Hypercholesterolemia has been recognized as a risk factor for atherosclerosis, and is now emerging as a contributing factor for the progression of renal disease 1. A high-cholesterol diet (HCD) and inadequate physical activity that characterize our modern lifestyle contribute to the development of hypercholesterolemia 2.
Evidence has been growing on the possible contribution of dyslipidemia in the development and progression of chronic kidney disease 3,4. A significant association between dyslipidemia and the deterioration of renal function in patients with pre-existing renal disease has been reported in previous research 5.
Many drugs that lower circulating cholesterol levels are available, but they are frequently associated with severe side effects 6. The consumption of functional foods or dietary supplements for lowering serum cholesterol levels has gained vast acceptance from the general public 7.
Many clinical trials have proven that various (poly)phenol-rich foods have protective effects against chronic diseases, including cardiovascular disease, neurodegeneration, and cancer. Conclusions regarding their preventive potential could not be drawn until now because of several limitations in existing studies 8.
Raisins are rich in phenolic compounds, flavonoids, and phenolic acids. Flavonoids are not only potent antioxidants but also have a multitude of functional capabilities, which may have an effect on health. Raisins are also an excellent source of polyphenols, which are powerful antioxidants that protect cell constituents against oxidative damage. They chelate metals, modulate enzymatic activity, inhibit cellular proliferation, and alter signal transduction pathways 9.
Few, if any, studies have dealt with the effect of raisins on hypercholesterolemia of the kidney. Therefore, this study was conducted to investigate the possible protective effect of raisins on the kidneys of adult Wistar rats fed an HCD, through biochemical and histopathological examination.
Materials and methods
Animals and diets
This experimental study was approved by the Biomedical Research Ethics Committee, Faculty of Medicine, King Abdulaziz University (Jeddah, Saudi Arabia). It was performed on 30 male albino Wistar rats, weighing 250–350 g, purchased from the animal house of King Fahd Medical Research Center. The animals were housed in stainless-steel cages and maintained on a 12-h light–dark cycle at a room temperature of 27±1°C under hygienic conditions. Rats were randomly divided into four groups: group I (n=5) served as the control group and received only saline through a nasogastric tube; group II (n=5) received 0.5 g of raisin homogenate through a nasogastric tube for 13 weeks, according to the method of Spiller et al. 10 and Bruce et al. 11 after adjusting the dose these authors used to the animal weight adopted by Laurence 12; group III (n=10) was fed an HCD, which was rat chow supplemented with 4% cholesterol and 1% cholic acid, as per Thiruchenduran et al. 13, for 13 weeks; and group IV (n=10), rats were fed an HCD along with raisin homogenate at the same dose.
Raisins, imported from Yemen, were obtained from nut stores in Jeddah. They were verified by a senior botanist at the Biology Department at the Faculty of Science, King Abdulaziz University (Jeddah, Saudi Arabia). The raisins were transformed into a homogenate using a sterilized blender with a small amount of water, packed into small blocks of 1 g each, and stored in the refrigerator until ready for use.
Metabolic and biochemical assessment
Food and water consumption and body weight were measured at the start, during, and end of the experiment. Weight gain and food efficacy were calculated. Blood samples were collected at the start, during, and end of the experiment for assessment of blood glucose and insulin levels, lipid profile, and kidney function.
At the end of the experiment, the animals were euthanized and the kidneys were dissected out, weighed, and processed for obtaining paraffin blocks. Paraffin sections of 5 μm thickness were stained with H&E, periodic acid-Schiff (PAS), and Masson trichrome, according to the method of Bancroft and Gamble 14. Verhoeff-Van Gieson stain was used to show the elastic fibers (stained black) in the wall of the renal blood vessels, as well as the surrounding collagen fibers (stained red) 15.
Small pieces of the renal cortex (1 mm) were fixed in 4% glutaraldehyde, postfixed in osmium tetroxide, processed, and embedded in Epon. Semithin sections (0.5–1 mm) were stained with toluidine blue and examined by means of a light microscope. Ultrathin sections (500–800 Å) of the glutaraldehyde-fixed specimens were counterstained with uranyl acetate and lead citrate according to Reynolds 16 and examined with a transmission electron microscope (JEM-100 Cx11; Jeol, Assuit, Egypt) belonging to the Transmission Electron Microscopy Unit of Assiut University (Egypt).
