High cholesterol diet (HCD) plays an important role in the initiation and progression of hypercholesterolemia-induced atherogenesis . Numerous studies suggested complex interrelations between hypercholesterolemia and progression of renal damage, showing an association between hypercholesterolemia and the degree of glomerular injury [2,3].
Several previous studies were concerned with the effect of hypercholesterolemia on renal tissue for long periods with the end result of focal glomerulosclerosis and proteinuria that progress rapidly to renal failure . However, there was little information about the early effect of hypercholesterolemia on renal injury .
The oil derived from deep-sea shark livers contains high levels of squalene, diacylglyceryl ethers, and triacylglycerols and minor levels of free fatty acid, sterol, and sterol ester . Shark liver oil (SLO), as a health supplement, is suggested to be used in the treatment of infectious diseases  and in cancer therapy . SLO is considered the richest source of squalene, which is reported to be hypolipidemic  and an antioxidant . No available information has been obtained regarding the effect of SLO on hypercholesterolemia-induced renal damage.
Therefore, the aim of this study was to investigate the effect of a short-term HCD (for 40 days) on the structure of the renal cortex and the level of serum lipids in adult male albino rats and to evaluate the possible protective effect of dietary supplement with SLO in two different doses against the HCD-induced deleterious effects.
Materials and methods
A total number of 40 adult male albino rats (4–6 months old, 200–250 g) were used in this study. They were obtained from the animal house of Faculty of Medicine (Assiut University, Egypt). The animals were isolated in clean and properly ventilated cages under normal conditions with free access to food and water throughout the whole period of the experiment.
The animals were randomly divided into two main groups. Group 1 (control group) included 10 rats that were fed the normal rat chow provided from the animal house for 40 days. Group 2 (hypercholesterolemia group) included 30 rats and were further subdivided into three subgroups (10 rats each). Group 2a rats (nontreated hypercholesterolemia) were fed a HCD for 40 days. HCD consisted of normal rat chow +1 g brain/rat/day. Cow's brain was purchased from the butcher and was mixed thoroughly with equal volume of water to form an emulsion. The freshly prepared emulsion was administered to rats by an oral tube at a dose of 1 g brain/rat/day . Group 2b rats (10% SLO-treated hypercholesterolemia) were fed a normal rat chow supplemented with 10% SLO +1 g brain/rat/day by an oral tube for 40 days. Group 2c rats (20% SLO-treated hypercholesterolemia) were fed a normal rat chow supplemented with 20% SLO +1 g brain/rat/day by an oral tube for 40 days.
SLO was purchased from Kraftsatim ehf Company (Iceland) as 100% pure natural arctic SLO extracted from the liver of the Greenland Shark (Somniosus microcephalus). SLO was added at a percentage of 10 and 20% to rat diet in groups 2b and 2c, respectively. The selected doses were based on the reported safety of SLO given orally to rats at doses of 1000–2000 mg/kg/day, which are 100–200 times greater than recommended for human consumption .
At the end of the experiment, blood samples were collected from the animals of different groups for biochemical study. Then, animals were killed and tissue specimens were taken from the right kidney and processed for histological study of the renal cortex.
General histological examination
- (1) Tissue specimens were fixed in 10% neutral formalin and processed for Harris hematoxylin and eosin stain for general histological examination according to Drury and Wallington ;
- (2) Tissue specimens were fixed in 4% gluteraldehyde, postfixed in osmium tetroxide, processed, and embedded in epon. Semithin sections (0.5–1 μm) were stained with toluidine blue  and examined by a light microscope.
Expression of desmin (a muscle cell marker used as an indicator of podocyte injury) was detected in formalin-fixed paraffin-embedded sections. Sections (6 μm) were deparaffinized in xylene and rehydrated in alcohol. They were processed according to the manufacturer's instructions using the universal kit [Ultravision Detection System, antipolyvalent, HRP/DAB, (Thermo Fischer Scientific, Fremont, California, USA)]. Immunohistochemical staining was demonstrated using the labeled streptavidin–biotin immunoperoxidase technique.
