Histological and Ultrastructural Evidence for Protective Effects on Aluminium-Induced Kidney Damage by Intraperitoneal Administration of α -Tocopherol

Abstract

The nephrotoxic actions of aluminium (Al) arise from its accumulation in the kidneys, with the resultant degeneration of the renal tubular cells. It has been suggested that Al generates reactive oxygen species that cause the oxidative deterioration of cellular lipids, proteins, and DNA. To test this hypothesis, we have here investigated the potential for a protective role of α-tocopherol (vitamin E) during short-term exposure of rats to Al. Al was administered intraperitoneally either alone or in combination with vitamin E at a different point of abdomen, and the alterations in the kidney tissue were analyzed histologically. The results reveal that significant light microscopical and ultrastructural damage is caused by Al, whereas with the immediate coadministration of vitamin E, there is a protective effect against this damage to the kidney tissue. In Al-alone group, the glomeruli and proximal tubuli and the Bowman capsules had swellings, adherence, hemorrhage, increase in mesengial matrix, and marked interstitial tissue fibrosis, indicating severe damage. In the Al and vitamin E immediate coinjected group, renal tubule cells were almost of a normal appearance. A slight stenosis was seen in the capsular area in the Malpighi corpuscules. The tubular organization and the cytoplasmic basophilia were also much the same as in the control group, with the lumen clearly visible in most of the cortical tubuli. The results highlight the need to reduce exposure to Al, with particular attention being paid to the known sources of Al. At the same time, the maintenance of a diet that is rich in vitamin E should be beneficial in the alleviation of Al toxicity.

Aluminium (Al) is a ubiquitous element that is found in every food product. Particular sources of Al include corn, yellow cheese, salt, herbs, spices, tea, cosmetics, and Al cooking utensils and containers (El-Demerdash 2004; Yousef 2004). In addition, Al is present in medicines and is added to drinking water for purification purposes (Ochmanski and Barabasz 2000; Nayak and Chatterjee 2001).

The promotion of renal failure through Al accumulation in the kidney and the subsequent systemic toxicity is well known (Alfrey 1980; Ecelbarger, MacNeil, and Greger 1994; Mahieu et al. 2005), with Al also seen to accumulate in a number of other mammalian tissues, including the brain, bone, and liver (Stein et al. 1987; Bertholf et al. 1989; Wills et al. 1993; Ferreyra-Moyano and Barragan 1994; Sahin, Varol, and Temizer 1994; Anand et al. 2002). Al accumulation in the kidney promotes the degeneration of the renal tubular cells, inducing nephrotoxicity (Ebina et al. 1984; Stein et al. 1987; Cacini and Yokel 1988; Bertholf et al. 1989; Roy, Talukder, and Sharma 1990; Somova, Missankov, and Khan 1997; Sargazi, Roberts, and Shenkin 2001; Anand et al. 2002; Mansour, Alan, and Norman 2006).

These toxic effects of Al have been suggested to be due to the generation of reactive oxygen species (Prakash and Rao 1995; El-Demerdash 2004), which results in the oxidative deterioration of cellular lipids, proteins, and DNA (El-Demerdash 2004; Mansour, Alan, and Norman 2006). Its nephrotoxicity arises through the formation of renal lesions that are confined to the renal tubules, e.g., necrosis and atrophy of proximal tubules, tubular ectasia, and the presence of grey material in the tubular epithelium (Bertholf et al. 1989; Anand et al. 2002), with further morphological changes seen in the altered tight junctions and brush borders of these cells (Sargazi, Roberts, and Shenkin 2001; Wills et al. 1993).

At the physiological level, although Al accumulation in renal tissue does not result in important changes in global renal functions, it has been seen to affect cellular metabolism, promote oxidative stress, induce alterations in renal tubular p-aminohippuric acid transport and renal tubular phosphate reabsorption, and to impair the sodium and water balance (Mahieu and Calvo 1998; Mahieu et al. 2003; Mahieu, Millen, and Elias 2004).

