To study the effect of monosodium glutamate on histomorphometry of cortex of kidney in adult albino rats

Abstract

Thousands of chemicals are being used recently in our new high tech foods like ready to eat Japanese, Chinese, packaged and tinned foods. Most food additives act as either preservatives or flavor enhancers like monosodium glutamate (MSG), a sodium salt of glutamic acid. The present study investigated the effect of intraperitoneally administered MSG on cortex of the kidneys of adult albino Wistar rats and compared with control group. The histomorphometry done by calibrating with ocular micrometer on kidney tissue of control and experimental group revealed a significant difference in glomeruli with increase in length, size of bowman’s capsule with an increase in bowman’s space. The size of renal tubules could not be compared as the cells of these tubules in experimental group were disintegrated and distorted. In the experimental group (rats treated with 4 mg MSG/g body weight), the cortex of the kidneys developed variable pathological changes, which were patchy in distribution with intervening normal areas. There was distortion of renal cytoarchitecture. Many glomeruli (66.4%) showed hypercellularity, i.e., cellular proliferation of mesangial or endothelial cells and infiltration of inflammatory cells. The capillary membrane showed thickening as was evident on PAS stain. Since MSG, as a food additive, was found to be toxic on various organs of the body by various researchers, it should perhaps be stopped from being used as a food additive. This may be a suggestion which needs validation in human studies.

• Histomorphometry
• monosodium glutamate
• kidney
• Wistar rats

Introduction

Monosodium glutamate

Thousands of chemicals are being used recently in our new high tech foods like ready to eat, Japanese, Chinese, packaged and tinned foods. Most food additives act as either preservatives or flavor enhancers.

Monosodium glutamate (MSG), a food ingredient was invented in 1908 in Japan, by Kikunae Ikeda. MSG, also known as AJINOMOTO, VETSIN, ACCENT, sodium salt of naturally occurring non-essential amino acid, glutamic acid, is one of the many food additives being used. MSG contains 78% glutamic acid, 22% sodium and water. Glutamate is one of the most commonly occurring amino acid found in nature, also produced in the body, forms the main component of proteins and peptides of various tissues and plays an important role in human metabolism. In general, the natural glutamic acid found in food does not cause problems, but the synthetic free glutamic acid formed during industrial processing is a toxin.

When MSG is added to food, it provides a flavoring function through stimulation of orosensory receptors and by improving the palatability of meals.1,2 It is stated that the taste quality elicited by MSG and other related substances was unique and was not some combination of primary taste qualities, namely, sweet, sour, salty, bitter.3 MSG was once being made from wheat gluten but in recent times it is being made by fermentation of carbohydrates through bacterial or yeast species such as Brevibacterium, Arthrobacter, Microbacterium and Corynebacterium. MSG is a stable colorless solid-like sodium salts of other amino acid existing ad stereoisomers but only naturally occurring l-glutamate form is used as a flavor enhancer. MSG also influences appetite positively and induces weight gain.4

Toxicity

Despite its taste stimulation and enhancement of appetite, many studies have reported MSG toxicity to humans and experimental animals.5 The interest in adverse reactions of MSG was developed because of its association with Chinese restaurant syndrome. The symptoms included headache, flushing, sweating, facial pressure or tightness, numbness, tingling or burning in face, neck and other areas, rapid fluttering heart beats (heart palpitations), chest pain, nausea, and weakness. People differ in their tolerances to MSG, but generally suffer similar reactions each time they ingest amounts of MSG that exceed their tolerances for the substance. Use of alcohol or exercise prior to, during, or after an MSG containing meal may exacerbate reaction to MSG in many people. Kwok first reported that MSG was neurotoxic (killing brain cells, causing retinal degeneration, and endocrinal disorder) and was also associated with other pathological conditions.6 The toxic effect of MSG further corroborated by the work is significant oligozoospermia.7

Aim of present study

In humans, majority of drugs administered are eliminated by a combination of hepatic metabolism and renal excretion. Since the kidney is involved in the excretion of many toxic metabolic waste products, particularly the nitrogenous compounds, it would therefore be worthwhile to examine the effects of MSG on the kidney of adult albino Wistar rats. The aim of the present study was to report the normal histomorphometric characteristics of the kidneys in adult male albino rat and to observe the effects of MSG on the histomorphometry of cortex of the kidneys in adult albino rat treated with MSG.

Materials and methods

Experiment

The study was done on inbred adult albino Wistar rats (weight 150–200 g) in Department of Anatomy. It was a STS project submitted to Indian Council of Medical Research (ICMR) and was a part of ongoing thesis work in the same department.

