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. 2008 Jun;212(6):845–852. doi: 10.1111/j.1469-7580.2008.00902.x

The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats

Muhammed Eyüp Altunkaynak 1, Elvan Özbek 1, Berrin Zuhal Altunkaynak 1, İsmail Can 1, Deniz Unal 1, Bunyami Unal 1
PMCID: PMC2423405  PMID: 18510511

Abstract

To characterize the kidney in a high-fat-induced obesity model, we examined the renal structure of adult Sprague–Dawley rats fed a control diet or a high-fat diet for 3 months. Ten adult female Sprague–Dawley rats were fed a diet consisting highly of fat (30%) for a period of 3 months. Ten control rats were maintained with standard rat chow. All animals were weighed every 10 days for 3 months. At the end of the experiment, the naso-anal length of the anaesthetized rats was measured to calculate body mass index, and subsequently whole kidneys of intracardially formalin-perfused animals were removed. Quantitative features of the kidney were analysed with the Cavalieri and physical dissector methods applied to serial paraffin sections. Kidney samples were also examined histologically. The body mass indices of the control and treatment groups were 4.528 ± 0.242 and 5.876 ± 0.318 kg m−2, respectively. The difference between the body mass indices of the two groups was statistically significant (P < 0.01, Mann–Whitney U-test), suggesting that the animals fed with a high-fat diet may be overweight. Stereological examination of the kidneys revealed differences in kidney weight, total kidney volume, volume of cortex, medulla, glomeruli, proximal and distal tubules, and numerical density of glomeruli and glomerular height in the treatment group compared with the control group. Light microscopic investigation showed a dilatation in blood vessels and Bowman's space, mononuclear cell infiltration, degeneration in nephrons, including glomerulosclerosis and tubular defects, and an increase in the connective tissue in the kidneys in the treatment group. We concluded that a fatty diet is responsible for the rats’ obesity and may lead to renal deformities as a result of histopathological changes such as dilatation, tubular defects, inflammation and connective tissue enlargement of the kidney.

Keywords: high-fat diet, kidney, light microscopy, obesity, stereology

Introduction

Obesity is currently one of the most frequently encountered medical problems. Among the complications associated with the pathological aspects of disease, renal disease is a significant issue and its pathophysiological mechanisms are not fully known (Kramer & Luke, 2007). For example, hypertension, hyperlipidaemia and insulin resistance affect renal function, each one in a different way (Liu et al. 2007). Obesity seems to be a condition in which kidneys demonstrate morphological and functional alterations, while hormones and growth factors play an important role (Papafragkaki & Tolis, 2005).

Evidence from human and animal studies suggests that malnutrition can induce many diseases such as hypertension, liver failure, cardiovascular diseases, kidney diseases and even some cancer types (Watanabe et al. 2007). A model of dietary imbalance whereby administration of a diet rich in animal fat causes the development of dyslipidaemia, abdominal obesity (Innis, 2007), fatty liver disease (Altunkaynak, 2005), hepatomegaly (Oldenburg & Pijl, 2001) and splenomegalia (Altunkaynak et al. 2007) has been previously described.

Stereology is used to recreate or estimate the properties of geometric objects in space. Applying stereological methods to a tissue or organ section allows us to estimate the geometric properties of the objects contained in the sections (Sarsilmaz et al. 2007). Space has three dimensions, and objects within it have properties for each possible number of dimensions. Objects have a volume (three dimensions), a surface (two dimensions), a length (one dimension) and a number (zero dimension). Each of these properties can be estimated by stereological methods (Gundersen, 1986; Basoglu et al. 2007).

In addition, these methods have been used to quantify renal morphology (Medeiros et al. 2006; Razga & Nyengaard, 2007). Parameters including kidney volume, surface area, volume of tubular structures, volume of mesangium, cortex, medulla or glomerulus, number of glomeruli and also the length, surface area and number of glomerular capillaries, etc., can be easily estimated. Methods for obtaining data for average glomeruli as well as individual glomeruli have been described (Bertram, 1995; Mayhew, 1999).