In addition, despite low gastrointestinal absorption, the accumulation of nontoxic levels of Al alone can cause dose-dependent neurotoxic effects in brain and cytotoxic effects in kidney (Somova, Missankov, and Khan 1997). Indeed, different forms of Al have been shown to have different gastrointestinal absorption rates. In particular, Al sulphate is generally used in animal experiments due to its high absorption. Similarly, different forms of Al can have different accumulation and excretion rates and toxicities. Thus, it has been shown that rats respond differently to Al chloride than Al citrate, where the kidneys of rats following intravenous administration of Al citrate contained much higher concentrations of Al than those that received Al chloride (Spencer et al. 1995). Significant accumulation of Al also occurs in liver and kidney more than in heart or lung tissue (Bertholf et al. 1989). Finally, when Al was given as the citrate, the urinary excretion was significantly higher than when given as the chloride or sulphate (Lote et al. 1995). However, despite these various studies on Al toxicity, the mechanism of Al-originated damage remains unclear (Sargazi, Roberts, and Shenkin 2001).

Vitamin E (α-tocopherol) is a naturally occurring antioxidant nutrient that has an important role in animal health through the inactivation of harmful free radicals that are produced during normal cellular activity and under various stress conditions. The antioxidant function of this micronutrient could also, at least in part, enhance immune reactions by maintenance of the functional and structural integrity of the all-important immune cells (Yousef, Abdallah, and Kamel 2003; El-Demerdash et al. 2004). Indeed, with the health problems that can be induced by many environmental pollutants, efforts have been made towards the evaluation of the relative antioxidant potential of vitamin E (El-Demerdash 2004).

In terms of the absorption of vitamin E after oral intake, there have been few studies reported in the literature. The α-tocopherol excretion in the feces has been seen to increase significantly with increased intake, with the absorption rate of α-tocopherol being inversely proportional to the level of long-term oral supply (Elmadfa and Walter 1981). Furthermore, it has been seen that free tocopherol (as α- or γ -tocopherol) absorption requires the presence of bile salts (Traber et al. 1986).

There are three specific situations when a vitamin E deficiency is likely to occur. It is seen in people who cannot absorb dietary fat, it has been found in premature, very-low-birth-weight infants (birth weights less than 1.5 kg, or 3.5 pounds), and it is seen in individuals with rare disorders of fat metabolism. Therapeutic doses by both parenteral and oral routes are the same in humans, implying that there are no absorption differences between these routes, with the mean daily available intake being 0.1 to 17.5 μg in humans (Cadorniga 1982; Kohlschütter et al. 1988).

The aim of the present study was thus to determine the potential for a protective role of vitamin E against the effects of Al exposure on rat kidney tissue, as seen under both light and electron microscopy.

MATERIALS AND METHODS

Adult male albino Wistar rats (180 to 200 g; supplied by Pamukkale University Experimental Animal Care Unit & Research Center, Denizli, Turkey) were housed in the Experimental Animal Care & Research Center of the University of Pamukkale, Denizli, Turkey. They were reared under the supervision of a veterinarian, and kept in a well-ventilated, noiseless environment, with free access to food and water. The male rats were selected randomly and divided into four groups of seven.

The treatment schedule provided for the intraperitoneal injection of saline in the first (control) group (2 ml/200 g body weight). The second group were similarly injected with the Al (Al₂(SO₄)₃; 1 mg/200 g body weight) (Sigma-Aldrich) dissolved in saline (maintaining 2 ml saline/200 g body weight, throughout). The third group had immediately sequential in-traperitoneal injections of the same level of Al followed by vitamin E (100 mg/200 g body weight) (α-tocopherol Asetat, 300 mg/dl; Aksu Farma, Turkey) dissolved in saline, with the fourth group injected with the α-tocopherol in saline alone. The injections were performed three times per week over a period of 2 weeks, and at the end of the second week the rats were sacrificed while under xylazine and ketamin anaesthesia (xylazine [Alfasan, Turkey] 5 mg/kg intramuscularly [IM]; ketamin [Richterpharma AG, Wels, Austria] 50 mg/kg, 10% IM).

The kidney tissues of each rat were collected and processed for routine histology and electron microscopy. Briefly, for the former, this involved sectioning of the kidney tissue (5 μm thick), staining with hematoxylin-eosin dye, and analysis under light microscopy (Olympus BX 51 Microscope); the sections were photographed using a VITEC CDC camera (Bilgi Elektronik, Istanbul, Turkey). The specimens were also processed for routine electron microscopy. Briefly, the tissues were fixed with 2.5% gluteraldehyde and 1% OsO₄, dehydrated through increasing concentrations of ethanol, and embedded in gelatine capsules. Thin sections were taken, stained with uranyl acetate and lead citrate, and examined and photographed under electron microscopy (LEO 906E).

