Renal Cortical Structural Alterations in Atorvastatin-treated Rats and the Possible Protective Mechanisms of L-Carnitine

*Corresponding Author:

Sahar Youssef
Anatomy Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo
E-mail: sahar_sayed@yahoo.com

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

Abstract

The present study investigated the structural changes in rat kidney following administration of atorvastatin and evaluated the possible protective role of L-carnitine. Rats were divided into four groups, group I- control group, group II received L-carnitine at 100 mg/kg/d, group III received atorvastatin at 80 mg/kg/d and group IV received atorvastatin plus L-carnitine. Hematoxylin and Eosin, periodic acid-Schiff, inducible nitric oxide synthase, proliferating cell nuclear antigen expression and electron microscopy investigation were performed. Light microscopy results of atorvastatin group showed severe atrophic glomeruli with widened glomerular spaces. Some renal tubules showed cytoplasmic vacuolations, loss of brush borders, hemorrhage and mild congested blood capillaries. Strong positive inducible nitric oxide synthase and proliferating cell nuclear antigen immunostaining in the tubules were observed. Ultrastructurally, the proximal convoluted tubules had small electron dense nuclei, numerous lysosomes, partial loss of microvilli and vacuolations. The podocytes revealed united foot processes and increase in the thickness of the glomerular basement membrane. L-carnitine preserved the structure of renal cortex, decreased inducible nitric oxide synthase and increased proliferating cell nuclear antigen reaction. In conclusion, atorvastatin exerted a deleterious effect and L-carnitine improved most of the histological changes produced by atorvastatin.

Keywords

Atorvastatin, kidney, PAS, iNOS, PCNA, ultrastructure

Statins are a class of the most recommended drugs prescribed for various diseases such as hypercholesterolemia, ischemic heart disease, and cerebrovascular accidents[1]. Hyperlipidemia has adverse effect on neurological outcomes that could lead to disability. Statins can reverse this risk of disability in patients treated with a lower dose[2]. High dose of statins has been recognized to be more successful in cardiovascular diseases than the lower dose. However, much concerns of threats of rhabdomyolysis, acute kidney injury, diabetes mellitus, and memory loss have been encouraged some researchers to identify more accurately the side effects of statins[3]. Statins are 3-hydroxy3-methyl-glutaryl-coenzyme A reductase inhibitors existent in several forms including atorvastatin (Lipitor)[4]. Studies have reported that atorvastatin, a lipid lowering agent, decreased vascular risk in patients with coronary artery disease by reducing plasma levels of cholesterol[5]. It has been reported that administration of atorvastatin is associated with an imbalance between pro-oxidant mechanisms and the antioxidant defences, contributing to oxidative stress. Thus, reducing intracellular glutathione level, and increasing intracellular lipid peroxidation leading to oxidative damage to proteins and nucleic acids[6].

Much concern has been focused on the defensive effects of antioxidants against oxidative stress damage. Investigators reported that L-carnitine is a compound biosynthesized from the amino acids methionine, and lysine in kidney and liver. It presents at a high concentration in skeletal and cardiac muscles. L-carnitine assists as a cofactor necessary for the transport of long chain fatty acids into the mitochondria for energy production in the peripheral tissues[7]. Previous study reported that L-carnitine acts as an antioxidant and protectant against oxidative damage in diverse organs including kidney[8]. L-carnitine used as a protectant against drug induced nephrotoxicity such as the aminoglycosides, the anticancer drugs, and the contrast medium agents[9]. It has been reported that L-carnitine inhibited free radical generation, prevented the damage due to fatty acid β-oxidation in mitochondria and protected tissues from impairment by renovating oxidized membrane lipids[10]. The current study was performed to assess the structural changes in the renal cortex following atorvastatin treatment and the possible protective mechanisms of L-carnitine.

Materials and Methods

Atorvastatin (Lipitor) 40 mg tablets were obtained from Egyptian International Pharmaceutical Industries Company (EIPICO), Egypt. Tablets were ground and freshly dissolved in distilled water. L-carnitine was obtained from Arab Co. for Pharmaceuticals and Medicinal Plants (MEPACO-MEDI Food), Egypt.

Experimental animals:

Male Wistar rats weighing 200±3 g were used in this study. Rats were obtained from the Central Animal House, Faculty of Medicine, Assiut University. Rats were housed and controlled according to the techniques in agreement to the National Institute of Health protocol and Institutional Ethics Committee of Assiut University. Before the beginning of the experiment, rats were set for two weeks for acclimatization to their new environment. Then they were housed in the stainless steel cages at a suitable temperature (24±1°) and humidity controlled room with 12-h light/dark cycle. They were retained under good illumination. Animals were fed with standard rat pellet, and had free access to water.

