Histological, ultrastructural and immunohistochemical studies on the ameliorative role of Cinnamon zeylanicum against high cholesterol diet-induced hypercholesterolemia in the kidney of adult male albino rats.

Cholesterol is an important type of lipid as it is involved in the structure of cell membrane, synthesis of steroid hormones, bile acid and vitamin D. Many diseases affect various mammalian organs, including the kidney, as a result of high cholesterol levels (hypercholesterolemia). Cinnamon zeylanicum (C. zeylanicum) proves its efficiency as it has anti-inflammatory and antioxidant prosperities. This study aimed to investigate the possible ameliorative role of C. zeylanicum on hypercholesterolemia-induced the renal toxicity in albino rats. Forty adult male albino rats were equally divided into four groups. The first group served as the control one. The second group was supplemented with C. zeylanicum powder (15% w/w) with the standard diet. The third group was fed high cholesterol diet (HCD) to induce acute hypercholesterolemia. The fourth group was fed HCD provided with C. zeylanicum powder (15% w/w). At the end of the experiment (8th weeks), kidneys were removed and prepared for histological, immunohistochemical and ultrastructure studies. Rats-fed HCD showed degenerated glomeruli and tubular cells with vacuolated or coagulated cytoplasm and pyknotic nuclei. Moreover, the renal cortex ultrastructural examination showed degenerated podocytes, parietal and mesangial cells, as well as the proximal and distal tubular cells appeared with rarified cytoplasm, degenerated mitochondria, large fat vacuoles and complete damaged microvilli. The same group showed a significant increase in the expression of desmin and inducible nitric oxide synthase. On the other hand, animals fed HCD provided with C. zeylanicum showed an obvious improvement in the observed histological, ultrastructural and immunohistochemical changes. The architecture of the renal cortex appeared mostly similar to the control one. This study concluded that C. zeylanicum has a promising role in treating the nephron-toxicity of HCD due to its natural constituent that is responsible for its pharmaceutical effects.

Introduction

Cholesterol is an important type of lipid (sterol) as it is involved in the structure of cell membrane, synthesis of steroid hormones, bile acid and vitamin D (Damodaran et al., 2007). Hyperlipidemia is characterized by increasing levels of cholesterol and triglycerides (Villegas Vílchez et al., 2022), very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol (Riyad et al., 2022), free fatty acids as well as reduced levels of high-density lipoprotein cholesterol (Ismail et al., 2022). Riyad et al. (2022) described hyperlipidemia as having decreased levels of high-density lipoprotein cholesterol and increased levels of free fatty acids, very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides (Ismail et al., 2022; Villegas Vlchez et al., 2022). Fast food or cholesterol-rich food contains saturated fat or trans-fat may increase the levels of cholesterol and cause hypercholesterolemia (Chiras, 2013). Furthermore, Lima Rocha et al. (2022) indicated that being fed hypercholesterolemic diet for 8 weeks caused lipid accumulation besides cytoplasmic degeneration and thin fibrosis on the central-lobular vein wall in the hepatocytes of rats as well as increased the malondialdehyde level and decreased superoxide dismutase activity.

Excess cholesterol caused several diseases including atherosclerosis (Hall, 2016), infertility (Mahdavi et al., 2021) and toxicity in different organs as the liver (Yu et al., 2021), thyroid gland (Rasekh et al., 2021) and kidney (Kaur et al., 2021). Zălar et al. (2022) reported that rats-fed a high-fat diet for 4 weeks showed an increase in oxidative stress, pro-inflammatory markers as well as induced signs of endothelial dysfunction in aorta sections.

A few decades ago, when compared to synthetic drugs, medicinal plants are widely used throughout the world, due to their potent pharmacological activities, not expensive and economic viability (Pracheta et al., 2011). C. zeylanicum is one of these plants which have medicinal and nutritional properties for both humans and animals due to its richness with many functional bioactive compounds.

