1.0 INTRODUCTION/LITERATURE REVIEW
Diabetes mellitus (DM) is an endocrine disorder associated with poor secretion of insulin or resistance to insulin actions by peripheral tissues (Wild et al., 2004; Ali et al., 2014; Shah and Khan, 2014). The multifaceted etiology of DM has been described elsewhere (El-Missiry and El-Gindy, 2000; Nagappa et al., 2003; Jung et al., 2006; Filippi and von Herrath, 2008;Gwarzo et al., 2010; Trna et al., 2012). Studies have established a connection between Type 1 DM and compromised activities of reactive oxygen species (ROS) antagonists and scavenging enzymes (Kesavulu et al., 2000; Yue et al., 2003; Shah and Khan, 2014), which engender disturbances in metabolism (Evans et al., 2002; Kumar et al., 2013) with attendant oxidative stress induced tissue damage (Ahmed et al., 2010; Ali et al., 2014) and complications such as retinopathy, microangiopathy, ketoacidosis, neuropathy and nephropathy (Rameshkumar et al., 2004; Pop-Busui et al., 2006; Yim et al., 2007; Singh et al., 2011). Molecular events leading to β–cell dysfunction and insulin resistance are connected with stress-sensitive signaling pathways, which are progenitors of DM pathology and complications (Evans et al., 2002; Malviya et al., 2010; Ali et al., 2014). Since alloxan or streptozotocin causes selective oxidative damage to pancreatic β–cells, intra-peritoneal injection of their salt solutions is commonly used to induce Type 1 DM in experimental animals (Takasu et al., 2001; Szkudelski, 2001; Yim et al., 2007; Shah and Khan, 2014).
Oxidative stress is currently suggested as a mechanism underlying diabetes and diabetic complications (Halliwell and Gutteridge, 1989). Free radicals are continually produced in the body as the result of normal metabolic processes and interaction with environmental stimuli. Under physiological conditions, a wide range of antioxidant defenses protects against the adverse effects of free radical production in vivo (Halliwell and Gutteridge, 1989). Oxidative stress results from an imbalance between radical-generating and radical scavenging systems, that is, increased free radical production or reduced activity of antioxidant defenses or both these phenomena. In diabetes, protein glycation and glucose autoxidation may generate free radicals, which in turn catalyze lipid peroxidation (Mullarkey et al., 1990; baynes, 1991). Moreover, disturbances of antioxidant defense systems in diabetes were shown: alteration in antioxidant enzymes (Strains, 1991), impaired glutathione metabolism (McLennan et al., 1991), and decreased ascorbic acid levels (Jennings et al., 1987; Young et al., 1992).
The kidneys regulate blood ions and pH levels as well as water balance. In addition, the kidneys serve as principal organ for the elimination of metabolic waste products. The functional unit of the kidney is the nephron and each kidney contains approximately two million of these structures.
Sodium glutamate or monosodium glutamate (MSG) is a major dietary component, which intensifies the savory flavor in foods worldwide (Eweka and Iniabohs, 2007; George et al., 2013; Nwajei et al., 2015). It has a daily consumption rate of 300–4000 mg/day in developed countries (Sharma et al., 2013; Husarova and Ostatnikova, 2013). The toxicity concerns and secondary physiologic effects following intake of MSG have been controversially discussed (Maluly, 2013; Boonnate et al., 2015). LD50 of sodium glutamate in rats ranges between 15000 and 18000 mg/kg of body weight (Walker and Lupien, 2000; Kolawole, 2013).
Meanwhile, certain chemical agents and dietary components perturb blood physiologic homeostatic parameters, such as distortion of plasma testosterone/estrogens concentrations with attendant hormonal imbalance and reproductive disorders, alterations in blood lipid profile associated with the development of atherogenicity as well as provoking the overwhelming level of oxidative stress (Ojo et al., 2006; Svalheim et al., 2008; Bitter et al., 2009; Ibegbulem and Chikezie, 2012; Deavall et al., 2012; Asare et al., 2014). Furthermore, dietary component may alter the weights of visceral organs, which is diagnostic for the atrophic or hypertrophic dysfunctional organs (Amresh et al., 2008).
Accordingly, the present study was carried out to investigate the functional status of renal tissues of alloxan-induced diabetes mellitus rats treated with monosodium glutamate/ascorbic acid.