Animal groups:

In this experimental study, 32 male rats were divided into 4 equal groups as follows, group I served as the control group received distilled water. Group II received L-carnitine, 100 mg/kg[10]. Group III received atorvastatin dissolved in distilled water at 80 mg/kg/d. Group IV received both L-carnitine and atorvastatin at the same doses as given to group II and III rats. The human therapeutic dose of atorvastatin is ranged from 10-80 mg/d[11]. The maximum human therapeutic dose of 80 mg/kg/d was used in the current work. The equivalent therapeutic dose for adult male rat was 1.44 mg/200 g, which was calculated using the formula of Laurence and Bacharach[12]. All drugs were administered orally and daily for 30 d.

Histological technique:

At the end of the experiment, kidneys from different groups were excised and the right kidneys were fixed in 10 % formalin solution for 24 h. The specimens were processed to prepare 5 μm thick paraffin sections and stained with Hematoxylin and Eosin (H and E) and Periodic acid-Schiff (PAS) stains[13].

Histopathological study:

To determine the changes or damage in kidney morphology, the H and E sections were investigated by blinded pathologist to determine the presence of glomerular and tubular damage. Five pieces of kidney section from each group were observed and scored under light microscope. The severity of renal injury score (0-4) was performed by assessing the percentage of glomerulus and cortical renal tubules that revealed abnormalities such as increases of Bowman’s space, pyknotic nuclei and tubular dilatation, The histopathological assessment was performed as follows: 0: none; 1: <5 %; 2: 5-25 %; 3: 25-75 % and 4: >75 % . All scorings were summed and represented as mean values on the graph[14].

Immunohistochemical technique:

Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) was directed using the avidin-biotin peroxidase according to a previously published technique[15]. Three mictometer thick sections obtained from paraffin embedded tissues were deparaffinised, rehydrated and rinsed in tap water. The endogenous peroxidase activity was removed by treatment with methanol comprising 0.03 % hydrogen peroxide for 20 min. Before incubation with primary antibody, sections were incubated with normal serum for 20 min to block nonspecific antibody. Immunohistochemical staining was carried out using a primary antibody to PCNA, (Clone PC10), mouse monoclonal, Thermo Fischer Scientific, 1:200). Next, the biotinylated goat antimouse IgG secondary antibody (Abcam, Cambridge, USA, 1:200) was used to detect the primary antibody to PCNA. The primary antibody for iNOS is antiiNOS antibody, rabbit polyclonal antibody (Abcam, Cambridge, USA, 1:200). Subsequently, sections were incubated with biotinylated goat antirabbit IgG secondary antibody (Abcam, Cambridge, USA, 1:100). All sections were stained successively with biotinylated IgG for 10 min and avidinbiotin horseradish peroxidase complex for 15 min. Visualization of the reaction was carried out by adding the chromogen diaminobenzidine (Dako, Glostrup, Denmark) to slides, which produces a dark brown colour that is detected by light microscopy. Then the slides were washed with distilled water and sections were counterstained with Mayer’s haematoxylin. Kidney sections were dehydrated, reduced transparent with xylene, and cover slipped. Slides were observed under a light microscope. The negative controls were run regularly in parallel by omitting the primary antibody. Normal kidney tissues were used as positive controls. The investigation and the photography were conducted at the Mycology and Biotechnology Unit, Al-Azhar University, Cairo, Egypt.

Transmission electron microscopy technique:

Specimens for electron microscopic study were cut into 1 mm³ pieces and fixed in 2.5 % glutaraldehyde for 24 h. The kidney specimens were then washed in three to four changes of cacodylate buffer (pH 7.2) for 20 min each. Post fixation was performed in 1 % osmium tetroxide for 2 h. Then, they were washed in four changes of cacodylate buffer for 20 min each. Dehydration was carried out using ascending grades of alcohol. They were cleared in propylene oxide and then embedded in Epon 812 using a gelatin capsule. The samples were kept in an incubator at 35° for one day, at 45° for another day and finally at 60° for 3 d[16]. The semithin sections, 0.5-1 μm were prepared using LKB ultramicrotome and stained with toluidine blue. Ultrathin sections (50 nm thick) from selected areas of trimmed blocks were prepared and collected on copper grids then the ultrathin sections were stained with uranyl acetate for 10 min and lead citrate for 5 min. Finally, these sections were examined under an electron microscope (Jeol JEM 1010, Japan) at the electron microscopic unit of Faculty of Medicine, Assiut University.

