In the present study, STZ-injected rats demonstrated typical characteristics of diabetes mellitus such as hyperglycaemia, polyuria, and growth retardation. Also, there is an evident increase in the urine albumin excretion of diabetic rats compared to control rats. This is in agreement with other reports [28,29] that show a significant increase in oxidative stress that can enhance kidney damage in rats and lead to a progressive increase in albumin excretion in urine. The results of this investigation also revealed that the mean Zn and Mg levels in the renal tissues of diabetic rats were considerably lower than those in the control group. The mean levels of Fe and Cu were found significantly higher in the kidneys of the diabetic rats, in comparison to the control rats, in agreement with our previous [30] results and those of Greń et al.[31].
In the present study, however, the levels of Zn and Mg were also found significantly higher, while the level of Cu was significantly lower in urinary excretion with STZ-diabetic rats. We were not able to measure the iron values of the rats' urine due to limited amounts of the collected urine.
Impaired metabolism of trace elements is observed in diabetic patients. It has been reported that the urinary excretion of calcium, zinc and magnesium is increased in two types of diabetes mellitus, causing a decrease in blood levels of these elements from these patients [30,32,33].
We also found a significant increased oxidative stress in kidneys of STZ-diabetic rats as compared to the control rats. STZ treatment caused many changes in renal antioxidant levels, including an increase in MDA, a reduction in GSH, and antioxidant enzymes like SOD, which represent the existence of excess peroxides and hydroxyl radicals in diabetic kidneys and are responsible for impaired renal function.
The increased lipid peroxidation and reduced activity of antioxidative enzymes in the kidneys of STZ-diabetic rats revealed in this study match previous findings and support the hypothesis that free radicals play a substantial role in the pathophysiology of diabetic nephropathy. [3,34].The decrease of antioxidants may be due to the deficiency of kidneys zinc and magnesium levels in diabetic rats. These observations are supported by the findings that Zn and Mg have antioxidant activities because not only do they constitute the active sites and/or stabilize the conformation of several antioxidant enzymes, but they also compete for iron- and copper-binding sites and can provide protection against transition metal-mediated and free radical-induced injury[35]. Previous researchers have suggested an interrelationship between diabetes and various micronutrients, including Mg, Cr, Fe, Zn, Cu, etc. Moreover, several studies have shown an increased lipid peroxidation in clinical and experimental diabetes [7,36, 37]. Our previous study results show that some elements’ impaired ion metabolism may have a contributing role in the progression of diabetic oxidative complications [30]. Critical elements are essential components of various important enzymes in intracellular antioxidant defense and may have protective or scavenging effects. Their lack or excess may lead to a shift in the pro-oxidant/antioxidant balance, resulting in the emergence of additional problems as the illness progresses. [38].
Hyperzincuria and poor intestinal zinc absorption in diabetic patients indicate that they are more prone to zinc insufficiency, which might be caused by hyperglycemia, reduced intestinal absorption, or increased urine Zn loss. [30, 39]. Increased intracellular oxidants and free radicals, as well as deficits in intracellular zinc and zinc-dependent antioxidant enzymes, may be linked to certain diabetic problems. [40]. Zinc reduces the severity of diabetes-related problems in a variety of animal models. It also plays a crucial role in controlling the function of the Langerhans islets [41] and their protective impact against diabetic kidney tissue damage by stimulating metallothionein production and regulating oxidative stress[42].
Magnesium is a cofactor of several enzymes involved in carbohydrate oxidation and plays a vital role in the cell membrane's glucose transport mechanism. Magnesium salts have been shown to be effective in the treatment of non-insulin-dependent diabetes. [43]. Prabodh et al.[44] stated that Mg had a significant negative relationship with diabetic nephropathy cases.
Changes in the concentrations of metals in the body occur as a result of the action of reactive oxygen species, but on the other hand, some metals may induce free radical formation which can take part in diabetes development [45]. The transition metals Fe and Cu are integral parts of important enzymes involved in vital biologic processes. There are suggestive evidences that Fe plays a pathogenic role in diabetes and its complications such as atherosclerosis. Excess Fe has been implicated in the pathogenesis of diabetes and its complications [46]. The increased levels of Cu, a transition metal that is redox-active and catalyzes lipid peroxidation, may enhance oxidation of low-density lipoproteins, causing increased level of TBARS in diabetic patients as reported by Heinecke et al.[47].
In the excess of certain metals (particularly Fe or Cu ions), a hydroxyl radical, which is the most powerful ROS, can be produced via the Fenton or the metal-catalyzed Haber-Weiss reaction [48]. These two chemical reactions appear to account for most of the hydroxyl radical production in biological systems and explain, at least in part, why metals such as Fe and Cu produce oxidative stress and ROS-induced injury in cells. Tissue accumulation of transition metal ions is extremely toxic, leading to many pathologic conditions consistent with oxidative damage to biologic membranes and molecules [49].
Angiotensin-converting enzyme inhibitors are a type of drug that has been shown to protect the kidneys. They may decrease the advancement of diabetic nephropathy by regulating hemodynamics in the renal glomerulus, reducing proteinuria, and slowing the course of diabetic nephropathy. Similarly, despite the absence of the bradykinin impact, AT1 receptor antagonists have the similar effect by decreasing proteinuria and delaying the course of diabetic nephropathy. [19,21,22]. However, the mechanism of their actions has not been fully elucidated. Because these drugs are commonly used for the treatment of diabetic nephropathy, their effect on Zn, Mg, Cu and Fe concentration may be additive, resulting in a more pronounced concentration of these elements in the diabetic rat tissues.
To the best of our knowledge, no prospective studies have investigated the effect of treatment with the ACEIs and ARBs on Zn, Mg, Cu and Fe concentrations in the kidney tissues of STZ-diabetics.