1.2 LIETERATURE REVIEW
1.2.1 MONOSODIUM GLUTAMATE
Monosodium glutamate (MSG) is a commonly-used additive in processed food and Asian cuisine to increase palatability. However, several studies in animals have shown that MSG is toxic to the various organs such as the liver, brain, thymus, and kidneys (Diniz et al., 2004; Farombi and Onyema, 2006; Pavlovic et al., 2009). Published data indicate that renal fibrosis is associated with the chronic consumption of MSG (Sharma et al., 2013) and oxidative stress is the main cause of kidney injury (Sharma et al., 2014). Oxidative stress is caused by the excessive production or a decreased elimination of free radicals in cells, the majority of which are oxygen radicals and other reactive oxygen species (ROS) (Bashan et al., 2009). Nutrition metabolism and several extracellular and intracellular factors such as hormones, cytokines, and detoxification processes contribute to the oxidative stress (Sundaresan et al., 1995; Corda et al., 2001; Stankiewkz et al., 2002). Therefore, excessive renal metabolism of glutamate as in chronic MSG intake can be a source of ROS. Decreased levels of major anti-oxidant enzymes and increased lipid peroxidation have been demonstrated in the kidneys of chronic MSG-exposed rats (Thomas et al., 2009; Paul et al., 2012). Also, high doses of glutamate have been shown to induce significant toxicity in renal culture cells (Leung et al., 2008). The abundance of long-chain polyunsaturated fatty acids in the composition of renal lipids makes kidney susceptible to damage by ROS (Kubo et al., 1997).This makes kidney tissues prone to damage by different mechanisms such as the promotion of lipid peroxidation, protein modification, and DNA damage, leading to cell death (Richter et al., 1988; Rubbo et al., 1994; Stadtman and Levine, 2000).
Accordingly, the involvement of ROS has been reported in glomerular, tubular, and tubulo-interstitial alterations (Klahr, 1997; Vielhauer et al., 2001). A host of studies have explained glutamate-induced oxidative damage in tissues like brain or neurons, where α- ketoglutarate dehydrogenase, glutamate receptors and cystine-glutamate antiporter are the vital players (Murphy et al., 1989; Jahr and Stevens, 1993; Zundorf et al., 2009).These molecules can contribute to the oxidative stress through, different mechanisms but little is known about their involvement in MSG-induced renal oxidative stress.
The increased level of α-ketoglutarate dehydrogenase has been found in the kidney of MSG-fed rats (Sharma et al., 2014) and accordingly, a strong consensus is being developed against α- ketoglutarate dehydrogenase, glutamate receptors, and cystine-glutamate antiporter for their potential role in the MSG-related renal oxidative stress.
220.127.116.11 MSG-induced kidney damage
The association between dietary factors, including MSG and the risk of kidney disease, has been hypothesized in numerous studies. The kidneys are highly sensitive to ischemia, toxic insults, and other chemicals. As such, processes leading to direct or indirect disturbances of renal cell energy metabolism will result in cell injury and acute renal insufficiency (Pfaller et al., 1990).
A summary of chronic MSG-induced renal alterations is illustrated in Fig. 1. MSG can induce changes in the renal cytoarchitecture, increase glomerular hypercellularity, infiltration of inflammatory cells in the renal cortex, edema of tubular cells, and eventually degeneration of renal tubules (Dixit et al., 2014). Although infiltration of inflammatory cells points towards a pathology, the exact pathophysiology is not fully understood. Cellular dysfunction is considered as an important cause of the subsequent development of most of the morphological alteration, regardless of the toxic principle acting upon the kidney. Therefore, ultra-structural examination of the kidney in experimental models with chronic MSG treatment could contribute to a better understanding of the mechanism of derangements during renal injury.
1.2.2 Oxidative stress
The formation of ROS in the kidney exposed to MSG was seen as a major contributor to their nephrotoxic effects leading to cellular and functional damage (Ortiz et al., 2006). MSG supplementation either by injection or oral intake has been shown to alter renal antioxidant system markers, including lipid peroxidation byproducts and kidney function in rats (Ortiz et al., 2006). Paul et al. (2012) found reduced activities of superoxide dismutase, catalase, glutathione-S-transferase and glutathione (GSH) in the kidney after MSG administration. They also reported that markers for lipid peroxidation such as malondialdehyde (MDA) and conjugated dienes were increased in MSG treated renal tissue. It is possible that MSG leads to the excessive production of free radicals and endogenous antioxidants are insufficient to meet the demand. The up-regulation of heat shock cognate 70, an indicator of oxidative stress, and the down-regulation of glutathione-S-transferase in MSG-treated kidneys further strengthens the findings (Sharma et al., 2014). Moreover, some studies have found the ameliorating effect of vitamin C, E, and quercetin on MSG-treated kidneys (Farombi and Onyema, 2006). The mechanism whereby these antioxidants exert such effects is yet to be fully elucidated. However, these antioxidants do seem to play a key role against renal inflammatory responses through a diminution of the activity of inflammatory enzymes (Ozaki et al., 1999) and cytokines secretion, or by inhibiting the activity of NF-ĸB (Massy et al., 1999; Bowie and O’Neil, 2000).