Monoclonal mouse antibody to desmin (muscle cell marker) (Lab Vision Corp, NeoMarkers Inc/Lab Vision, Fremont, California, USA) was used at 1 : 100 dilution overnight at 4°C .
Sections were then incubated with biotinylated goat antipolyvalent secondary antibody for 10 min at room temperature. The reaction was then visualized using diaminobenzidine for 10 min, and counterstained using hematoxylin.
Negative control consisted of kidney sections processed routinely without the primary antibody. Sections from the appendix were used as positive control .
Ultrathin sections (500–800 A˚) of gluteraldehyde-fixed specimens were counterstained with uranyl acetate and lead citrate  and examined with JEOL (JEM-100 cx11, Egypt) the transmission electron microscope present at the Electron Microscope Unit of Assiut University, Egypt.
Blood samples were collected from the animals by a retro-orbital puncture (optic vein) and processed for the determination of the following:
- (1) Serum triglyceride level;
- (2) Serum total cholesterol level;
- (3) Serum high-density lipoprotein (HDL) cholesterol level.
Estimation of triglycerides in serum
Triglyceride (TG) concentrations were measured colorimetrically with commercially available kits (Triglycerides test, Eli Tech Diagnostics, France), according to Bucolo and David .
Estimation of total cholesterol in serum
Total cholesterol concentrations were measured colorimetrically with commercially available kits (cholesterol C test, Eli Tech Diagnostics, France), according to Allain et al. .
Estimation of high-density lipoprotein cholesterol in serum
HDL-cholesterol concentrations were measured colorimetrically with commercially available kits (HDL-cholesterol test, Eli Tech Diagnostics, France), according to Lopes-Virella et al. .
Estimation of LDL and VLDL cholesterol in serum
The concentration of LDL cholesterol was calculated according to the equation of Friedewald et al.  as follows:
The quotient TG/5 is used as a measure of very low-density lipoprotein (VLDL) cholesterol concentration. The TG:VLDL cholesterol ratio is constant at approximately 5 : 1 .
General histological results
In control animals (group 1), the renal cortex was characterized by the presence of renal corpuscles, proximal convoluted tubules (PCT), and distal convoluted tubules (DCT). Each renal corpuscle consisted of a glomerular capillary tuft surrounded by capsular space and Bowman's capsule. The PCT was lined by a high cuboidal epithelium with deeply stained acidophilic cytoplasm and rounded nuclei. The DCT was lined by cuboidal cells with pale acidophilic cytoplasm and rounded nuclei (Fig. 1). In semithin sections, the glomerular capillaries were defined by the prominent glomerular basement membrane. Podocytes invested the capillary loops and had pale nuclei and pale stained cytoplasm. The parietal layer of Bowman's capsule was lined by simple squamous epithelium. The mesangial cells were identifiable by their densely stained nuclei (Fig. 2). The cells lining the PCT had an apical brush border and well-defined basal striations. Their nuclei were central and pale with prominent nucleoli. The cells of DCT had no apical brush border and their nuclei appeared apical in position (Fig. 3).
The renal cortices of nontreated HCD-fed animals (group 2a) exhibited structural changes mainly involving the renal corpuscles with some affecting PCT and DCT. Many renal corpuscles showed attachment of the glomerular tufts to Bowman's capsule with a noticeable reduction of the urinary space. Both PCT and DCT showed numerous intracellular vacuoles (Fig. 4). In semithin sections, many podocytes had intensely stained nuclei. Mesangial cells appeared relatively unchanged (Fig. 5). Some nuclei of the tubular epithelial cells appeared intensely stained. Many PCTs showed partial loss of their apical brush membranes and basal striations (Fig. 6).