In the present study, we attempted to determine the effects of a high-fat diet (HFD) on the kidneys of female rats. Both the qualitative and the quantitative histological features of the kidney samples were analysed with conventional histopathological methods at the light microscopic level and by modern stereological methods.

Materials and methods

Animals and diets

Twenty adult female Sprague–Dawley rats (175 ± 10 g) from the Experimental Research and Application Center (Atatürk University, Erzurum, Turkey) were randomly allocated into two equal-sized groups. In the control group, rats were fed a commercial rat diet (7–10% fat, 68–70% carbohydrates, 18–20% protein, 1–2% vitamins and minerals; 210 kcal 100 g−1 day−1) for 3 months, and rats in the HFD group were fed a high-fat diet (30% calories as fat) for the same period of time. To prepare the fatty diet, the commercial rat diet in powder form was mixed with melted animal abdominal fat so as to provide 30% of the total energy from fat. This mixture, in a dough-like consistency, was shaped to match the commercial rat diet, dried and subsequently used for feeding animals of the HFD group. The rats were housed in plastic cages (two animals per cage), maintained under standardized conditions of light (12/12-h light/dark cycle) and room temperature (22 ± 2 °C), with free access to food and tap water. After 3 months, anaesthesia was induced by inhalation of 2–3% sevoflurane (Sevorane® Liquid 250 ml, Abbott, Istanbul, Turkey) in 100% oxygen, and white adipose tissue samples were removed. This study received ethical approval from the Experimental Research and Application Center of Atatürk University.

Microscopy

Kidney samples for light microscopic examination were fixed in 10% formaldehyde, dehydrated in a graded alcohol series and cleared in xylene. After dehydration, specimens were embedded in fresh paraffin (Agar, Cambridge, UK). Sections were cut using a microtome (Leica, Germany). Each paraffin block was serially cut to 5-µm thickness. Approximately 100 sections were obtained from each tissue block. The sections were stained with haematoxylin-eosin (H-E) for light microscopic examination and stereological analysis.

Volume of cortex, volume of medulla and volume of glomeruli and tubules

We used 15–20 sections obtained for application of the Cavalieri method to estimate the volume of the cortex, medulla, glomeruli, and the distal and proximal tubules, and their fraction of whole kidney volume (Cruz-Orive & Weibel, 1990). For this purpose, two different point counting test grids were used (see Fig. 2). The point density of the point-counting grids, which are designed to hit a minimum of 1000 points per region of interest (cortex, medulla, glomeruli, and tubules), for each animal was appropriate to obtain a significant coefficient of error (CE) (Gundersen et al. 1988; Tunc et al. 2006). CE and coefficient of variation (CV) were estimated according to Gundersen & Jensen (1987). Two different point-counting grids were composed of dense and loose points according to the area of interest (Fig. 1). Grids with a systematic array were randomly placed on the PC screen; the hitting point of the grid on all subjects of interest was counted. The loose points were used to estimate the volume fraction of the cortex and medulla (Fig. 1a,b), and the dense points were used for the volume fraction of glomeruli, Bowman's space and tubules (Fig. 1c,d). Sampled areas were chosen in a systematic random manner by means of a motorized stage. The total number of points that were superimposed on each area of interest was counted.

Fig. 2.

Fig. 2

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Stereological procedures for sampling and applying the physical dissector. Images (b) and (d) are reference and (c) and (e) are look-up sections. rp, reference point, used for localization of dissector pairs. Black arrows (thick) show glomeruli hitting the inclusion lines or located inside the frame in both the reference and the look-up sections. Black arrows (thin) show section profiles of glomeruli in the reference section. Transparent arrow was counted as a dissector particle if its profile was not seen in the look-up section. Black arrowhead indicates that the profile of the particle was not seen in the look-up section. Scale bars = 100 µm.

Fig. 1.

Fig. 1

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Stereological procedures for applying the Cavalieri principle. d, distal tubule; p, proximal tubule.