C. zeylanicum belongs to the family Lauraceae (Vinitha and Ballal, 2008). Its bark contains a lot of phytochemicals like flavonoids, coumarins, alkaloids anthraquinone, steroids, tannins and terpenoids (Shihabudeen et al., 2011). Moreover, there are many essential oils that are found in C. zeylanicum included trans-cinnamaldehyde (E)-cinnamaldehyde, cinnamyl acetate, eugenol, L-borneol, caryophyllene oxide, β-caryophyllene, L-bornyl acetate, E-nerolidol, α-cubebene, α-terpineol, terpinolene, and α-thujene (Tung et al., 2010). Khan et al. (2018); Al-Qulaly et al. (2021) found that rats treated with 200 mg/kg of cinnamon extract did not show any negative side effects. Additionally, the same authors found that cinnamon extract at different doses of (50, 100, and 200 mg/kg) were effective in treating acute renal damage and improved kidney function. Cinnamon oil administration (50–200 mg/kg) ameliorate the kidney damage induced by acetaminophen in rats via improving the histological picture, reducing urea, uric acid and creatinine levels and increasing the activities of superoxide dismutase, and catalase (Alshahrani et al., 2021). Moreover, cinnamons extract reversal the pancreatic ultrastructure and insulin signaling pathway in diabetic rats after two months of treatment (Faruk, 2021). Adding cinnamon to the basal diet of rats (5%) improved liver function, kidney function and lipid profile in diabetic rats (Hussei et al., 2022).

C. zeylanicum has proved to exhibit useful biological activities including antipyretic (Zaino et al., 2014), antibacterial (Gupta-Wadhwa et al., 2016), antifungal (Goel et al., 2016), ameliorates both hepatic (Eidi et al., 2012) and renal toxicity (Safdar et al., 2016), as well as testicular toxicity (Okdah and Kandil, 2018), cardiac toxicity (Sedighi et al., 2018) and used in tumor treatment (Kubatka et al., 2020). Many studies have relied on the change in the biochemical parameters, or the histological architecture and a few ones carried on the ultrastructural changes of the kidney. Therefore, this study aimed to investigate the effect of C. zeylanicum on hypercholesterolemia-induced renal toxicity in albino rats through histological, ultrastructural and immunohistochemical studies.

Materials and methods

Cinnamomum zeylanicum

Dried C. zeylanicum bark was purchased from the local market of agricultural herbs and medicinal plants (Cairo, Egypt). The studied specimen was identified by matching with herbarium specimens deposited in Orman garden herbarium (Giza, Egypt). It was grinded using an electric mixer into a fine powder and then added to animal's diet 15% weight by weight (w|w) daily for eight weeks (Rahman et al., 2013).

Induction of hypercholesterolemia

Hypercholesterolemia was induced by feeding rats a high cholesterol diet composed of wheat (34g), casein (10g), mineral mixture (3.3g), vitamin mixture (1g), cellulose (4.7g), sucrose (15g), corn starch (15g), Cholesterol (2g) and Cocoa butter (15g) for 100g of diet. Materials used for preparing the HCD (cholesterol, sucrose, cellulose, etc.) were purchased as a pure powder, from El Gomhorya Co, Egypt.

Formulas for different diets used in these experiments

The different food compositions employed in these tests are listed in Table 1. Additionally, El-Gomhouria Co. Cairo, Egypt, supplied the raw components (cholesterol, sucrose, cellulose, etc.) utilized to prepare the HFD (Arisha et al., 2020b).

Table 1

The formula of different diets used in these experiments (g/100g of diet).

Contents	standard diet 100 g	HCD 100 g	Standard diet + cinnamon 100g	HCD + cinnamon 100g
Wheat	76	34	61	19
Casein	10	10	10	10
Mineral mixture	3.3	3.3	3.3	3.3
Vitamin mixture	1	1	1	1
Cinnamon	-	-	15	15
Cellulose	4.7	4.7	4.7	4.7
Sucrose	-	15	-	15
Corn starch	5	15	5	15
Cholesterol	-	2	-	2
Cocoa butter	-	15	-	15

Animals

Forty adult male albino rats (Rattus norvegicus), three months old, weighing from 120 to130 g were purchased from Egyptian Company Nile for pharmaceuticals & chemical industries (Amiriya, Cairo, Egypt). The rats were randomly separated and put in special plastic rodent cages under restrained temperature (23 ± 2 °C) with reverse natural dark/light cycle 12/12 h s for 10 days before starting the experiment. Animals were maintained on a standard rodent diet and water were available ad libitum. The protocol of this study was approved by the Ethics Committee of Faculty of Science, Menoufia University (Approved No. MUF/F/HI/3/21), Egypt according to the National Institutes of Health guide for the care and use of laboratory animals (NIH publication No. 8023, received 1978).