An Olympus Microscope BX-51 (Olympus, Los Anglos) with a digital camera connected to a computer was used for photographing. The glomerular cross-sectional surface area and the diameter of the glomerular capillaries were measured in 30 glomeruli from each kidney, according to the method of Langheinrich et al. 17. PAS staining was performed to evaluate the presence of mesangial expansion, and Masson trichrome staining was used for quantification of tubulointerstitial fibrosis according to Balarini et al. 18. A total of 30 glomeruli were used to calculate the percentage of the stained area of each kidney using ProPlus Image Analysis Software, USA (version 6.0).
Immunohistochemical staining was performed on neutral-buffered, formalin-fixed, paraffin-embedded tissue sections (4 μm). A standard immunohistochemical staining procedure was performed as done previously, with a little modification for each specific antibody according to the references in Table 1. Briefly, deparaffinization was performed using xylene and ethanol. Antigen retrieval was achieved by boiling tissue slides with 0.01 mol/l citric buffer in a microwave for 5 min. Hydrogen peroxide was used to quench the endogenous peroxidase activity. After blocking with 10% serum–Tris buffer for 20 min at room temperature, the sections were incubated with the primary antibody at the dilution given in Table 1 at room temperature for 120 min. Corresponding biotinylated-conjugated secondary antibody from a Dako (Glostrup, Denmark) staining system was used. Slides stained with secondary antibody only were used as negative controls. The nuclei were counterstained with hematoxylin.
Labeling intensity (mean intensity) and extension of the reaction (area percentage) of α-smooth muscle actin (ASMA), desmin, and endothelial nitric oxide synthase (eNOS) were assessed in 30 glomeruli in each rat kidney with ×40 objective lens and ×10 ocular lens using ProPlus Image Analysis Software (version 6.0). CD68 expression was seen as cell membrane and cytoplasmic staining. The patterns of proliferating cell nuclear antigen (PCNA) expression were defined as nuclear staining. The number of CD68-positive and PCNA-positive cells was counted in both the renal cortex and the medulla, and the median was calculated.
Data were analyzed using statistical package for the social sciences (SPSS, version 16; SPSS Inc., Chicago, Illinois, USA). For nonparametric data, analysis of variance Kruskal–Wallis, followed by a post-hoc test (based on the Dunn procedure), was used to analyze each pair of groups, thereby avoiding a multiple-comparison effect. For parametric data, the different groups were compared using analysis of variance (f-test), followed by a Bonferroni post-hoc test. A P value of less than 0.05 was considered significant.
The chemical composition of the 100 g of raisins used in the study was analyzed in the Analytical Chemistry Unit (Table 2).
It was found that the weight gain of the rats that received the HCD was significantly higher compared with the control group, and the addition of raisins significantly reduced this weight gain. Food and water intake, as well as food efficacy, during the experiment is shown in Table 3. It was observed that the relative weight of the kidney in the rats that received the HCD was significantly lower compared with both the control and treated groups, as illustrated in Table 3.
The blood glucose and insulin levels in the rats that received the HCD had significantly increased at the end of the experiment compared with the levels at the start of the experiment, as well as with the levels in the control group. Administration of raisins significantly decreased both blood glucose and insulin levels at the end of the experiment compared with the levels in rats that received only HCD (Figs. 1a and b).
Rats that received the HCD showed a significant increase in the cholesterol, triglycerides, and low-density lipoprotein levels, and a significant decrease in the high-density lipoprotein level at the end of the experiment compared with their levels at the start of the experiment and with the levels of the control group. These parameters significantly decreased, with the exception of high-density lipoprotein, which increased, in rats that received the HCD and raisins compared with those that received only the HCD (Figs. 2a–d).
As regards the renal function tests, it was observed that the HCD significantly increased the creatinine and urea levels in the blood, whereas the blood urea nitrogen level did not significantly increase compared with the control group. Raisins could significantly decrease levels of creatinine and blood urea nitrogen, but failed to do so for the urea level (Table 3).
This study revealed that the renal corpuscles located in the renal cortex of the kidney of control rats and of rats that received raisins have intact structure with glomerular capillary tufts and surrounded by Bowman’s capsule. The renal corpuscle of rats that received HCD showed congestion of the glomerular capillaries with aneurysmal dilatation of some of these capillaries, widening of the Bowman’s capsule, and mesangial expansion, which resulted in a significant increase in glomerular surface area compared with the control group. The corpuscles of rats that received HCD with raisins showed less mesangial expansion compared with those that received only HCD, although most of the glomerular capillaries appeared congested but not dilated. Morphometrical measurements showed a significant increase (P<0.001) in the glomerular capillary diameter of the renal corpuscle of rats that received HCD and a significant decrease (P<0.001) in rats that received HCD with raisins (Figs. 3a–d, i, and j).
