cells of the proximal volume of the nephron and the volume of the cavity of the Bowman's capsule (r = 0.82).
The elemental homeostasis was evaluated with the use of the blood and hair of laboratory animals as biosubstrates.The study showed the development of diselementosis under the influence of intoxication, which expressed in a decrease concentration of Mg, P, As, Fe, Cu, Si, Zn in the blood, and increase concentration of Cd (Table 2).Our results are consistent with those of other authors.It is known that a deficiency of phosphorus may occur during intoxication with ethanol.A number of authors (Rebrov & Gromova, 2008) point out that alcohol increases the loss of magnesium through the kidneys and alcoholics are often found deficient in this element.The effect of ethanol on the increase in nephrotoxicity of cadmium in rats was proposed in the work (Brzoska, Moniuszko-Jakoniuk, Piłat-Marcinkiewicz, & Sawicki, 2003).Clinical studies confirm the toxic effects of cadmium on renal function (de Burbure, C. et al., 2006).Thus, it can be assumed that people who abuse alcohol are at greater risk of kidney damage when exposed to cadmium.The content of iron, zinc and copper were lower in the blood of animals from the test group.Low iron concentration may be a result of its intense consumption by hepatocytes during chronic intoxication with ethanol, which may be due to the increased number of ferritin receptors (Jurczuk, M., Brzóska, M. M., Rogalska, J., & Moniuszko-Jakoniuk, J., 2003).Low levels of zinc is apparently a consequence of the high content of lead and cadmium, which are antagonists.
The results of studying of the elemental composition of rats' hair are presented in the Table 3.Among the macroelements it was only potassium which concentration in rats' hair significantly differed between the control and the experimental groups.Its concentration in the hair of rats from the control group was increased by 30% in comparison with the experimental.Among the essential and conditionally essential elements, the concentrations of arsenic, cobalt, chromium, iron, nickel and zinc were significantly different between the groups.
The concentration of all these elements except for zinc was lower in the hair of animals from the experimental group: arsenic was decreased by 2.3 times compared to control (p ≤ 0,01), cobalt was decreased by 1.8 times (p ≤ 0.05), chromium was decreased by 1.7 times (p ≤ 0, 05), iron (p ≤ 0,01) and nickel (p ≤ 0.05) were decreased by 1.5 times, zinc was increased by 1.5 times.There were no significant differences among the concentrations of other elements in the two groups.The significant differences in the content of the toxic and potentially toxic elements were obtained for cadmium and lead.The concentration of cadmium was 86 times (p ≤ 0,001) higher and the concentration of lead was 25 times (p ≤ 0.05) higher in the hair of animals from the experimental group than in the hair of animals from the control group.
In this study we examined the changes in renal tissue during simultaneous exposure to multiple toxicants (ethanol and cadmium) on the body of laboratory animals.Taking into consideration the abundance of drinking alcohol and tobacco smoking among the population of the Russian Federation, the combined effect of the two substances is not uncommon (Onishchenko, 2003).Our results indicate the presence of clearly distinguishable morphological and functional changes in the kidney of rats under effect of ethanol and cadmium.Since ethanol increases the permeability of biological membranes for cadmium (Pal, Nath, & Gill, 1993) people who use alcohol are more susceptible to the effects of this heavy metal.

Conclusion
It was revealed that the rats of the experimental group had about 65% of nephrons exposed to destruction with microinfarctions, 80% of kidney cells reduced their volume, while at all levels of the nephron the neprocytes had an increased volume.The volumes of glomerulus and renal corpuscles were the leading structures in the kidney of animals of the experimental group, the volume of the proximal part of nephron epithelial cells showed correlation only with the volume of the cavity of the Bowmans' capsule, indicating the desynchronization of correlation structures of the system (in comparison with the control group), expressed in the kidney failure.The investigated content of chemical elements in the blood of laboratory animals indicates the development of changes of the element status under the influence of the consumption of ethanol in combination with cadmium.

Table 1 .
Indicators of histological structure of kidneys of rats in the control and experimental groups, (M ± m), mkm

Table 2 .
Averages content of the chemical elements in the blood of laboratory animals (M ± m), mg / kg

Table 3 .
Average content of chemical elements in the hair of laboratory animals (M ± m), mg / kg