Fig. 1 An outline of chronic MSG-induced renal alterations in the kidney. Alkaline urine and oxidative stress due to chronic MSG intake may damage the kidneys by unknown mechanisms. Urolithiasis can also contribute to the interstitial fibrosis by producing inflammatory cytokines and ROS
Furthermore, studies using thiol antioxidants such as Nacetylcysteine (NAC) and lipoic acid have demonstrated therapeutic protection against glutamate-induced neurotoxicity (Han et al., 1997; Penugonda and Ercal, 2011). Although there is no experimental evidence available supporting the protective effect of these molecules in MSG-induced renal oxidative toxicity, NAC has been shown to reduce kidney MDA levels in a diabetic mouse model (Ribeiro et al., 2011). In cultured human proximal tubular epithelial cells, NAC reduced lipid peroxidation and maintained mitochondrial membrane potential, thereby preventing apoptosis following hydrogen peroxide administration (Ye et al., 2010).
Also, lipoic acid has been effective in protecting kidneys from oxidative stress and mitochondrial dysfunction (Cimolai et al., 2014). In a different context, the ameliorating effect of selenium on MSG-induced testicular oxidative toxicity has been demonstrated (Hamza et al., 2014). These important findings add further prospective to the therapy of MSG-induced renal oxidative stress using antioxidants.
1.2.3 Urolithiasis and interstitial fibrosis
Obstructive nephropathy due to chronic dietary MSG has been reported in adult rats probably due to alkaline urine and decreased levels of stone inhibitors such as magnesium and citrate in the urine (Sharma et al., 2013). The mechanism behind MSG-caused urine alkalization is still unknown but this effect was first reported by de Groot et al. (1988). It is likely that MSG-treated animals may generate higher catabolic products of glutamate in kidney cells and its carbon skeleton is converted into carbon dioxide and then to bicarbonate anions (Vercontere et al., 2004).
The generated bicarbonates are then absorbed back into the blood circulation and ultimately to the kidneys for excretion of the extra-alkali, resulting in alkaline urine (Hediger, 1999). Alkaline urine can influence the kidneys capacity in terms of secreting or reabsorbing metabolites that can contribute to stone formation, whereas inhibitors of stone formation play a major role in natural defense. An elevated ion activity product of calcium phosphate in the alkaline urine of MSG-fed mice indicates the risk of calcium-phosphate stone formation (Sharma et al., 2013). Furthermore, ROS can cause damage to the cells leading to cell death and formation of membrane-bound vesicles which support crystal nucleation (Khan et al., 2002; Talham et al., 2006). With this background, hydronephrosis with major changes such as fibrosis in the tubulo-interstitial compartment has been reported in MSG-treated rat kidneys by Sharma et al. (2013).
The mechanical disturbance resulting from complete ureteral obstruction causes tubular injury, resulting in a pro-inflammatory cytokines and tubulo-interstitial fibrosis (Ricardo and Diamond, 1998). Accordingly, in an experiment with a ureteral obstructed rat model, the investigators found increased 4-hydroxynoneal (4-HNE) stain for ROS products in the renal tubulo-interstitial compartment (Yeh et al., 2011). It can therefore be surmised that urolithiasis and oxidative stress due to MSG can cause fibrosis in the kidney, as ROS can induce the transformation of fibroblasts to myofibroblast (Sampson et al., 2011). Tubular interstitial fibrosis is highly associated with the progress of renal diseases (Barnes and Gorin, 2011).
1.2.4 MSG-induced ROS generation in kidney
The possible mechanisms of MSG-induced ROS production in the kidney are illustrated in Fig. 2. ROS arises as a by-product of aerobic metabolism (Coyle, 1993). The main sites of ROS production are the mitochondrial electron transport system, peroxisomal fatty acid, cytochrome P-450, and phagocytic cells (Beckman and Ames, 1998; Aguilaniu et al., 2003). One study suggested that the mitochondrial electron transport chain is a major source of ROS in oxidative glutamate toxicity (Tan et al., 1998) and that extracellular glutamate level increases the formation of hydroxyl radicals (Yang et al., 1995). Most cellular ROS arise due to leakage of electrons from the mitochondrial respiratory chain. In normal physiological conditions, ROS produced as a byproduct of metabolic processes are completely inactivated by cellular and extracellular defense mechanisms. Nutrient metabolism can affect the production of oxidative stress in the kidney by altering energy metabolism. In this scenario, α-ketoglutarate dehydrogenase (α -KGDH) is the primary site of the control of the metabolic flux through the Krebs cycle.