The small renal blood vessels in the cortex of the rats that received HCD showed interrupted internal elastic lamina and a few showed a dissected aneurysm in the wall with an increase in the surrounding collagen fibers, whereas those of rats that received HCD with raisins appeared intact as in the control rats apart from a little vacuolation in the wall (Figs. 3e–h).
It was observed that the renal corpuscles of rats that received HCD and those of rats that received HCD with raisins showed no difference in the intensity of PAS reaction in the glomerular and Bowman’s capsule basement membrane compared with the control rats. This was statistically confirmed as there was insignificant difference in the PAS-stained area in the renal corpuscles of the studied groups (Figs. 4a–d and i). In addition, there was an insignificant difference in both the glomerular and periglomerular fibrous tissue among the studied groups (Figs. 4e–h and j).
Glomeruli of the renal corpuscle of control rats and of rats that received raisins showed weak ASMA and desmin expression, whereas those of rats that received HCD showed a strong to moderate ASMA expression and moderate desmin expression. In contrast, glomeruli of rats that received HCD with raisins showed a moderate to weak ASMA and desmin expression (Figs. 5a–h). Only the glomeruli of rats that received HCD showed positive PCNA staining among all the studied groups (Figs. 5i–l). This semiquantitative analysis showed a significant increase in the mean intensity and area percentage of ASMA and desmin immunoexpression in the renal glomeruli of rats that received HCD compared with the control group, whereas those of rats that received HCD with raisins were significantly reduced compared with rats that received only HCD (Figs. 5m–p).
The glomeruli of the control rats and of rats that received raisins showed strong expression of eNOS in the endothelial cells of the glomerular capillaries and the arterioles, whereas rats that received HCD showed weak expression and rats that received HCD with raisins showed a moderate to weak expression. The mean intensity and area percentage of eNOS expression were significantly reduced (P<0.001) in rats that received HCD compared with the control group, whereas both parameters were significantly increased in the glomeruli of rats that received HCD with raisins compared with those that received only HCD (Fig. 6). Very few CD68 positive macrophages were observed in the glomeruli, periglomerular area, and interstitium of both control rats and rats that received raisins. The mean number of these cells significantly increased (P<0.001) in both the glomeruli and interstitium of the rats that received only HCD compared with the control, and it significantly reduced in rats that received HCD with raisins compared with rats that received only HCD (Fig. 7).
Electron microscopy findings
The glomeruli of control rats and of those that received raisins showed a normal ultrastructure. The podocytes had irregular nuclei and extensive cytoplasm with their secondary processes and were surrounded by glomerular capillaries and rested on a thin regular basement membrane, whereas the mesangial cells had darker nuclei, less cytoplasm and were surrounded by mesangial matrix (Fig. 8). In contrast, the glomeruli of rats that received HCD showed excessive mesangial matrix deposition around the mesangial cells, thickening and irregularity of the glomerular basement membrane, and areas of fusion of foot processes of the podocytes. In other areas of the glomeruli the minor foot processes of the podocytes were lost with obliteration of filtration slits (Fig. 9).
Glomeruli of rats that received HCD with raisins showed a regular glomerular basement membrane with average thickness in almost all areas and it appeared thickened and irregular in a few areas. Regular foot processes with restoration of the filtration slits was also observed. Increased deposition of the mesangial matrix was still observed but with a lesser extent compared with that of rats that received only HCD (Fig. 10).
Although many human intervention trials have provided evidence on the protective effects of various (poly)phenol-rich foods, including red grape extract and raisins, against chronic diseases, they are still in need of better in-vivo intervention and in-vitro mechanistic studies to fully understand their interaction with the physiological and pathological processes of humans 8. Therefore, this study was conducted to investigate the histopathological changes resulting from simultaneous administration of an HCD and raisins on the kidneys of adult rats, and to correlate these findings with the biochemical changes observed.
In this study, the raisins significantly decreased the HCD-induced elevated blood glucose and insulin levels. This finding is supported by the observation of Rankin et al. 22 who reported a similar finding during their clinical trials on obese subjects consuming raisins. This hypoglycemic activity of raisins could be attributed to its flavonoid constituents 23. These constituents can preserve the insulin-secreting capacity and viability of pancreatic β cells as was described by Yugarani et al. 24 during their studies on polyphenolic natural products rich in flavonoids. Plasma cholesterol levels were significantly increased after HCD ingestion as expected and raisins significantly reduced it, a finding that is supported by the results of some previous human studies conducted by Bruce et al. 11, Spiller et al. 10, Gardner et al. 25, and Puglisi et al. 26. This hypolipidemic effect of raisins is attributed to its polyphenols, which may interfere with cholesterol absorption 27. The renal function impairment indicated by a significant increase in the levels of creatinine and urea in rats that received raisins was observed in this study as well as in previous studies by Vaziri et al. 28 on apolipoprotein E (ApoE)-deficient mice, and by Balarini et al. 18. In contrast, Joles et al. 29 reported that dietary hypercholesterolemia had no effect on either plasma creatinine or creatinine clearance.