Animal groups

The experimental animals were equally divided into four groups (10 rats each): the first group served as a control group and received a standard diet only, for 8 weeks. The second group was supplemented with a standard diet containing C. zeylanicum powder (15% w/w) for eight weeks. The third group was fed HCD (for 8 weeks) to induce acute hypercholesterolemia. The fourth group was fed HCD provided with C. zeylanicum powder (15% w/w) for eight weeks. At the end of the 8th weeks, rats were fasted overnight before dissecting. Kidneys were removed and prepared for histological, immunohistochemical and ultrastructure studies.

Ultrastructural study

Small pieces (not more than 1–2 mm3) of the kidney from both the control and experimental groups were handled as described by Reynolds (1963) for transmission electron microscopic study. The ultrathin sections were examined and photographed for required magnification with a 1400 plus-JSM transmission electron microscope (Tokyo, Japan) at Electron Microscope Unit, Faculty of Science, Alexandria University (Alexandria, Egypt). Chemicals used in the histological and ultrastructural analysis were purchased from Sigma-Aldrich Corp (St Louis, MOUSA).

Histological study

Kidneys were cut into small pieces and instantly fixed in 10% neutral formalin for 24 h. The fixed tissue was dehydrated in ascending series of ethyl alcohol, cleared in two changes of xylene and embedded in molten paraffin wax (mp.54-56°c). Sections of 4 microns thickness were cut using a rotary microtome and mounted on clean slides. These slides were stained with Ehrlich's hematoxylin and counter-stained with eosin. Slides were examined and photographed by Olympus microscope (BX41 TF, Olympus Corporation, Shinjuku City, Tokyo, Japan).

Immunohistochemical investigations

Kidney sections of 4 microns were processed to determine the immunohistochemical expression of desmin and induced nitric oxides according to Hsu et al. (1981). The intensity of the reaction of both desmin and induced nitric oxides expressions in kidney sections were quantified by using the image j program.

Statistical analysis

The obtained data were expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed by statistical package for social science (SPSS) program, IBM SPSS Statistics for Windows, version 20 (IBM Corp, Armonk, N.Y, USA).

One-way analysis of variance (ANOVA) test was done for comparing data. The values were considered significant at P ≤ 0.05.

Results

Histological observations

Examination of the semi-thin sections of kidney obtained from the control rats and animals that received C. zeylanicum with their standard diet showed normal architecture of the renal cortex. The nephron is the structural and functional unit of the kidney, composed of Malpighian corpuscles, convoluted renal tubules, and collecting ones. Most parts of the nephron are found in the cortex (Malpighian corpuscle, distal and proximal convoluted tubules) except its lope of henle's. The Malpighian corpuscle consists of a cup-shaped structure called Bowman's capsule which is lined with a single layer of simple squamous epithelial surrounded by a tuft of capillaries called glomeruli. The proximal convoluted tubule is lined with simple cuboidal epithelia with rounded, central nuclei and provided with a long brush border. Moreover, the distal convoluted tubules have widened lumen than that of the proximal ones and are lined with cuboidal epithelial cells containing rounded apical nuclei and have a few brush borders (Figure 1a and b).

Figure 1

Photomicrographs obtained from kidney of (a): the control rats showing normal Bouman's capsule (arrow) glomeruli (G), proximal and distal convoluted tubule (PCT and DCT, respectively). (b): rat-fed normal diet with C. zeylanicum showing normal structure of the renal cortex. c-e): rats-fed HCD (c): showing different size of Malpighian corpuscles (short arrows) with partially separation of its lining epithelial cells (long arrow), widened Bowman's space (arrowheads), shrunk glomeruli (G) and dilated renal tubules (∗) hyaline cast (blue arrows). (d): showing hemorrhage (H), degenerated tubules (∗) with vacuolated cells (arrowheads) and pyknotic nuclei (arrows). (e): showing atrophied glomeruli (G), dilated Bowman's space (blue arrows), leucocytic infiltration (LI), hemorrhage (H), hyaline cast (arrowheads), pyknotic nuclei (arrows) and karyolitic nuclei (O). (f): rats-fed HCD and C. zeylanicum showing approximately normal glomeruli (G), Bowman's spaces (arrows), proximal and distal convoluted tubules (PCT and DCT, respectively).