Subsequently, the volumes of the structures of interest in each section were estimated from the following equation: Inline graphic where V is the volume of interest (kidney, glomerulus, tubules) in one section plane, t is the section thickness, a/p is the interpoint area, and ΣP is the number of points hitting the object of interest in that section. After this formula was applied to other sections, the total volume to be estimated was obtained from:

total volume =V1+V2+ … +Vn.

The volume fractions for each region were estimated by dividing the total point number that was superimposed on the related region by the sum of the points superimposed on the whole kidney:

volume fraction(region/kidney)=P(region)/P(kidney)

where P is the number of points hitting the region of interest or whole kidney in that section.

Numerical density of glomeruli and total number of glomeruli

Selection of the physical dissector pairs was performed as described by Sterio (1984). Based on the findings obtained from a pilot study, the first chosen section and its adjacent section, called a dissector pair, were separated by a distance of 30 µm (thickness of seven sections: 35-µm distance) as a rule of the physical dissector. According to this rule, the distance between the sections pairs must be about 30–40% of the average projected height of the object of interest to be estimated (Fig. 1); in this way, approximately 15–20 section pairs were obtained and evaluated. This number is in an acceptable range for stereological analysis (Unal et al. 2004; Kaplan et al. 2005). Two consecutive sections were mounted on each slide. Photographs of adjacent sections were taken with a digital camera at a magnification of ×400. An unbiased counting frame (Unal et al. 2004) was placed on the reference and the look-up sections on the screen of the PC, to perform the counting according to the dissector method. The bottom and the left-hand edges of the counting frame were considered to be the forbidden (exclusion) lines together with the extension lines. Other boundaries of the frame that are the top-right edges were considered to be inclusion lines, and any particle that hit these lines or was located inside the frame was counted as a dissector particle (Fig. 2). The size of the unbiased counting frame was adjusted to count ∼600 glomeruli from each sample.

The dimension of the counting frame on the PC screen was 20 × 20 cm, and the real dimension of this counting frame (1 cm2) was estimated by the following formula:

real dimension = screen size of frame/total magnification of microscope.

Glomeruli seen in the reference section but not in the look-up section were counted (Kaplan et al. 2005). The mean numerical density of glomeruli [Nv(glomeruli)] per cm3 was estimated using the following formula:

graphic file with name joa0212-0845-m2.jpg

where Inline graphic is the total number of glomeruli counted in the reference section, t is the mean section thickness (5 µm), and A is the area of the unbiased counting frame.

Thus, the total number of glomeruli (TN(glomeruli)) in a whole rat kidney was estimated by the following equation:

TN(glomeruli)=Nv(glomeruli) × kidney volume

where Nv(glomeruli)is the numerical density of glomeruli per cm3, TN(glomeruli)is the total number of glomeruli in the whole kidney calculated by using Nv(glomeruli) and the kidney volume results estimated by the Cavalieri method.

Finally, histopathological examinations were carried out on images of the same sections.

Statistical analysis

Differences in volumetric data, numerical density, total number or glomerular height between the two groups were tested using the independent samples t-test (two tailed, with a significance limit of P = 0.05 in this test). All statistical calculations were performed using SPSS 13.0 software for Windows.

Results

Stereological results

All stereological results are summarized in Tables 1 and 2.

Table 1.

Mean volume and volumetric fraction data of the kidney, cortex, medulla and tubules in the control and high-fat diet (HFD) groups

Group
Estimation Control HFD
Mean volume of kidney (cm3) 1.7 ± 0.212 2.256 ± 0.343
Mean volume of cortex (cm3) 1.34 ± 0.072 1.57 ± 0.125
Mean volume of medulla (cm3) 0.363 ± 0.090 0.686 ± 0.144
Mean volume of distal tubule (mm3) 0.0011 ± 0.00118 0.0036 ± 0.0025
Volume of total distal tubules (mm3) 0.24 ± 0.0103 0.29 ± 0.0152
Mean volume of proximal tubule (mm3) 0.0029 ± 0.0032 0.0018 ± 0.0018
Volume of total proximal tubules (mm3) 0.374 ± 0.0020 0.323 ± 0.0034
The volume fraction ratio of total distal tubules to all kidney 0.139 ± 0.086 0.128 ± 0.0051
The volume fraction ratio of total proximal tubules to all kidney 0.219 ± 0.0098 0.143 ± 0.0127
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Table 2.