In this study, mesangial expansion in the glomeruli of rats that received HCD as well as congestion and dilatation of the glomerular capillaries was observed during the light microscopic examination, and mesangial matrix deposition and increased thickness and irregularity of the glomerular basement membrane were observed during examination of the ultrastructure of the same group. These findings are in line with some previous studies of dietary hypercholesterolemia models 30,31 and in a study conducted in hyperlipidemic ApoE-null mice by Bruneval et al. 19, whereas Balarini et al. 18 found that the glomerular tuft and Bowman’s capsule were not affected by hypercholesterolemia in ApoE mice. Mesangial expansion might result from accumulation of lipids in the macrophages to form foam cells 32,33. Although it was statistically insignificant, glomerular and periglomerular fibrosis was observed in rats that received the HCD in this study, as well as in the previous study by Balarini et al. 18 and Chade et al. 34, who attributed this fibrosis to an increase in extracellular matrix deposition and reduced matrix degradation. In dyslipidemia, circulating lipids bind to extracellular matrix molecules and undergo oxidation, with a subsequent increase in the formation of reactive oxygen species, such as superoxide anion and hydrogen peroxide. The oxidized low-density lipoprotein could be responsible for increased matrix deposition 35,36.
Administration of raisins together with the HCD ameliorated these histopathological changes. There were no studies dealing with the effect of raisins on hypercholesterolemic kidneys available for comparison.
The ASMA expression level is used as a marker of mesangial cell activation, which could be a target cell of hyperlipidemia and significantly contributes to the extracellular matrix production 18. In this study, the increased ASMA expression in the glomeruli of the hypercholesterolemic rats indicated mesangial cell activation, which could account for increased thickness and irregularity of the glomerular basement membrane observed with the electron microscopic examination. This study, as well as that of Bruneval et al. 19, failed to detect proliferation in any of the glomerular cell types as demonstrated by PCNA; hence, cell proliferation was excluded as a cause of mesangial expansion.
The expression of desmin, an intermediate filament protein, is often upregulated in various glomerular diseases in which podocyte damage is involved 37,38. In the present work, areas of fusion of foot processes of the podocytes or loss of these processes with obliteration of filtration slits in other areas as well as the increased desmin expression in the glomeruli of hypercholestermic rats indicated podocyte injury. This was in accordance with the study by Joles et al. 29 and Attia et al. 39. Greiber et al. 40 attributed hyperlipidemia-induced podocyte injury to oxidative stress. In contrast, Bruneval et al. 19 suggested that the podocyte is not a target cell in cholesterol-induced glomerular injury.
eNOS has an important role in maintaining normal renal hemodynamics 41. In the present study, hypercholesterolemic rats showed weak expression of eNOS in the glomerular capillaries and arterioles. Attia et al. 39 also demonstrated that hypercholesterolemia increased podocyte stress, which was associated with eNOS deficiency. Abrass 36 reported that dyslipidemia causes a reduction in the actions of endothelium-derived vasodilators/growth inhibitors, such as prostacyclin and nitric oxide. In the present study, the HCD resulted in a significant increase in the number of CD68 positive macrophages in the glomeruli and periglomerular area. Similar findings were reported by Hattori et al. 42 and Joles et al. 29 in their studies on different dietary hypercholesterolemia models. The preferential homing of Mø to the glomeruli might be mediated by local activation of the endothelial cells of the glomerular capillary 19.
In conclusion, raisins protected the kidney from hypercholesterolemia-induced injury. It significantly reduced mesangial cell activation as evidenced by reduced ASMA expression, podocyte injury denoted by decreased desmin expression, and glomerular inflammation denoted by the reduced number of monocytes. In addition, raisins significantly increased eNOS expression in the glomerular capillaries that secured the endothelial-derived vasodilator effect of nitric oxide. Consumption of raisins or its pharmaceutical preparations is advised particularly for those who consume a high-fat diet.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 The Egyptian Journal of Histology
hypercholesterolemia; immunohistochemistry; kidney; raisins; rat; ultrastructure