In the renal cortex of 10% SLO-treated HCD-fed animals (group 2b), most renal corpuscles showed an absence of adhesions between the glomerular capillary tufts and Bowman's capsule. Less vacuoles were observed in the cytoplasm of tubular epithelial cells in comparison with those of the renal cortex of group 2a animals (Fig. 7). In semithin sections, most podocytes and tubular epithelial cells exhibited pale stained nuclei (Fig. 8).
The renal cortices of 20% SLO-treated HCD-fed animals (group 2c) showed more preservation of the structure of most renal corpuscles and tubules (Fig. 9). Semithin sections of the renal cortices of this group showed a relatively normal appearance of most podocytes and the preservation of apical brush borders and basal striations in most PCTs (Fig. 10).
Regarding desmin staining, which is a marker of podocyte injury, it was virtually absent in all the glomeruli of the renal cortex of group 1 animals (Fig. 11).
In group 2a animals, de novo staining of desmin was observed in most glomeruli, particularly at the edges of the glomerular tufts (Fig. 12).
In the glomeruli of the renal cortex of groups 2b and 2c, desmin staining was decreased to appear relatively similar to normal control glomeruli (Figs 13 and 14).
Electron micrographs of group 1 animals revealed the normal ultrastructure of the renal cortex. The podocyte was stellate in shape with irregular nucleus and extensive cytoplasm. Its secondary processes surrounded the glomerular capillaries as regularly spaced small foot processes (Fig. 15). The lining cells of PCT exhibited rounded euchromatic nuclei and profuse long microvilli on their apical surfaces. The cytoplasm showed numerous elongated mitochondria and some apical vacuoles (Fig. 16). In the DCT, the lining cells had rounded nuclei, few short apical microvilli, and basal infoldings that surrounded the elongated mitochondria. The cytoplasm contained few small vacuoles (Fig. 17).
Examination of ultrathin sections of the renal cortex of group 2a animals revealed ultrastructural changes in the renal corpuscles and the renal tubules. In most renal corpuscles, the podocytes contained vacuoles in their cytoplasm and processes. Few podocytes showed cell body attenuation and pseudocyst formation with increased electron density of their nuclei and cytoplasm. Podocyte foot processes frequently appeared flattened and fused around the obviously thickened distorted glomerular basement membrane (Figs 18 and 19). Many of the tubular epithelial cells lining PCTs exhibited partial loss of apical microvilli. The mitochondria were disorganized and exhibited variable degrees of degeneration. Many mitochondria showed partial or complete loss of their cristae. Some mitochondria had increased electron density (Fig. 20). Many of the epithelial cells lining DCTs showed vacuolization of the cytoplasm, degenerative changes of the mitochondria, and marked reduction of their basal infoldings (Fig. 21).
In the renal cortex of group 2b animals, the podocytes exhibited few vacuoles in their cytoplasm and processes. Their foot processes showed regular alignment around the glomerular basement membrane (Fig. 22). The luminal surfaces of many PCTs showed uninterrupted layer of apical microvilli. Most mitochondria had intact cristae with increased electron density of their matrices (Fig. 23). In the DCTs, less cytoplasmic vacuolization and less mitochondrial degenerative changes were observed in their tubular epithelial cells in comparison with those of group 2a animals (Fig. 24).
In the renal cortex of group 2c animals, a reduction in the thickness of the glomerular basement membrane with regular arrangement of podocytes foot processes around it was observed in most renal corpuscles of this group compared with those of group 2a renal cortices (Fig. 25). In the PCT, there was a noticeable restoration of the integrity of apical microvilli of their cells. Some mitochondria had a relatively normal ultrastructure and electron density. Other mitochondria exhibited increased electron density (Fig. 26). Most of the tubular cells lining the DCTs showed relatively normal electron density of most mitochondria and restored basal infolding (Fig. 27).
The data presented in Table 1 showed the effect of adding SLO at two different doses to rat diets on serum lipids in HCD-fed rats.
In group 2a (HCD-fed rats), there were elevated levels of triglycerides, total cholesterol, LDL cholesterol, and VLDL cholesterol. The highest increase was in LDL cholesterol (57.38%), then triglycerides and VLDL cholesterol (56.19%), whereas total cholesterol increased approximately by 34.35%. However, HDL cholesterol declined approximately by 20.74% in this group.