Fig. 2 A proposed model of MSG-induced ROS production in the rat kidney. Glutamate upon chronic MSG exposure may raise the activity of α-ketoglutarate dehydrogenase, a potential ROS generator. Additionally, an increased intracellular calcium level via glutamate receptors can stimulate free radical generation and lipid peroxidation. Inhibition of cystine uptake leads to decreased GSH levels that may further promote ROS-mediated renal cell damage
1.2.5 α-Ketoglutarate dehydrogenase: an ROS generator
High glutamate concentration may increase the mitochondrial proton gradient as a result of the over production of the electron donor by the Krebs cycle, which may in turn increase the production of mitochondrial superoxide. This proposed mechanism is supported by evidence from brain tissues where α-KGDH is a potential site of ROS generation against glutamate (Zundorf et al., 2009). The E3 subunit (lipoamide dehydrogenase) of α-KGDH can activate oxygen, resulting in the production of superoxide and/or hydrogen peroxide (Massey, 1994; Starkov et al., 2004; Tretter and Adam-Vizi, 2005). α-KGDH is a key and arguably the rate-limiting enzyme in the Krebs cycle. The enzyme is inhibited by its own product, succinyl-CoA, or by a high NADH/NAD+ ratio, as well as by a high dihydrolipoate/lipoate ratio, thereby playing an important role in cellular redox regulation (Bunik, 2003). However, an increased level of succinyl CoA ligase in the MSG-treated kidney tissue (Sharma et al., 2014) may favor the activation of α-KGDH by consuming succinyl CoA, an inhibitor. In addition, during the oxidative stress a segment of the Krebs cycle is maintained by glutamate through α-ketoglutarate (Yudkoff et al., 1994).
1.2.6 Glutamate receptors
Most studies in the literature link oxidative stress and tissue damage through glutamate receptor (N-methyl –Daspartate, NMDA) via calcium (Ca2+) in MSG-induced renal toxicity. There are two categories of receptors available to glutamate: ionotropic and metabotropic receptors (Willard and Koochekpour, 2013). Nearly all of the known glutamate receptors and many of their interacting proteins have been detected in the kidney (Rastaldi et al., 2006; Puliti et al., 2010; Gu et al., 2012). Most of the functional studies of the kidney have examined NMDA receptors, a subtype of ionotropic receptor, and group 1 metabotropic glutamate receptors (mGluRs).
NMDA receptors are Ca2+ favoring glutamate gated ion channels, whereas mGluRs are coupled to G protein cascades (Aramori and Nakanishi, 1992). The functional significance of these receptors for normal kidney physiology is not well understood. But, increased NMDA receptor subunit NR1 and NR2C expression correlates with the renal damage in a rat model of gentamicin nephrotoxicity (Leung et al., 2004). Furthermore, a study applying NMDA receptor agonists (glycine, glutamate) and antagonists (MK 801, CPP) in renal culture cells has demonstrated that an excessive stimulation or blockade of the renal NMDA receptor results in cell death (Leung et al., 2008). Sustained activation of these receptors induces changes in cellular Ca2+ dynamics that can trigger numerous cellular reactions, including the activation of nitric oxide synthase and protein kinase C (Said et al., 1996; Lan et al., 2001). These in turn can activate free radical generation and lipid peroxidation (Baba et al., 1994), leading to cell damage. This mechanism of excitotoxicity has been described not only in neurons but also in lung (Said et al., 1996). However, there is no direct evidence in the literature of studies investigating the role of glutamate receptors against MSG-induced renal cell damage; experiments with the blockade of NMDA receptor to prevent MSG-induced toxicity could be conclusive.
1.2.7 Cystine-glutamate antiporter
The cystine-glutamate antiporter, designated as system xc; exchanges extracellular cystine for intracellular glutamate in a variety of cells (Bridges et al., 2012). The uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. Under the condition of oxidative stress, the transport activity of this carrier appears to be up-regulated (Miura et al., 1992; Kim et al., 2001).
Considering the fact that the system xc is strongly expressed in the kidney (Burdo et al., 2006) and the decreased GSH levels are prominent in MSG-induced renal toxicity, our group investigated the expression level of system xc in acute and chronic MSG-treated kidney. However, there are other minor transporters for cystine intake into the cell as well.
This research seeks to ascertain the functional status of tissues of alloxan-induced diabetes mellitus rats treated with monosodium glutamate/ascorbic acid (200mg/kg and 400mg/kg).