• Control – 10 animals

• Study – 10 animals

Animals were group housed under natural light and dark cycle with ad libitum access to food and water. The body weight of all the animals was recorded at the beginning and at completion of experiment. The experimental group of animals was treated daily subcutaneously with 4 mg/g body weight of MSG (Ajinomoto) dissolved in sterile normal saline for 7 d. The control animals received same amount of sterile normal saline by the same route. All the animals were sacrificed 30 d after the completion of the experiment by perfusion with formal saline under anesthesia.

Dissection and block making

The kidneys were dissected out carefully and dehydrated in ascending grades of alcohol (70%, 90%, and absolute), cleared in xylene and embedded in paraffin wax. Paraffin wax blocks were made using L moulds. Sections of 8 µm thickness were obtained using rotatory microtome. The sections were then taken on slide already having a fixative (egg albumin) and stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) stains.

H&E stain

After immersion of the deparaffinized sections (by dipping in xylene), the tissue was rehydrated with descending grades of alcohol (absolute, 90%, 80%) and washed with water. Staining with hematoxylin was done for 7–10 min, and then washed in running water for 2–3 min, examined under microscope for preliminary examination of stain. Remove excess of stain by differentiating in acidified alcohol (0.5–1% HCl in 70% alcohol). Then blueing of hematoxylin was done by washing in alkaline tap water for 5 min. Counter staining was done with 1% aqueous Eosin for 2–3 min and excess stain washed off with water. The slide is then examined under microscope for staining. Dehydration with alcohol was done followed by clearing with xylene. Slide was then mounted (mounting medium DPX) and examined.

PAS staining procedure

Preparation of Schiff’s reagent

Distilled water (200 cm³) was boiled and 1 g of basic fuchsin was added to it. The solution was then cooled and filtered which then became clear and transparent red colored. It was kept in a dark bottle overnight and then treated with 1 g activated charcoal, shaken and filtered. The solution was then stored in refrigerator until further use.

Methodology

The sections were brought to water and then oxidized with 1% periodic acid, then washed in running water for 5 min and rinsed in distilled water. Stain with Schiff’s reagent for 10–20 min and excess stain was washed off for 10 min in running water. Stain nuclei with Harris hematoxylin, washed with water and differentiated with acidified alcohol, dehydrated in alcohol, cleared in xylene. The slide was then mounted.

Histomorphometry

The sections of both the stains were observed under light microscope examined under 4×, 10×, 20× and 40× and desired areas were photographed using Carl Zeiss photomicroscope. Structural changes of the cortex of kidneys were observed and morphometric studies of different cells were carried out using computerized measurement system.

In each section, glomeruli present per field were counted and abnormal glomeruli out of these were observed. Area of each glomeruli in the fields observed was determined by using calibrated ocular micrometer. In addition, area of glomerular tuft was measured for each of these glomeruli. Diameter of renal tubules was also calculated.

Statistical analysis

The findings of control and experimental groups were compared and statistically analyzed by SPSS 17 (Delhi, India). Experiment was carried out after obtaining clearance from the Institute ethical committee for animal experimentation.

Observations and results

Various structural changes in the experimental group were compared with the control group in the cortex of the kidneys. Both qualitative and morphometric observations were recorded.

Observations in control group

In the control group the glomeruli, renal tubules (both proximal and distal) and collecting ducts were examined in the cortex of the kidney under 4×, 10×, 20× and 40×. Most of the glomeruli appeared normal in shape and size on both H&E and PAS stain. The glomeruli consisted of anastomosing network of capillaries lined by fenestrated endothelium invested by two layers of epithelium. The visceral epithelium was intimately related to the capillary wall, separated from endothelial cells by a basement membrane. The parietal epithelium (which was simple squamous epithelium) situated on the Bowman’s capsule lined the urinary space (the cavity in which plasma filtrate is first collected). Most of the renal tubules were regular in shape, having brush border epithelium with euchromatic nuclei and a nucleolus. The cytoplasm stained pink. The distal convoluted tubules were also lined by cuboidal epithelium. Some of the glomeruli appeared to be shrunken, showing a diffuse coloration which on higher magnification denoted accumulation of homogenous material, which could have been exudation of contents from the capillary wall. On still higher magnification, epithelium of Bowman’s capsule and capillary wall were indistinguishable, unlike the normal glomeruli. Such abnormal glomeruli were 10% of the total in the microscopic fields examined in H&E stain. While PAS stained slides showed 8.04% of abnormal glomeruli (Table 1, Figure 1). These changes occurring in control group could have been due to anesthesia used and perfusion of the animal with the formal saline during the experiment.