Table 1 showed reduction of the elevated serum lipids in SLO-supplemented groups compared with HCD-fed group with more reduction in 20% SLO-supplemented group. Thus, in group 2b (10% SLO-supplemented group), the reduction was 43.69% in LDL cholesterol, 21.93% in total cholesterol, and 21.12% in triglycerides and VLDL cholesterol. In group 2c (20% SLO-supplemented group), the reduction was 50.34% in LDL cholesterol, 23.60% in total cholesterol, and 28.20% in triglycerides and VLDL cholesterol compared with HCD-fed group. The serum level of HDL cholesterol was increased approximately by 49.46% in 10% SLO-supplemented group and 72.47% in 20% SLO-supplemented group compared with the HCD-fed group.
The results of this study demonstrated the deleterious effects of HCD feeding (for 40 days) on rat renal cortical structure and serum lipid profile.
The structural renal damage primarily involved the renal corpuscles, which revealed prominent podocyte injury as evidenced by de novo desmin staining, foot process effacement, cell body attenuation, and pseudocyst formation.
The podocyte injury observed in this study was in accordance with the study of Joles et al.  and Attia et al. , who investigated the effect of hypercholesterolemia-inducing diet in rats. However, in hyperlipidemic ApoE null mice (an experimental model for spontaneous hyperlipidemia) podocytes showed normal electron microscopic structure . This controversy may be due to the different rodent models used in these studies.
Desmin, an intermediate filament protein, has been long suggested as a podocyte injury indicator, the expression of which is often upregulated in various glomerular diseases in which podocyte damage is involved . Podocyte epithelial dedifferentiation was accompanied by the induction of mesenchymal markers such as desmin . It was hypothesized that podocytes might undergo epithelial dedifferentiation and mesenchymal transition due to upregulation of transforming growth factor-β 1in the diseased kidney and that de novo desmin expression in podocyte could be a reliable relevant marker for podocyte epithelial-to-mesenchymal transition .
Foot process effacement observed in this study represented a reduction in the complexity of cell–cell connections, which ranged from partial retraction of the foot process to a total disappearance of the usual interdigitated pattern . Foot process effacement could be viewed as a part of podocyte epithelial-to-mesenchymal transition that would affect their complex morphological architecture and their highly specialized functions, leading to the onset of proteinuria .
Occurrence of cell body attenuation and pseudocyst formation of some podocytes observed in this study was indicative of the prominent podocyte injury that would progress to detachment of podocyte from the glomerular basement membrane (GBM)  and thus could explain the attachment of glomerular tufts to the Bowman's capsule. When an area of denuded capillary came into contact with parietal cells of Bowman's capsule, the latter were triggered to attach to the capillary basement membrane, which represented the beginning of tuft attachment . Similarly, glomerular tuft adhesion to the Bowman's capsule was observed in rabbits fed a hypercholesterolemia-inducing diet for 12 weeks .
Thickening and distortion of GBM observed in HCD-fed rats in this study could be due to distortion of the native GBM matrix structure when reactive oxygen species attacked the GBM . Thickened GBM has been reported to be more permeable to proteins than normal GBM due to reduction of type 1 proteoglycan, which functions as an anionic barrier for macromolecules .
The mesangial cells did not appear to be the target cell in hypercholesterolemia-induced rat glomerular injury as they revealed a relatively normal light and electron microscopic appearance in HCD-fed animals, in agreement with the study of Joles et al. . However, it seemed likely that longer durations of hypercholesterolemia might be associated with mesangial matrix expansion . In addition, Joles et al.  explained that in the absence of immune-mediated mesangial injury or glomerular hypertension, the mesangium did not seem to play a pivotal role in the development of proteinuria and progression to glomerulosclerosis.