Animals fed HCD showed massive tissue abnormalities in the renal cortex. The Malpighian corpuscles have different sizes, and its lining epithelial cell is partially separated from its basement membrane. Some glomeruli were atrophied, shrinkage or fragmented (lobular segmentation) with widened Bouman's space and other glomeruli showed decreased cellularity. The epithelial cells of the renal tubules appeared degenerated with vacuolated or coagulated cytoplasm, the nuclei of these cells appeared either pyknotic, karyorrhexis or karyolitic. The lumen of the degenerated tubules appeared dilated and filled with the hyaline cast. Moreover, hemorrhage and leucocytic infiltration were also noticed (Figure 1c-e).

When rats were fed HCD and C. zeylanicum, an obvious improvement in the kidney architecture was observed. Healthy glomeruli with nearly a normal size and structure appeared almost like the control ones. Moreover, the renal tubules lined with cuboidal cells containing a normal nucleus, and homogenous cytoplasm expects a few degenerated ones (Figure 1f).

Ultrastructure observations

The transmission electron microscopic examination of the renal cortex of the control rats revealed the normal structure of both Malpighian corpuscles and tubular epithelia. The glomerular capillary is lined with flat endothelial cells resting on the basement membrane and has an elongated nucleus. The Bowman's capsule has two faces, including the urinary space in between. The parietal face (the outer one) is lined with a single layer of simple squamous epithelia and the visceral one (the inner one) is composed of modified, specialized epithelial cells called podocytes. The Podocyte is a triangular cell with a normal a large euchromatic nucleus and long cytoplasmic processes extended from its body, giving them a star shape appearance, called primary processes from which the secondary processes called pedicles fused with foot processes on the glomerular basement leaving narrow slits in between them called filtration slits. The filtration barrier appeared with its three structures; fenestrated endothelium, basement membrane (formed by the fusion of glomerular capillary membrane and basal lamina of the podocyte) and the slit diaphragm in between the podocyte's foot processes (Figure 2a). Glomerular capillaries are connected and supported by irregularly shaped cells called mesangial cells that have small irregular heterochromatic nuclei and produce the mesangial matrix which provides structural support for the mesangial (Figure 2b).

Figure 2

Electron micrographs obtained from renal cortex of the control animals showing (a): part of renal corpuscles with glomerular capillary (GC) endothelial cells (E), podocyte (P) with normal nucleus (N) and filtrating barrier with its three layers structure; fenestrated endothelial (arrowhead), basement membrane (red arrow) and foot process (FP) of podocyte including filtrating slit (blue arrow) in between. (b): normal mesangial cells (MC) with indented nucleus (N) and normal chromatin distribution. (c): part of normal proximal convoluted tubules lined with normal cuboidal cells with a thin basement membrane (white arrow), normal nucleus (N), some lysosomes (L), mitochondria (M) and continuous, regular microvilli (Mv). (d): distal tubular cell with folded basement membrane (thin arrow), nucleus (N), mitochondria (M) and a few micro projections (thick arrow).

The proximal tubular cells appeared to rest on a thin basement membrane and have homogenous cytoplasm containing central, oval and euchromatic nucleus and an ordinary number of mitochondria and lysosomes. The apical surface is characterized by a prominent, continuous, regular and intact brush border (Figure 2c). The distal convoluted tubular cells have a basement membrane with basolateral infoldings associated with the longitudinal mitochondria. The nuclei of these cells are rounded, apical with normal chromatin distributions. The apical portion of the cell has a little short micro projection (Figure 2d).

On the other hand, examination of the ultrathin sections of the renal cortex obtained from rats-fed HCD showed massive cellular changes. The endothelial cell lined the glomerular capillaries appeared partially separated from its underlying basement membrane and its irregular nucleus showed degenerative features and deep invagination in its nuclear envelope. Moreover, the podocyte appeared degenerated with severe indented or shrunk nuclei contained condensed chromatin materials. The degenerated mesangial cells appeared with clumped chromatin and rugged (irregular) nuclear membrane (Figure 3a-c). The filtration barrier was also severely affected; its fenestrated endothelial layer disappeared, the basement membrane became thick, and the foot processes of podocytes were fused which led to the disappearance of the filtrating slits. Secondary lysosomes and dilated smooth endoplasmic reticulum were also noticed (Figure 3c). Moreover, the proximal tubular epithelia showed many degenerated features including phago-nucleus or shrunken one with clumped chromatin materials. These cells have a thick or irregular basement membrane rarified cytoplasm, degenerated mitochondria of different sizes, a large number of lysosomes than the control, large fat vacuoles, dilated endoplasmic reticulum, and complete damaged microvilli (Figure 3d-f).