In this prospective study, we examined the hypothesis that irbesartan, an AT1 receptor blocker and perindopril, an ACE inhibitor, may have an effect on element metabolism by studying the effects of these drugs on Zn, Mg, Cu and Fe levels in the kidney tissues of STZ-diabetic rats. We have applied the 15 mg/kg dose of irbesartan which is an AT1 receptor antagonist because it is shown in the other studies [17,50] that this amount of dose reduces systemic blood pressure, regulates the changes in renal hemodynamics and decreases intraglomerular pressure. On the other hand, we have applied the 6 mg/kg dose of perindopril, an ACEI, in other STZ-diabetic group. Kelly et.al. [51] and Yao et.al. [52] shown that, in this dose ranges, perindopril was reduced systemic blood pressure, decreasing the urinary albumin excretion rate and protecting the renal function in normotensive early diabetic nephropathy and diabetic rats.
Our data show that there are statistically significantly modifications regarding kidney tissues and urine Zn, Mg, Cu and Fe concentrations.
In the current study, the concentrations of Zn and Mg in the kidneys of the diabetic rats treated with perindopril and irbesartan were found significantly higher than in the untreated STZ-diabetic rats, while Cu and Fe concentrations were significantly lower in treated rats in comparison with all the observed tissues of the untreated STZ-diabetic rats. The levels of Zn and Mg were also found significantly lower, while the level of Cu was significantly higher in urinary excretion with perindopril and irbesartan treatment.
The loss of these minerals might be attributed to the impaired absorption and/or the excess excretion of these metals in urine in diabetic patients, which may induce a deficiency or marginal state of these minerals in the blood of diabetic patients [53]. We suppose that the increased kidney tissue levels of Zn and Mg may be due to the decrease in urinary excess by these drugs as well as a possible action of this on the renal cation transporters. Tubek [54] showed that the excretion of Zn via urine is normalzed in primary arterial hypertensive patients after a treatment with perindopril.
In our study, the effect of ACE inhibitor and of AT1 receptor blocker on the investigated parameters of oxidative stress in the diabetic kidney tissues were also studied. Our studies revealed that perindopril and irbesartan significantly decreased MDA content and increased SOD activities and GSH levels after 4 weeks of therapy.
These findings are in agreement with the results of Kedziora–Karnatowska [12] who showed that both enalapril and losartan decrease lipid peroxidation in the kidneys of diabetic rats during the early stages of the development of diabetic nephropathy. ACE inhibitors have been also shown to reduce lipid peroxidation in diabetic rat tissues [13] and enhance antioxidant defenses in mouse tissues [14]. Irbesartan administration attenuated the increased lipid peroxidation and decreased the antioxidant defence mechanism along with altering renal glomerular filtration rate. This is in line with other observations that chronic treatment with candesartan and losartan, AT1 receptor antagonists, can attenuate oxidative stress and alter renal function of experimentally induced diabetic animals [15]. Mastan et.al. [55] have reported that Angiotensin-II blockage by the ACEIs (i.e Captopril, Enalapril, Lisinopril) have been shown to increase total antioxidant status (TAS) and reduce total oxidative status (TOS) in the serum during the diabetes, especially in the captopril treated diabetic group. It is also reported that enalapril, an ACEI, has beneficial effects on the increased oxidative stress in the diabetic rats.To investigate the relationship between RAS an antioxidant defenses, Cavanagh et.al. [56] have been administered enalpril, an ACEI, to STZ-induced diabetic rats. The results of this study have shown that, in the heart, kidney and liver of enalpril-treated rats, total glutathione and the activities of antioxidant enzymes were higher than untreated diabetic rats.
As previously described, STZ rats' kidneys displayed morphological alterations such as glomerular basal membrane thickening, hypertrophic glomeruli, tubular degeneration, and dilatations. [57].The results revealed that diabetic animals treated with irbesartan and perindropril showed a significant improvement in renal function coupled with attenuation of increased renal oxidative stress and morphological derangement. It has been reported in another study that treatment with irbesartan can prevent the changed morphology in diabetic kidneys [18, 58]. This finding is consistent with previous research showing that the ACE inhibitor enalapril and the AT1 receptor blocker valsartan can prevent renal impairment and reduce oxidative stress in diabetics. [59,60].
The data of our present study confirms the role of oxidative stress in the formation of diabetic nephropathy in the first stage of diabetes development and indicates the possible antioxidant mechanism of the nephroprotective effect of both the angiotensin receptor antagonists and the angiotensin-converting enzyme inhibitors.
In conclusion, the results of the present study indicate an imbalance in amounts of some elements including Cu, Fe, Zn and Mg in kidney tissues in STZ-diabetic rats. In addition, oxidative stress parameters like the MDA level are found higher in kidney tissues, while the activity of antioxidant enzyme SOD and non-enzymatic antioxidant GSH levels are reduced in the tissues. The disturbed oxidative balance in tissues might be affected by the increases in Fe and Cu as well as the decreases in Zn and Mg levels in kidneys of STZ-induced diabetic rats. Therefore all these findings may contribute to explain the role of impaired ion metabolism of some elements in the progression of diabetic oxidative complications.
In the present study, we demonstrated in an experimental diabetic animal model that treatment with both irbesartan, an AT1 receptor blocker and perindopril, an ACE inhibitor, effectively regulated the level of these elements (Zn, Mg, Cu and Fe) and ameliorated the antioxidant levels in the kidney, thus resulting in retardation of the progression of diabetic nephropathy. Finally, the present results suggest that modulation of the same elements (Zn, Mg, Cu and Fe) either by ACE inhibition or by AT1 receptor blockage may be beneficial in reducing lipid peroxidation and increasing antioxidants levels in diabetic rat kidneys.