Morphometric and statistical analysis:

All parameters were measured Using Leica 500 software image analyser (Leica image system Ltd, Cambridge, England). The Mean area percent of PAS positive reaction of the brush borders and basal laminae of the proximal and distal convoluted tubule was measured. The parameters of mean area percent of iNOS and PCNA positive immunoreaction were also measured. Randomly, twenty fields were taken at x400 magnification. Obtained data of the morphometric results were organized and analysed. Data were summarized as mean±standard deviation (SD) and compared using one-way ANOVA followed with posthoc Tukey’s test for multiple comparisons between groups using SPSS software 16 (SPSS, Chicago, Illinois, USA). The differences were statistically significant when p<0.05.

Results and Discussion

H and E stained sections obtained from the control rats (group I) revealed normal appearance of the renal cortex (fig. 1a). The renal cortex consists of the renal corpuscles and the tubules. The renal corpuscles comprised of tuft of capillaries (glomerulus) surrounded by Bowman’s capsule. The capsule consists of two layers, the outer parietal and inner visceral layers. The urinary space or Bowman’s space exists between the two layers of the capsule. The proximal convoluted tubules are lined by acidophilic cuboidal epithelium. The proximal convoluted tubules with apparent apical brush border and a narrow lumen were observed. The cytoplasm was acidophilic and granular. The nuclei were rounded, basal, and vesicular. However, the distal convoluted tubules were lined with acidophilic cuboidal epithelium and they have wide lumen (fig. 1a).

H and E stained sections obtained from L-carnitinetreated group (group II) revealed that the renal corpuscles consist of tuft of glomeruli and Bowman’s capsule (fig. 1b). Intact glomeruli with thin walled Bowman’s capsules were observed. The proximal convoluted tubules were lined with rounded nuclei, apical brush border and narrow lumen. The distal convoluted tubules were lined with cubical cells, rounded central nuclei and wide lumen (fig. 1b). H and E stained sections of atorvastatin-treated group (group III) showed some renal corpuscles with atrophied glomeruli and widening of Bowman’s space. Some cells of the renal tubules with pyknotic nuclei and vacuolated cytoplasm can be seen. Hemorrhage was observed in the interstitium (figs. 1c-e). H and E stained sections of atorvastatin and L-carnitine-treated group (group IV) showed that most of the glomeruli, the proximal and distal convoluted tubules appeared nearly normal. Clearly, conservation of renal tissue and decrease of tubular impairment were observed (fig. 1f).

Semiquantitative analysis showed the result of kidney injury in different studied groups. The atorvastatin treated group results were supported by the significant increase in renal injury score when compared with control (p<0.05; fig. 1g). Atorvastatin plus L-carnitine treatment led to a decrease in these morphological changes as indicated by a significant improve in renal injury score when compared with atorvastain group alone (p<0.05; fig.1g).

In control and L-carnitine sections, glycogen exist in the cytoplasm of tubular epithelial cells, the brush borders and the parietal layer of Bowman's capsule (figs. 2a, b), respectively. Rats treated with atorvastatin showed a decrease in PAS positive reaction in the renal tubular cells and the brush borders and an increase in the parietal layer (fig. 2c). Rats given atorvastatin and L-carnitine revealed an increase in PAS positive reactivity in renal tubules with nearly normal brush border. Nearly normal PAS positive basal laminae of parietal epithelium of Bowman's capsules were observed (fig. 2d). Morphometric results of PAS reaction are illustrated in (fig. 2e).

Renal iNOS expression was evaluated by using an immunohistochemical technique. The current results showed strong iNOS immunopositive reaction in atorvastatin-treated group that expressed in most of the renal tubular cells (fig. 3c). Mild iNOS immunopositivity was found in kidney section of atorvastatin and L-carnitine treated group (fig. 3d). In contrast, a weakly iNOS immunopositive reaction was shown in the kidney of control group (fig. 3a) and L-carnitine group (fig. 3b). These findings were confirmed by morphometric analysis of iNOS positive staining in the renal tissue (fig. 3e).