Figure 3

Electron micrographs obtained from renal cortex of animals fed HCD showing (a): capillary lumen (CL) with degenerated endothelial cell (E) partially separated from glomerular wall (arrowhead) with irregular nucleus (N) and deep indented nuclear membrane (red arrow), damage podocyte (P) with invaginated, fragment nucleus (black arrow). (b): degenerated mesangial cell (MC) with shrunk nucleus (N) contained clumped heterochromatin (arrow), podocyte (P) with pyknotic nucleus (N). (c): enlarged portion from the previous figure showing abnormal filtration barrier, with disappeared fenestrated endothelial (thin arrow), thick basement membrane (thick arrow), fused foot process (blue arrow), filtrating slits (arrowheads) disappeared, degenerated podocyte (P) with lobulated nucleus (N), secondary lysosomes (red arrow) and dilated endoplasmic reticulum (∗). (d-f): showing degenerated proximal tubular cells with thick basement membrane (blue arrow), pyknotic (N) nucleus, phago-nucleus (arrow), rarified cytoplasm (∗), degenerated mitochondria (M), vesicular endoplasmic reticulum (ER), lipid vacuoles (Li), and broken microvilli (Mv).

The epithelial cells of the distal tubule appeared more affected, contained many lipid vacuoles with degenerated basolateral infoldings not associated with pleomorphic mitochondria appeared a few in number. The nuclei of these cells appeared pyknotic with condensed chromatin or severe intended with abnormal chromatin distribution. Moreover, the luminal micro projections were destroyed (Figure 4a-d).

Figure 4

(a–d): Electron micrographs obtained from renal cortex of animals fed HCD showing part of distal convoluted tubule with degenerated tubular cells contain shrunk, intended nuclei (N) or pyknotic one (arrow) lipid vacuoles (Li), lysosomes (L), degenerated mitochondria (M), rarified or vacuolated cytoplasm (∗), and damaged micro projection (red arrows).

Examination of ultra-sections of the renal cortex obtained from animals received HCD and C. zeylanicum revealed obvious improvements in the renal cortex architecture. The Bouman's capsule, filtrating barrier, glomeruli, podocyte and mesangial cells appeared to some extent normal. The tubular cells were approximately similar to the control ones and appeared with normal nuclei, longitudinal mitochondria and a few numbers of lysosomes, and a continuous brush border. A few cells had vacuolated cytoplasm still appeared (Figure 5a-d).

Figure 5

Electron micrographs obtained from renal cortex of animals fed HCD and C. zeylanicum showing (a): part of Bouman's capsule with normal glomerular lumen (GL) lined with endothelial cell (E), podocyte (P) and mesangial cell (MC) both with normal nuclei (N). (b): enlarged portion of the previous figure showing podocyte (P) with normal nucleus (N), homogenous cytoplasm (∗), normal basement membrane (arrowhead) and normal mesangial cell (MC). (c): part of proximal convoluted tubules lined with normal cuboidal cell appeared with normal basement membrane (arrowhead), mitochondria (M), lysosomes (L), normal nuclei (N) and continuous microvilli (Mv). (d): showing distal convoluted tubules lined with normal cuboidal cell with almost normal mitochondria (M), lysosomes (L), nucleus (N) and a few micro projections (red arrow).

Immunohistochemical results

Data in table (2) and Figures 6 and 7 showed changes in the immunohistochemical expressions of desmin and induced nitric oxides (iNOS) in different experimental groups. Immunohistochemical results of the control and C. zeylanicum groups showed desmin expression in a few glomerular cells as a brown color (Fig. 6a, b). While iNOS expression was appeared as brown color in a few tubular cells (Fig. 7a, b). There was a non-significant difference in the percentage area of desmin and iNOS expressions between C. zeylanicum and the control groups. Rats-fed HCD showed an increase in desmin expression that appeared in a large number of glomerular cells (Figure 6c). and iNOS expression in a large number of tubular cells (Figure 7c). The same group recorded a significant increase in the percentage area of desmin and iNOS expressions when compared with the control group. On the other hand, animals fed HCD, and C. zeylanicum showed normal expression of desmin and iNOS in glomerular and tubular cells respectively which recorded a significant decrease in the percentage area of their expression comparing with the HCD group (Figures 6d, 7d, respectively).