Mean volume, volumetric fraction and numerical data of glomeruli and Bowman space in the control and high-fat diet (HFD) groups

Group
Estimation Control HFD
Mean numerical density of glomeruli (cm−3) 24 800.7 ± 798 19 782.16 ± 612
Total number of glomeruli 33 233.3 ± 987 31 058.4 ± 645
Mean volume of glomerulus (µm3)  698 400 ± 1921  564 700 ± 1644
Mean volume of Bowman capsule (µm3)  78.57 ± 2.98  94.05 ± 5.86
The volume fraction ratio of total volume of glomeruli to all kidney  0.114 ± 0.032  0.0414 ± 0.0013
The volume fraction ratio of total volume of glomeruli to cortex  0.345 ± 0.023  0.159 ± 0.0021
The volume fraction ratio of total volume of Bowman capsule to all kidney  0.00148 ± 0.00023  0.00126 ± 0.00012
The volume fraction ratio of total volume of Bowman capsule to cortex  0.00197 ± 0.00036  0.00184 ± 0.00019
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Volume of kidney

Volumes of kidney tissues were estimated by the Cavalieri principle on a series of H-E-stained sections. Estimates of the kidney volumes of the control diet (CD)- and HFD-fed rats are shown in Table 1. The volume of the kidney was approximately 1.7 cm3 for rats in the control group. By the 12th week of diet application, the volume of the kidney was estimated as approximately 2.256 cm3. Statistical analysis by independent samples t-test revealed a significant difference between these estimates (P < 0.01).

Volume of cortex and medulla

Volumes of cortex and medulla of kidney tissue were additionally estimated by the Cavalieri principle on a series of H-E-stained sections. The estimates of the volumes of the kidney cortex and medulla of control rats and those of HFD-fed rats are given in Table 1. The volumes of the kidney cortex and medulla were approximately 1.34 and 0.36 cm3, respectively, for rats in the control group. By the 12th week of diet application, the volumes of the kidney cortex and medulla were estimated to be 1.57 and 0.686 cm3, respectively. Statistical analysis by independent samples t-test revealed a significant difference between these estimates (P < 0.05).

Volume of distal and proximal tubuli

The mean distal and proximal tubule volume and also the mean volume fraction ratio of the distal–proximal tubules and total kidney volume results of both groups are shown in Table 1. There were significant differences between the HFD group and the CD group (P < 0.05) in terms of the mean volume of the distal and proximal tubule and also the mean volume fraction ratio of distal–proximal tubules and total kidney volume.

Volume of glomerulus and Bowman's space

The mean volume of the glomerulus, the volume fraction ratio of total glomeruli to mean cortex, and the volume fraction ratio of the total glomeruli to the total kidney were also estimated. Additionally, the same estimations were performed for Bowman's space (Table 2). The mean volumetric values of the glomerulus were 698 400 and 564 700 µm3 in the CD and HFD groups, respectively. The mean volume fraction ratios of total glomeruli to mean cortex were 0.345 in the CD group and 0.159 in the HFD group. The mean volume fraction ratios of the total glomeruli to the total kidney were 0.114 in the control group and 0.0414 in the HFD group. Mean volume values of Bowman's space were 78.57 and 94.05 µm3 in the control and HFD groups, respectively. The mean volume fraction ratios of the total Bowman's space to mean cortex were 0.0019672 in the CD group and 0.0018396 in the HFD group. The mean volume fraction ratios of total Bowman's space to the total kidney were 0.001476 in the CD group and 0.001262 in the HFD group. All values of the HFD and CD groups were significantly different from each other (P < 0.05).

Mean numerical density of glomeruli (Nv(Glomeruli))

The mean numerical densities of the glomeruli for both groups are shown in Table 2. There were approximately 24 800.7 glomeruli cm−3 in the kidney of the control rats. However, the numerical density of glomeruli in the kidney decreased significantly from the beginning to the end of the experiment. There were approximately 19 782.16 glomeruli cm−3 in the kidney of the HFD-fed rats. Specifically, the mean density of glomeruli in the HFD group was decreased by 20.3% relative to the CD group (P < 0.0001).