Increased positive PCNA immune reaction in renal tubular cells in atorvastatin-treated group was detected (fig. 4c). Further increased positive PCNA immunereactivity in renal tubular cells was observed in the atorvastatin and L-carnitine-treated group (fig. 4d). However, few PCNA immunostaining in renal tubular cells in the control group (fig. 4a) was seen. No changes were seen to PCNA immunoreactivity in L-carnitine group (fig. 4b) as compared with the control group (fig. 4a). These results were confirmed by the morphometric study of PCNA immunoreactivity staining in renal tissues (fig. 4e).

Electron microscopic examination of ultrathin sections of the renal cortex of the control group showed normal architecture of proximal tubular cells and renal corpuscles (fig. 5a-c). The proximal convoluted tubular cells appeared with euchromatic nuclei and numerous tightly packed apical microvilli (fig. 5a, b). The basal plasma membrane revealed infoldings with numerous elongated mitochondria (fig. 5a). The renal corpuscles showed the glomerular capillaries bounded by podocytes. The podocytes had long primary processes which branched to give the secondary foot processes. The later separated by the narrow slits membrane. The podocytes and the glomerular capillaries shared the smooth thinning basement membrane with trilaminar form (fig. 5c).

Electron microscopic examination of ultrathin sections of the renal cortex of the L-carnitine group (group II) revealed no tubular or glomerular damage (fig. 6a, b). The proximal convoluted tubules appeared with large, rounded or oval euchromatic nuclei. Many tubular elongated mitochondria between the basal infoldings of the cell membrane were observed. The cells rest on thin basal lamina (fig. 6a). The renal corpuscles revealed glomerular capillaries surrounded by podocytes. The arrangement of secondary processes and uniform thinning of the glomerular basement membrane was detected. The podocytes have large euchromatic nuclei (fig. 6b).

Electron microscopic examination of ultrathin sections of the renal cortex of Atorvastatin treated group (Group III) revealed tubular and glomerular injury (fig. 7a-d). Some proximal convoluted tubules showed electron dense shrunk nuclei. Apparently, increased number of lysosomes was observed. The cytoplasm showed numerous vacuoles of variable sizes and partial loss of microvilli. Clearly, loss of longitudinal arrangement of mitochondria and disorganized basal infoldings were detected (fig. 7a, b). The renal corpuscles revealed congested glomerular capillaries and vacuolated podocytes with irregular nuclei. Fusion of podocyte foot processes and regional thickening of the glomerular basement membrane were observed (fig. 7c, d).

Electron microscopic examination of ultrathin sections of the renal cortex of the atorvastatin- and L-carnitinetreated group (group IV) presented nearly normal structure of proximal tubular cells, podocytes and their processes (fig. 8a, b). Most of the proximal convoluted tubules revealed normal euchromatic nuclei and restoration of the numerous elongated mitochondria (fig. 8a). The renal corpuscle showed thinning of the glomerular basement membrane. The presence of slightly congested glomerular capillaries was seen (fig. 8b).

Principally, atorvastatin is a cholesterol lowering medication broadly recommended for primary and secondary prevention of cardiovascular disease. The kidneys have a remarkable regeneration capability and it is the most competent player amongst all tissues in the body. After kidney injury, some of the living renal tubular cells differentiated and then reenter the cell cycle to produce epithelial cells to renovate the structure and the function of the renal tubular cells[17].

Light microscopic examination of the kidneys of atorvastatin-treated rats revealed degenerative changes in the renal cortex. Many degenerative changes including atrophied glomeruli with widening of their Bowman’s spaces. This wide distance might be secondary to shrinkage of the glomerulus. This study illustrated that the proximal convoluted tubules showed vacuolation, pyknotic nuclei and dark acidophilic cytoplasm. In agreement with the present results, some investigators have been reported that the high dose of statin might lead to kidney tubular cell injury[18]. Similarly, other researchers suggested that tubular injury lead to tubular atrophy and ultimately end stage renal failure[19]. The cytoplasmic vacuolations could be attributed to increasing fluid uptake as a result of altered permeability of the cell membrane as reported in another study[20]. It has been reported that pyknosis is a histological feature of apoptosis[21].