Table 2

Immunohistochemical expression of desmin and iNOS in different experimental groups.

	Desmin	iNOS
Control	0.084 ± 0.001	0.127 ± 0.009
C. zeylanicum	0.156 ± 0.005	0.120 ± .004
HCD	4.796 ± 0.45∗	52.40 ± 3.25∗
HCD + C. zeylanicum	1.090 ± 0.23∗∗	28.60 ± 5.69∗∗

Data were expressed as mean ± standard deviation (n = 5). HCD: high cholesterol diet ∗significant comparing with the control at P ≤ 0.05.

∗∗significant comparing with the HCD group P ≤ 0.05.

Figure 6

Photomicrographs obtained from renal cortex of (a): the control rats showing expression of desmin protein in a few glomerular cells (arrow). (b): animals fed on C. zeylanicum showing normal expression of desmin protein (arrows). (c): animals fed HCD showing positive expression of desmin protein in a large number of glomerular cells (arrows). (d): animals fed HCD and C. zeylanicum showing positive expression of desmin protein in a few glomerular cells (arrow).

Figure 7

Photomicrographs obtained from renal cortex of (a): the control rats showing expression of iNOS protein in a few tubular cells (arrows). (b): animals fed on C. zeylanicum showing normal expression of iNOS protein in the tubular cells (arrows). (c): animals fed HCD showing positive expression of iNOS protein in a large number of tubular cells (arrows). (d): animals fed HCD and C. zeylanicum showing positive expression of iNOS protein in a few tubular cells (arrows).

Discussion

In the current study, the histopathological alterations in the kidney of rats-fed HCD showed degenerated Malpighian corpuscles, glomeruli and the degenerated tubules had widened lumen filled with hyaline cast and their cells had vacuolated or coagulated cytoplasm and pyknotic nuclei. Moreover, hemorrhage and leucocytic infiltration were observed. The ultrastructural results in the present work showed massive cytological changes in most glomeruli and renal tubules of animals fed HCD. Atrophied or fragmented glomeruli, degenerated podocytes, parietal and mesangial cells have appeared. Degenerated proximal and distal tubular cells appeared with the thick basement membrane, rarified cytoplasm, degenerated mitochondria, many lysosomes, large fat vacuoles and complete damaged microvilli. These alterations may be due to increasing the production of reactive oxygen species associated with HCD which results in stimulation of the chronic inflammatory responses.

Similarly, Savini et al. (2016) reported that obesity, hypercholesterolemia, usually associated with both metabolic and oxidative insult leading to enzymatic activation. The body fights these insults by increasing its antioxidant activities. The proposed mechanism of the renal pathological alterations induced by HCD included the rise in oxidative stress that in turn generates reactive oxygen species which destroyed cells, increased lipid peroxidation, and decreased the oxidant markers, causing abnormal lipid metabolism, lipotoxicity, inhibition of activated protein kinase, and endoplasmic reticulum stress (Szeto et al., 2016; Abdel-Zaher et al., 2020).

In parallel with our results, Koubaa-Ghorbel et al. (2020) found that mice fed HCD showed extensive foci of hemorrhage and some necrotic renal tubular cells associated with an impairment in kidney function which may be referred to as a defect in the cell membranes permeability and integrity. In agreement with the siting results, Al Saad et al. (2020) found that hypercholesterolemia caused glomerular basal membrane thickening, dilated glomerular capillaries lumens and mononuclear cells aggregation.

The leucocytic infiltration that appeared in this study may be attributed to an increase in the inflammatory cytokines in inflamed tissue. These results were confirmed by Al-Mayyahi et al. (2020) who attributed the appearance of inflammatory cells in the kidney of rats-fed HCD to the accumulation of lipid. The aggregation of leucocytic infiltration in renal tissue of mice fed HCD may be due to the elevation in the proinflammatory gene expression and high levels of the proinflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin -1β and nuclear factor kappa-light-chain-enhancer of activated B cells (protein complex that controls transcription of DNA, cytokine production and cell survival) (Al Saad et al., 2020). Low levels of interleukin-10 indicated that HCD-induced inflammatory response. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells increased by 92.2% in the liver of rats-fed HCD (Tuzcu et al., 2017).

The observed lipid accumulation in the present study may be attributed to disturbance in the hormones associated with lipid metabolism. Similarly, Sahin et al. (2013) found that lipid accumulation may be due to a decreased expression of insulin receptor substrate 1 which plays a key role in regulating carbohydrate and lipid metabolism in diabetic rats-fed HCD.