With regard to the renal tubules, variable degrees of damage were observed in the renal cortex of HCD-fed rats. These included intracellular vacuoles, variable degrees of mitochondrial degeneration, partial loss of apical microvilli in PCT, and loss of basal infoldings in DCT. It was reported that renal tubular damage occurred primarily by lipemic oxidative injury and cellular dysfunction, which was termed lipotoxicity . Weinberg  reported that lipotoxic cellular dysfunction and injury could result from accumulation of nonesterified (free) fatty acids and their toxic metabolites. Russo et al.  suggested that PCT might be particularly vulnerable to lipotoxicity due to its role in reabsorption of free fatty acid-bearing albumin. This was in agreement with Bobulescu et al.  who studied the effect of renal lipid accumulation on rat proximal tubular cells. They suggested that primary renal tubular damage might also occur as a result of lowering of urinary pH and impaired ammonium urinary excretion with altered mitochondrial function in proximal tubular cells . Renal tubular damage was also suggested to be secondary to hypercholesterolemia-induced glomerular damage and proteinuria leading to tubular luminal obstruction by protein casts .
With regard to serum lipids in this study, the levels of triglycerides, total cholesterol, LDL cholesterol, and VLDL cholesterol were increased, whereas serum HDL-cholesterol level was decreased in HCD-fed group. These results agreed with Joles et al.  and Sudhahar et al.  who reported increased serum lipid abnormalities in HCD-fed rats.
Hypercholesterolemia was reported to be associated with increased formation of oxidized LDL  and increased glomerular and tubulointerstitial generation of reactive oxygen species . Moreover, elevated serum VLDL and LDL cholesterol in HCD-fed rats could directly induce renal injury as LDL receptors were expressed on podocytes in biopsies  and cultured podocytes were reported to bind and take up VLDL and LDL .
In this study, SLO supplementation in two different doses (10 and 20%) revealed a noticeable reduction of hypercholesterolemia-induced renal structural changes in most renal corpuscles and tubules by light and electron microscopic examination. The use of the low dose (10% SLO) showed substantially low renal damage, whereas more preservation of renal architecture was observed following the use of the high dose (20% SLO). These protective effects of SLO on renal cortical structure were associated with a marked correction of plasma levels of lipids. The normalizing effect of SLO on the altered serum lipids was more pronounced in 20% SLO than 10% SLO-supplemented rats. The reduction in rat serum triglycerides in SLO-supplemented animals might be due to inhibition of hepatic triacylglycerol synthesis, and stimulation of hepatic peroxisomal β-oxidation . The lowered serum cholesterol levels in SLO-supplemented rats might be attributed to the high content of squalene in SLO. Chan et al.  suggested a cholesterol-lowering effect of squalene in patients with hypercholesterolemia. Squalene was reported to inhibit cholesterol synthesis in the liver and to increase fecal excretion of cholesterol . Sawada et al.  reported that squalene was directly involved in lipid metabolism as a feedback inhibitor for regulating cholesterol metabolism. In contrast, Zhang et al.  reported that squalene and SLO were hypercholesterolemic in hamsters. This discrepancy may be due to the differences in the studied species, the levels of squalene/SLO supplement, and the duration of the experiment. In addition, SLO contained moderate amounts of omega-3 polyunsaturated fatty acids, which had hypolipidemic and antithrombotic properties .
As a medicinal supplement, SLO is reported to have a wide safety and tolerability in its oral use . Previous studies reported that 60–85% of squalene is absorbed when administered orally and distributed to various tissues with no reported signs of toxicity for an increased intake of squalene .
It is to be mentioned that the scope of this study did not include the histological structure of renal blood vessels to be focused on in a later study.
In conclusion, SLO administration effectively restored most of HCD-induced deleterious effects, suggesting that this medicinal supplement can play a therapeutic role against renal cortical damage and disturbed serum lipids associated with dietary hypercholesterolemia. The effect was better with 20% than 10% SLO administration. The exact mechanisms of the prophylactic effect of SLO supplement with HCD feeding remain to be elucidated.
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