In the current study, nitric oxide was measured as an indicator of reactive oxygen species and oxidative stress. Nitric oxide is formed from L-arginine by three isoforms of nitric oxide synthase (NOS). They are the neuronal (nNOS), the endothelial (eNOS) and the inducible NOS (iNOS). iNOS is upregulated in pathological disorders, such as obesity[22]. Nitric oxide is considered as a paracrine mediator with several aims that contributed in several physiological and pathological mechanisms. The mechanisms involved the inflammation, immune system, apoptosis and gene regulation[23,24]. In this study, iNOS was expressed in the cytoplasm of renal tubule epithelial cells. The more intense expression of iNOS was detected in the atorvastatin-treated group as compared with the control and L-carnitine groups. The present findings were in agreement with those of previous investigators who observed that the iNOS has been recognized as a crucial player in the pathogenesis of metabolic kidney diseases[25,26]. Similarly, the upregulation of iNOS expression was observed in the septic kidney tissues[27]. Moreover, in rodent models, various investigations stated an elevation of iNOS expression of obesity and diabetes[28]. Indeed, nitric oxide produced by iNOS in the tubular cells might induce an increase in oxidative damage and consequently lead to tubular impairment. In our study, the upregulation of iNOS expression was inhibited by treatment with L-carnitine. Significantly, investigators noticed inhibition of iNOS provided an improvement of tubular function by suppressing proteinuria and oxidative stress production[29,30]. L-carnitine produced its antioxidant properties through diverse mechanisms, including the scavenging of free radical activity directly or by inhibition of its production. Consequently, keeping the competence of the mitochondrial electron transport chain and exciting the activation of the antioxidant enzymes[31]. Moreover, L-carnitine attenuated doxorubicin induced generation of free radicals and improved the functional and structural integrity of the myocardium[32].

PCNA expression is a significant index of renal regeneration. Scientists showed that the expression of PCNA linked with the rates of cellular proliferation and DNA synthesis[33]. In this study, the number of PCNA positive cells increased in atorvastatin group as compared with the control and L-carnitine groups. These results were consistent with other studies, which reported that the increased PCNA expression in tubular epithelial cells and interstitial infiltrate specified up regulation of their proliferative rate[34].

The current data revealed that PCNA immunoreactive positive cells further increased in atorvastatin and L-carnitine group. This suggests that kidney cells of rats after L-carnitine treatment have stronger replicative activity. In agreement with the current results, the number of PCNA positive cells was elevated five-fold in silymarin plus mycotoxin-treated mice as compared to mycotoxin group[35]. The same was observed by other investigators, who stated that the administration of hematopoietic stem cells derived from human umbilical cord significantly increased the number of PCNA positive cells, proposing that umbilical cord CD34+ strongly stimulated tubular cell proliferation however inhibiting cell apoptosis[36].

Evaluating brush border preservation in the proximal tubules is a histochemical marker for monitoring the function of proximal tubule cells. In this study, the proximal tubule cells of atorvastatin group showed partial loss of their brush border, which was proved in ultrathin sections by marked interruption of apical microvilli. Examination of PAS stained sections of this group verified partial or complete loss of brush border in most of the proximal convoluted tubular cells. Moreover, an increase in the number of lysosomes in the same group as compared with control. Definitely, acute renal injury enhanced lysosomal proliferation and activated the autophagy pathway in the proximal tubular cells[37].

Several renal ultrastructural changes in the glomeruli were detected following atorvastatin such as thickened glomerular basement membrane and fusion of podocytes foot processes. Thickening of the basement membrane is an indicator of injury produced due to reactive oxygen species. The current results are consistent with previous investigators, who reported similar results in kidney diseases associated with proteinuria and after cisplatin administration[38]. The findings of this study have shown that co-administration of L-carnitine reduced the harmful effect of atorvastatin. This was detected by both light and electron microscopes. Most of the glomeruli were more or less normal and restoration of microvilli was also observed. These findings were supported by PAS stained sections that exhibited nearly normal PAS positive brush border in most of the cortical tubules. A decrease of iNOS reaction and an increase in PCNA immunoreactivity were detected. These findings were in agreement with the previous investigators who reported that L-carnitine has a protective effect as an antiinflammatory and antiapoptotic[39]. L-carnitine has physiological functions on fatty acids transport across the inner mitochondrial membrane and it has the properties to scavenge reactive oxygen species[40].

Despite all the deleterious of atorvastatin found in this study as well as in other investigations, some studies deliberated that atorvastatin and other statins are safe to use even at high doses. Other researchers suggested that statins exert a defensive possession in the structures affected by diabetes such as the kidney[41]. Atorvastatin induced a nephrotoxic effect at the dose studied in this study, which indicate that it is appropriate to adjust the dose. Significantly, the current results brought the evidence that the mechanism of the defensive effect of L-carnitine is linked to improve cell proliferation and inhibition of iNOS in the kidney. Future investigations should be carried out using the molecular mechanism.

Conflict of interest:

No conflict of interest between any of the author.

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