In the present study, the ultrastructural mitochondrial alterations may be due to the increase in oxidative stress that results in mitochondrial dysfunctions. The mitochondrial alterations in the hyperlipidemic testicular tissues resulted in a mitochondrial dysfunction which finally raised oxidative stress (Henchcliffe and Beal, 2008). Nonalcoholic human fatty disease impaired oxidative phosphorylation and caused mitochondrial abnormalities (Ahishali et al., 2010). Moreover, excess lipids reduced mitochondrial size, damaged cristae and decreased mitochondrial matrix density in all renal cell types (Szeto et al., 2016). Stanchev et al. (2018) suggested that the structural alterations in the renal cells are incidental to a diminished number or unfunctional mitochondria.

In the current work, damaged microvilli in the proximal convoluted tubules may be attributed to the loss of cellular integrity of neighboring cells. Basile et al. (2012) found similar results and stated that the loss of the renal brush border is due to the breakdown of cytoskeletal elements.

Moreover, the ultrastructural alterations in the podocyte in the present results may be due to the severe inflammatory effect of HCD. As well as it has been reported that hypercholesterolemia triggers renal injury primarily via podocyte rather than via mesangial cell damage. Such podocyte injury is accompanied by tubulointerstitial cell activation and injury (Joles et al., 2000).

Concerning the immunohistochemical results, HCD increased the expressions of desmin and iNOS in renal cortex tissue. The rising in the expressions of desmin and iNOS protein after HCD confirmed the renal injury reported in the histological and ultrastructure results and may be due to alterations in the endothelial lining. In agreement with our results, desmin expression up-regulated in the kidney after different treatments as cisplatin (Karimi and Absalan, 2017) or diclofenac (Youssef and Salah, 2020) in rats. The authors added that the upregulation of desmin indicated podocyte injury. Moreover, the histological changes in the endothelial lining resulted in a decreased expression of endothelial nitric oxide synthase (eNOS) which generates enzymes that maintain the normal renal structure. While increasing the expression of iNOS resulted in increasing the production of enzymes responsible for renal damage (Bhatia et al., 2003; Zhou et al., 2000). It has been reported that upregulation of iNOS protein expression in rats-fed HCD occurred because of a reduction in the expression of eNOS (Abdel-Zaher et al., 2017; Luo et al., 2009; Rao and Sundaram, 2017). The upregulation of mRNA expression in the iNOS gene might be due to enhancing the production of superoxide, which then reacted with nitric oxide to form peroxynitrite and thereby decreasing nitric oxide levels as well as increased oxidized low-density lipoprotein which has the capacity of regulating iNOS activity via inhibiting Akt-mediated eNOS serine 1177 phosphorylation (Ricardo et al., 1997).

Microscopic examination of kidney cortex of rats-fed standard diet and C. zeylanicum did not show any changes in the histological, ultrastructure and immunohistochemical investigations as previously reported (Morgan et al., 2014; Hamouda et al., 2019; Hussain et al., 2019; Elshopakey and Elazab, 2021). In the current study animals fed HCD and C. zeylanicum revealed obvious improvements in the different parts of the renal cortex which appeared mostly normal in both histological and ultrastructural investigations. The positive effect of C. zeylanicum on the renal cortex architecture may be due to its lipolytic activity and its antioxidant power. The potential antioxidant activity of C. zeylanicum may be due to its flavonoids and polyphenols compound that acts as reactive oxygen and nitrogen species scavengers, redox-active transition metal chelators, and enzyme modulators (Elshopakey and Elazab, 2021). Pretreatment with C. zeylanicum improved necrosis, membrane thickening, hemorrhage in kidney structure induced by acetaminophen in mice because it has the ability to increase the level of the total antioxidant capacity and decrease the total oxidative stress level (Hussain et al., 2019). Concerning the lipid profile parameters, Arisha et al. (2020a) supported this study by their previous biochemical results when they studied ameliorative effect of C. zeylanicum against hepatotoxicity induced by the same diet and found that HCD caused severe changes in rats' lipid profile including the levels of cholesterol, high-density lipoprotein low-density lipoprotein cholesterol, and triglycerides and confirmed that adding cinnamon to HFD significantly alleviated dyslipidemia due to the presence of biological elements such as cinnamate.