Total number of glomeruli

Table 2 shows the total number of glomeruli in the kidneys of the CD- and HFD-fed rats. The control rats had 33 233 glomeruli in the kidney. By the end of the experiment, the total number of glomeruli in the kidney had decreased significantly to 31 058 Statistical analysis performed by independent samples t-test revealed that the total number of glomeruli in the kidney changed significantly from the beginning to the end of the treatment (P < 0.01) (Table 2).

Histological results

The renal cortex of the control rats contained glomeruli, vessels, tubules and interstitium (Fig. 3a). When evaluating these renal specimens by light microscopy on an H-E-stained section, the following glomerular features were seen: the overall cellularity of the glomerulus, the symmetry of the glomerulus and the thickness of the capillary walls (Fig. 3c). Renal tubules (the long and winding neck) formed as the proximal tubule, the loop of Henle and the distal tubule (Fig. 3b,c). Among tubular structures, there was very little interstitium in the cortex (Fig. 3b,c).

Fig. 3.

Fig. 3

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Light micrographs of kidney tissue from the control group. co, cortex; me, medulla; p, proximal tubule; d, distal tubule; gl, glomerulus. Scale bars = 80 µm.

Light microscopic findings of kidney sections from HFD-fed rats are summarized as follows. Dilatation in glomerular capillaries and other blood vessels was detected (Fig. 4a inset,  4b). The lumens of the tubules and Bowman's space were enlarged, similar to those of the control rats (Figs 4a and 6a–c). Mononuclear cell infiltration was seen in the renal cortices of the HFD-fed rats (Figs 4b and 5a,b). Degeneration in nephrons, including glomerulosclerosis, segmental necrosis and tubular defects, was found (Figs 5a,b and 6c,d). Thickening in the basal membrane of glomeruli and tubules was seen (Fig. 6a–c). There were many necrotic interstitial cells in the kidneys of the HFD-fed rats (Figs 4a and 5a). The epithelium of the tubules was more shortened than that of the control rats (Fig. 6c). Also, an accumulation of white adipocytes localized in the subcapsular region was defined in HFD-fed rats (Fig. 6d). Additionally, tubular necrosis was observed in these sections (Fig. 6d).

Fig. 4.

Fig. 4

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Light micrographs of kidney in the HFD group. bv, dilated blood vessels; p, proximal tubule; gl, glomerulus; e, enlarged Bowman's space; black arrow, necrotic mesengial cells; black arrowhead, distal tubules; transparent arrow, degenerated distal tubule. Inset reveals dilated glomerular capillaries (asterisk). Scale bars = 50 µm.

Fig. 6.

Fig. 6

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Light micrographs of kidney in the HFD group. White arrow, thickened basal membrane of glomerulus; black arrow (thick), thickened basal membrane of tubules; transparent arrow, sclerotic glomeruli; black arrow (thin), lipid droplets in distal tubule (inset); black arrowhead, degenerated border of proximal tubule; gl, glomerulus; asterisk, segmental necrosis of glomerulus; wa, subcapsular white adipocyte accumulation; tn, tubular necrosis. Scale bars = 50 µm.

Fig. 5.

Fig. 5

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Light micrographs of kidney in the HFD group. Mi, mononuclear cell infiltrations; n, necrotic mesengial cells. Scale bars = 50 µm.

Discussion

Obesity is a major health problem that affects up to 30% of the adult population in developed countries and has been linked to increases in dietary intake, especially fat intake, and a sedentary lifestyle (Kramer & Luke, 2007). It is well documented that obese patients are associated with serious mortalities, including a high incidence of type 2 diabetes, hyperlipidaemia, hypercholesterolaemia, cardiovascular disease and liver abnormalities, and more attention has been paid to the impact of obesity on renal functions recently (Hall, 1994; Yilmaz et al. 2002; Altunkaynak, 2005). Chronic administration of dietetic lipid can cause abdominal obesity and can significantly alter the renal cortical structure of rats (Aguila & Mandarim-De-Lacerda, 2003; Armitage et al. 2005). In the present study, an obesity model was performed by feeding with animal tallow, and the effects of this obesity on rat kidneys were studied.