Figure 1. Photomicrograph of kidney sections of the control group rats showing normal histology of (a) glomerulus and (b) renal tubule in the cortical region (H&E stain ×40).

Table 1. The percentage of abnormal glomeruli in total number of fields examined in control and study group for both H&E and PAS stained slides.

Abnormal glomeruli	Control group (%)	Study group (%)
H&E stain	10	34.4
PAS stain	8.04	66.4

Observations in experimental group

In the experimental group (rats treated with 4 m MSG/g body weight), the cortex of the kidneys developed variable pathological changes which were patchy in distribution with intervening normal areas. There was distortion of renal cytoarchitecture. Many glomeruli (34.4% in H&E stain, 66.4% in PAS stain) showed hypercellularity, that is cellular proliferation of mesangial or endothelial cells and infiltration of inflammatory cells. The glomerular tuft was showing cellular swelling and homogenous accumulation of material, which could have been due to matrix increase or hyalinosis or exudation of cellular contents out of the cells with damaged membranes. When extensive, this change contributed to obliteration of capillary lumen of glomerular tuft. There was also an increase in lobulation of glomerular tuft. The capillary basement membrane showed thickening as was evident on PAS stain. The changes seen in glomeruli were focal (i.e., involving a portion of glomerulus) and segmental (i.e., affecting only a part of each glomerulus) (Figure 2).

Figure 2. Photomicrograph of kidney sections of rats of experimental group treated with 4 mg MSG/g body weight for 30 d (PAS stain ×40). (a) Abnormal glomerulus, (b) hypercellularity, (c) degenerated renal tubule, (d) cellular debris.

The changes in the renal tubules were also patchy in distribution with structurally normal areas intervening between them. There was patchy cloudy swelling, edema of the renal tubular cells with indistinct outlines and narrow lumen. Some of the cells were seen as necrosed or disintegrated and cell debris was found in the lumen of the tubules. The degenerated tubules showed detachment of the cells from the basement membrane and exudation of cellular contents in the lumen with cytoplasmic vacuolations. In such tubules, the remaining cells were enlarged and polyhedral with slightly dark staining cytoplasm. The nuclei of these cells were either pyknotic or karyolyses. Very few focal hemorrhagic areas and inflammatory cells were seen in between the renal tubules. The capillaries present in between the tubules were also dilated and their basement membrane thickened as seen by PAS stain.

Main results

The histomorphometry done by calibrating with ocular micrometer on kidney tissue of control and experimental group revealed significant differences in glomeruli with increase in length, size of Bowman’s capsule with an increase in Bowman’s space (Table 2).

Table 2. The comparison of morphometry of controls and experimental group.

	Control group, mean ± SD	Study group, mean ± SD	p Value
Glomerulus
Area (µm^2)	0.016 ± 0.005	0.029 ± 0.001	0.067
Length (µm)	0.154 ± 0.021	0.224 ± 0.001	0.043
Width (µm)	0.134 ± 0.024	0.165 ± 0.007	0.222
Bowman’s capsule
Area (µm^2)	0.022 ± 0.004	0.047 ± 0.001	0.033
Length (µm)	0.190 ± 0.014	0.250 ± 0.023	0.091
Width (µm)	0.160 ± 0.014	0.212 ± 0.004	0.038
Bowman’s space
Area (µm^2)	0.006 ± 0.001	0.015 ± 0	0.012
Length (µm)	0.036 ± 0.007	0.052 ± 0.001	0.088
Width (µm)	0.026 ± 0.010	0.065 ± 0.005	0.039

The bold values indicate significant data.

The size of renal tubules of control and experimental group could not be compared as the cells of these tubules in study group were disintegrated and distorted.

Discussion

MSG, a sodium salt of amino acid glutamic acid being added to Chinese food, canned vegetables, soups and processed food, acted as a flavor enhances via stimulation of orosensory receptors, thereby improving the palatability of meals. Despite its taste stimulation and appetite enhancement, various researchers had reported that it was toxic to humans and experimental animals.

Comparison of present study with others

The present study showed that toxic effects of MSG on glomeruli of the kidneys were focal and segmental. There was shrinkage of glomeruli, increased cellular proliferation, exudation of contents of capillaries with obliteration of their lumen and perhaps hyalinization. The renal tubules also showed patchy cloudy swelling with cells being necrotic with karyolitic nuclei and cell debris seen in the lumen. The morphometric measurements (size of glomeruli, Bowman’s capsule and bowman’s space were increased) of the experimental group were also significantly different from the controls.