Moreover, C. zeylanicum aqueous extract ameliorated renal pathological features after bisphenol A and decreased glomerular congestion and hypercellularity associated with more or less normal renal tubules (Morgan et al., 2014). The author returns these improvements to phenolic and flavonoid contents that have an antioxidant effects. Moreover, the C. zeylanicum extract showed prominent improvement in the kidney after sodium valproate on both histological and cytological levels; the glomeruli were nearly normal, and the tubular epithelial cells appeared continuous in healthy tubules (Hamouda et al., 2019).

In the current results, the leucocytic infiltration was disappeared in the rats-fed HCD and C. zeylanicum which may be due to the anti-inflammatory effect of C. zeylanicum. The improving effect of C. zeylanicum extract may be due to its ability to reduce the expression of the proinflammatory cytokines nuclear factor kappa-light-chain-enhancer of activated B cells (Tuzcu et al., 2017). The anti-inflammatory effect of C. zeylanicum may be due to its active components like cinnamic aldehyde, cinnamyl aldehyde, tannins that inhibit tumor necrosis factor-α, interleukin-2 (Elshopakey and Elazab et al., 2021).

The improvement in the shape of podocytes and mesangial cells in rats-fed HCD and C. zeylanicum in our study was previously observed by Kreydiyyeh et al. (2000) who reported that C. zeylanicum can permeate the membrane and inhibit the sodium-potassium adenosine triphosphatase provides the driving force for many transport processes.

The progress in mitochondrial structure noticed in this work may be attributed to the active constituent of C. zeylanicum that had the ability to regulate the mitochondrial permeability. Similarly, Panicker et al. (2009) reported that C. zeylanicum polyphenols decreased oxygen-glucose deprivation that in turn minimized cell swelling as well as lowering the inner mitochondrial membrane potential by inhibiting the mitochondrial permeability transition. C. zeylanicum extracts possess strong lipolytic properties because it reduced triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels that in turn prevents hypercholesterolemia and hypertriglyceridemia (Mohammed and Abdel Fattah, 2018). Moreover, C. zeylanicum extract showed negligible fatty infiltration and granular degeneration in the liver of hyperlipidemic rats (Abdelgadir et al., 2020). The lipolytic role of C. zeylanicum in reducing hypercholesterolemia is attributed to its ability to decrease the expression of proteins, transcriptional fa their target genes like SREBP-1c (responsible for the induction of lipogenesis), LXRs (important regulators of cholesterol, fatty acid, and glucose homeostasis), ACLY (take part in the fatty acid biosynthesis), hepatic 5- hydroxy-3-methylglutaryl-coenzyme A reductase and peroxisome proliferator-activated receptors mRNA (Tuzcu et al., 2017; Lopes et al., 2015; Sheng et al., 2008). Another mechanism of the hypolipidemic effect of C. zeylanicum includes its hyperinsulinemia activity and its ability to decrease serum leptin levels due to its high content of polyphenols which inhibit the intestinal absorption of cholesterol with subsequent hypocholesterolemia activity (Shalaby and Saifan, 2014). C. zeylanicum contains quercetin (flavonoid) which proved to be effective in reducing hyperlipidemia (Mariee et al., 2012). Quercetin reduces de novo synthesis of fatty acids and consequently cholesterol biosynthesis and lipoproteins formation (Gnoni et al., 2009) Moreover, quercetin has a scavenger activity against oxygen-free radicals and is a good metal chelator that prevents oxidative injury and cell death (Ahmadi and Shahri, 2019).

The improvement in the immunohistochemical picture of both desmin and iNO expression in rats-fed HCD and C. zeylanicum may be due to the improvement in the histological picture of the kidney. Lee et al. (2002) revealed that cinnamaldehyde has a suppression effect on inducible nitric oxide synthase expression. As well as Uslu et al. (2018) found that C. zeylanicum administration down-regulates iNOS in the thoracic aorta.

Conclusions

This study concluded that the addition of C. zeylanicum to our diet has a promising effect in reducing renal toxicity which may result from feeding HCD due to its richness with pharmaceutical constituents that had many pharmacological activities, such as anti-inflammatory, antioxidant and lipolytic effects. Further studies should be carried out to confirm our results and to identify the compounds in C. zeylanicum responsible for its improving effect.

Declarations

Author contribution statement

Samah M. Arisha; Eman Kandil: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Mona E. Saif: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.