This study was approved by the Ethical Committee of the Pamukkale University Experimental Animal Facility.

RESULTS

Light Microscopy

Under the light microscope, the kidney sections from the control group of rats did not show any damage, with the glomeruli and tubuli having a normal appearance (Figure 1A). However, in the Al-injected group, the glomeruli and proximal tubuli were swollen, and the Bowman capsules had adhered to the glomeruli. There was also an increase in the mesangial matrix (Figure 1B). Some of the glomeruli showed crescent formation, dilatation in the Bowman space, and exudation of erythrocytes (Figure 1C). In the cortical tubuli of the kidneys of these Al-treated rats, there were slight swellings, and considerable damage and degeneration, with shape, position, and volume disturbances of the nuclei seen (Figure 1B). There were numerous renal tubule cells with very dense and obscure cytoplasmic details. On the other hand, some of the tubuli had a critically dilated appearance. Marked interstitial tissue fibrosis was seen among the damaged tubuli, with marked destruction of the tubule epithelial cells also evident (Figure 1C).

In the Al and vitamin E double-injection group, there were no significant histological abnormalities seen in comparison with the control group, and the renal tubule cells were of a normal appearance (Figure 1D, E). However, a slight stenosis was seen in the capsular area in the Malpighi corpuscules (Figure 1D). The tubular organization and the cytoplasmic basophilia were also similar to the control group (Figure 1D, E), with the lumen clearly visible in most of the cortical tubuli (Figure 1E).

Electron Microscopy

In the control group, the brush borders of the proximal tubuli were well developed and appeared intact, with long brush borders, numerous and deep basolateral invaginations, and large and elongated mitochondria positioned at the basal pole (Figure 2A). In the kidneys from the Al-injected group, the brush borders of the proximal tubuli were shorter and either irregular in height or focally absent (Figure 2B). There were fewer basolateral invaginations, which also showed fragmentation, and the mitochondria were smaller, heterogeneous in size, and randomly scattered throughout the cytoplasm (Figure 2C). Dilatation and vacuolization of the mitochondria was seen, with some evidence of necrosis (Figure 2D).

In the Al and α-tocopherol coinjected rats, the microvilli were similar to those in the control group and the cell nuclei were slightly more heterochromatic. Although the mitochondria were again randomly scattered throughout the cytoplasm, they were more homogenous in terms of their shapes and sizes, and the cristae structure appeared almost normal, with no observable damage (Figure 2E).

DISCUSSION

Al-induced damage to body organs has already been reported in several studies (Parkinson, Ward, and Kerr 1981; Wills and Savory 1983; Exley, Pinnegar, and Taylor 1997), and the accumulation of Al in the kidney has been related to a deterioration in renal function (Nesse et al. 1997). The daily intake of Al has been estimated to be 9 and 14 mg, with pharmacological doses of Al as antacids estimated to be 1 and 3 g daily (Roy, Talukder, and Sharma 1991). The absorption rate of Al via the oral route is low, at 0.1% to 1% (Sargazi, Roberts, and Shenkin 2001).

Acute proximal tubular necrosis has previously been seen in rats given daily intraperitoneal injections containing 5 mg Al per kg body weight (Ebina et al. 1984). Similarly, administration of 1.7 mg/100 g body weight of Al sulphate has been shown to result in a slight swelling of the tubules of the cortex, whereas 2.9 mg/100 g body weight led to the contraction of glomeruli and degeneration of the distal tubules, subcapsular necrosis, and dilatation and degeneration of the medulla. The further increase to 8.6 mg/100 g body weight induced hemorrhage and dilatation of the tubules, with 17.2 mg/100 g body weight causing marked degeneration of tubules (Roy, Talukder, and Sharma 1991). The present study shows that the kidneys of rats exposed to repeated Al dosing over a 2-week period show major histological alterations both at the light and electron microscopy levels, further implicating Al as a major toxicant in kidney tissue in the induction of renal failure.