Our findings were similar to those reported by Salam and Agha who treated the rats with 2–3 mg/g body weight.1 They also stated that the renal proximal convoluted tubules were more severely affected than other tubules with inflammatory infiltration and focal hemorrhagic areas. Similar results were also reported by Aughey et al., Kjellstom, Mitsumari et al. and Inkielewicz et al.8–11 Bopanna et al. also studied the changes produced by MSG in rats on atherogenic diet on kidney and liver and showed that there was glomerular mesangial proliferation with extensive damage and vacuolation of tubular epithelial cells and infiltrates of inflammatory cells.12 Eweka working on kidneys of adult Wistar rats, observed distortion of renal cortical structures with some degree of cellular necrosis due to MSG.3

Mode of injury

In general, the natural glutamic acid found in food was not problematic but synthetic free glutamic acid formed during industrial processing was a toxin. Salam and Agha suggested that glutamate was absorbed from the gut by amino acid-specific active transport system.1 Higher levels of glutamate which were transaminated increased the alanine levels in portal blood and led to release of large amounts of glucose, lactate, glutamine and other amino acids into systemic circulation. Bopanna et al. suggested that MSG in higher doses induced prolonged ischemia and caused irreversible injury leading to cell death.12 MSG was responsible for producing O₂ and O₂-free radicals (partially reduced O₂ species), which increased lipid peroxidation and also acted as important messenger for many pathological conditions by increasing cytosolic-free calcium. Sustained increase in the levels of calcium increased the membrane permeability. The swelling of cells took place perhaps because of these which caused a drop in aerobic respiration. To maintain ATP levels, glycolysis took place in the cells producing lactic acid and consequently causing the intra-cellular pH to drop. This led to dysfunction of sodium–potassium ATPase and consequent membrane damage, mitochondrial and lysosomal damage. Therefore, cell membrane damage was the most important factor in the pathogenesis of irreversible cell injury. Farombi indicated in his study that dietary antioxidants had protective potential against oxidative stress induced by MSG, which suggested that active oxygen species played an important role in its toxicity.13

Renal tubular epithelial cells were particularly sensitive to ischemia and toxins. Several factors predisposed the tubules to toxic injury including vast charge surface for tubular reabsorption, their higher metabolic rate, their oxygen consumption requirement and their vulnerable enzyme systems. Also, tubules came in contact with toxic chemicals during their excretion and elimination by the kidneys. The loss of polarity of polarized epithelia of proximal convoluted tubules due to its contact with toxins resulted in their ischemia, then eventual necrosis. Results of the present study suggested that the functions of the kidney could have been adversely affected due to distortion of renal cortical cytoarchitecture and cellular necrosis.

Effects of MSG on other organs as studied by other researchers

Various researchers in their studies have reported toxic effects of MSG on other organs of the body. Kwok reported that MSG was neurotoxic (killing brain cells, causing retinal degeneration, endocrinal disorder) and was also associated with other pathological conditions which was also in agreement to findings of Lucas and Newhouse.6,14 Dawson et al. in their study claimed that the mice became obese by administration of MSG, which was due to MSG-induced lesions in the hypothalamus.15 Yu et al. demonstrated that maternal oral administration of MSG in rats (2.5 mg or 4 mg/g body weight) resulted in its penetrating the placental barrier using glutamic acid as a tracer, which in turn damaged the embryonic tissues with more effects on fetal brain, while Bizzy et al. studied the neurotoxic effects induced by MSG in rodents and their relevance to man.16,17 Onakewhor et al. worked on effects of MSG on testes and stated that there was significant oligozoospermia and increased abnormal sperm morphology which was dose-dependent.7 Oforofuo et al. and Eweka et al. also established MSG as a cause of male infertility.18,19

Neuroscientists also stated that the infants and elderly were more at risk of MSG-induced neurotoxic effects because of leaky blood–brain barrier.

Since MSG as a food additive was found to be toxic on various organs of the body by various researchers, it should be perhaps stopped from being used as a food additive.

Conclusion

The present study demonstrated that MSG induced marked histopathological changes in the kidneys of the rats, suggested it to be toxic, which was also corroborated by other researchers, so the use of MSG in foods remains controversial.

Declaration of interest

The authors report no conflicts of interests. The authors alone are responsible for the content and writing of this article.