The mean numerical density, total number, height and volume of glomeruli and volume of whole kidney, cortex, medulla, Bowman's space, distal and proximal tubules, and their fraction of the renal cortex and of the whole kidney of the rats from both groups were investigated using stereological methods (Sterio, 1984; Unal et al. 2004). Stereological quantifications and histopathological examinations were conducted on light microscopy sections. The results from the two approaches confirmed each other. Findings from the HFD group are discussed below. Results from quantitative methods, which provide numerical density or geometric data, contribute substantial information on activity of that structure. Previously, only two papers have used histological methods to estimate the diameter of the glomerulus and other structures in the kidneys of obese rats. In these studies, a few stereological parameters such as the number and volume of glomeruli, the volume of tubules, and the volume of the renal cortical interstitium were investigated (Aguila & Mandarim-De-Lacerda, 2003; Armitage et al. 2005). However, neither of these studies investigated the effects of obesity on the kidney. In the present study, many structures in the kidneys of obese rats were analysed with stereological and histological methods.

According to our stereological results, the numerical density of glomerulus and total number of glomeruli in the HFD group was significantly decreased in comparison with the CD group; this indicates that the HFD resulted in glomerular atrophy. A decrease in the mean volume of glomerulus and volume fraction ratio of all glomeruli to the renal cortex and to the whole kidney in the HFD group was also found. Quantifications of the volume of the kidney based on only two-dimensional sections and with a two-dimensional approach have suggested that the mean volume of the kidney in terms of both the cortex and the medulla is increased owing to vasodilatation, connective tissue enlargement or inflammation in the HFD group as compared with the control group at the light microscopic level. To the best of our knowledge, no previous study has investigated the volume of Bowman's space and its volumetric fraction to the total kidney. Our data are thus the first in this regard. The mean volume of Bowman's space was increased in the HFD group compared with the control group. However, the volume fraction of Bowman's space to both the whole kidney and the cortex was decreased due to an increased kidney volume. According to other volumetric data, both distal and proximal tubule volumes were increased in the HFD group. However, volume fractions of neither distal nor proximal tubules were increased. These quantitative results may indicate glomerular atrophy and functional loss of glomeruli and tubules.

In our histological study, dilatation of glomerular capillaries and large blood vessels and subcapsular adipocyte accumulation were seen. In addition to tubular deformations, histologically detected glomerular atrophy and necrosis supported our stereological data. The increase in the volume of the kidneys of HFD-fed animals may have resulted from oedema due to mononuclear cell infiltrations among the tubules. And it is clearly understandable that dilatation may lead to a volumetric increase in the kidney. Early reports listed obesity as a risk factor for mortality from ‘chronic nephritis’, and the subsequent recognition of the more common association of obesity with diabetes, hypertension and heart disease altered this and questioned its being a risk factor for kidney disease (Eknoyan, 2006). Popov et al. (2003) showed that a high-caloric saturated fat intake induced diabetes in hamsters. Their model was also associated with the development of a range of pathologies characteristic of human diabetes, including nephropathy and defects in vasculature.

Much quantitative data can be obtained more easily using recently developed stereological methods (Huang et al. 2006; Marcos et al. 2006). However, there have been no stereological studies showing the effects of obesity on quantitative alterations to the kidney. Our quantitative findings are consistent with previous reports showing that HFD-dependent obesity is associated with many kidney alterations (Handa & Kreiger, 2002; Jiang et al. 2005).

In conclusion, the results of the present study suggest that there is a significant relationship between fatty diet intake and structural changes to the kidney such as a decrease in the numerical density of glomeruli, tubular deformations, prominent dilatation of the renal vessels and tubules, glomerular necrosis and atrophy, and basal membrane thickening.

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