In agreement with previously published data (Squibb, Pritchard, and Fowler 1984; Condron, Schroen, and Marshall 1994; Herak-Kramberger, Brown, and Sabolic 1998; Sargazi, Roberts, and Shenkin 2001; Sabolic et al. 2002), we have here confirmed the occurrence of specific morphological damage in the cortical convoluted tubules, which includes a shorter and irregular brush border membrane, fragmented mitochondria and vacuolization of the cytoplasm. In the Al-injected group, numerous proximal renal tubule cells showed swelling, tubule epithelia damage and morphological changes in epithelia cell shape. This was accompanied by occasional tubule dilatation, tubule interstitial fibrosis, and lymphocyte infiltration into some of the interstitial areas. These findings are also in agreement with previously reported studies (Roy, Talukder, and Sharma 1991; Wills et al. 1993; Damek-Poprawa and Sawicka-Kapusta 2003; Hanafy and Soltan 2004).

The alterations seen to the glomeruli in our Al-injected group also paralleled previously reported findings (Talukder and Sharma 1991; Wills et al. 1993; Damek-Poprawa and Sawicka-Kapusta 2003; Roy, Sabolic et al. 2006). These have included glomerulus swelling and capsular dilatation, which in some glomeruli resulted in their half moon–like appearance. In addition, there was an increase in the glomerulus mesengial matrix in the samples from the Al-injected rats, as was also seen in some previous studies, although the granular grey-blue accumulation that has been seen previously in mesengial cells of the glomeruli was not observed in the present study (Wills et al. 1993). These differences may be related to the different Al dosing used across these and the present study. Thus, it can be seen that this histological appearance of the kidney tissue is indeed due to the Al toxicity.

Of note, some of our findings in the Al-injected group parallel what has also been seen previously in the kidney tissue from rats that were injected with cadmium, another heavy metal that is known to be toxic to the kidney (Sabolic et al. 2006). These included a decrease in the basolateral infoldings of the proximal tubule cells, and alterations in mitochondria shape and location, i.e., seen as rounded structures within the cytoplasmic areas outside of these infoldings, when they are normally seen as elongated structures in between these infoldings.

Katyal et al. (1997) reported that Al is implicated in the pathogenesis of several clinical disorders, including renal dysfunction, and thus the main aim of this study was to investigate the ability of vitamin E to protect against such Al-induced kidney damage. Lipid peroxidation is one of the main manifestations of oxidative damage, and it has been found to have important roles in the toxicity and carcinogenicity of many xenobiotics (Anane and Creppy 2001). Antioxidants are known to reduce oxidative radical–induced reactions (Somova, Missankov, and Khan 1997; El-Demerdash 2004). Antioxidative agents have also been used against Al and/or cadmium toxicity, including selenium and ascorbic acid. A lack of maternal and developmental toxicity in mice given high doses of Al hydroxide and ascorbic acid during gestation has been shown (Colomina et al. 1994), with a protective role of ascorbic acid seen towards Al-induced changes in hematobiochemical parameters, lipid peroxidation, and enzyme activities in male rabbits (Yousef 2004). Effects of selenium on the functional state of rat kidney in Al/cadmium poisoning have also been shown (Rudenko et al. 1998).

α-Tocopherol, or vitamin E, is an important antioxidant in biological systems, and it has been shown to inhibit the peroxidation of membrane lipids by scavenging lipid peroxyl radicals, with the production of a tocopheroxyl radical as a consequence (Arita et al. 1998). Chinoy and Memon (2001) demonstrated that α-tocopherol has a very significant bearing on the protective effects against Al toxicity in humans. Thus, in the present study, we also immediately coinjected the rats with α-tocopherol, along with Al, with a significant protective effects against the Al-alone kidney tissue damage seen in this group. Thus, there was no tubule damage, interstitial fibrosis, or lymphocyte infiltration; further, as with the control samples, the glomeruli and the capsular space appeared normal.

In conclusion, with Al known to have adverse effects on human health, the present study demonstrates that the administration of α-tocopherol in combination with Al minimizes these hazards. Thus, whereas all efforts should be made to reduce the human exposure to Al, with particular attention being paid to sources of Al in foods, water, and personal care products, the use of a diet that is rich in vitamin E should also be beneficial in alleviating and protecting against Al toxicity.