DN occurs ultimately between 20-40% of patients with diabetes and it is one of the main causes of renal failure requiring dialysis and deaths because of microvascular diabetic complications . Endothelial dysfunction, tubular fibrosis, glomerular hypertrophy, podocyte injury, and progressive proteinuria are the hallmarks of the disease [2,3]. DN has five different phases, as early hypertrophy-hyper function stage, glomerular lesions without clinical disease stage, microalbuminuria stage, overt DN stage and terminal stage of renal failure . Despite treatment, the number of people with diabetic kidney disease continues to increase. Physiopathological mechanisms of DN is not fully understood and the reason why not all diabetic patients develop this complication is unknown.
The underlying mechanisms which contribute to the evolution of DN are extremely complex, and metabolic, genetic, and several hemodynamic factors have been identified . Among the metabolic factors, ROS are thought to play an important role in the development of DN . ROS are cytotoxic to renal cells and lead to activation of various pathways leading to glomerular hypertrophy, decline in glomerular filtration rate resulting in nephropathy [7,8]. Studies in rat models have shown that the increase in free oxygen radical production due to oxidative stress may be responsible for the development of DN [9,10]. The ROS producing sources contribute to diabetic complications remains fully understood. Mitochondrial oxidative enzymes are major intracellular ROS generating sources . MAOs are a family of enzymes that catalyze the oxidation of monoamines and located bound to the outer membrane of mitochondria in most cell types in the body. Oxidative deamination of monoamines by MAOs result in formation of ROS which might cause diabetic nephropathy contributing to unbalanced kidney redox state [12-17].
Recently, it has been suggested that the increase in angiotensin II activity is responsible for the pathogenesis of DN. Angiotensin-II mediates the constriction of the efferent artery, enhances glomerular pressures, induces hypertrophy and increases tissue oxidative stress. In addition, Angiotensin II increases MAO activity in rat kidneys by activating AT1 receptor. Diabetic animal models, clinical trials, and meta-analysis have clearly demonstrated the effectiveness of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers to improve glomerular and tubulointerstitial damage, reduce proteinuria, and decrease progression of chronic kidney disease [18-21]. According to the above description, MAO levels might be found increased in DN and this state might contribute to reducing the natriuretic effect of dopamine and to unbalanced cell redox state. However, the pathophysiological mechanisms associated with angiotensin II in DN are not fully understood. Studies show that the pathological process of DN is irreversible and resistant to clinical treatment. Therefore, it is necessary to identify new therapeutic drugs to delay the development and progression of DN. Is the increased renal MAO-A and/or MAO-B activity can be responsible for the pathogenesis of DN? One of the aims of this study is to shed light on the answer to this question with current knowledge about the pathogenesis of DN, and the other aim is to help develop new treatment strategies to prevent the genesis and progression of nephropathy in diabetics.
Material and Method
Male Wistar albino rats with body weights ranging from 220-250 g were fed with commercial rodent food, water, and ad libitum diet, under standard laboratory conditions with seven animals per cage. Animals were acclimated to the laboratory for 1 week prior to the start of theexperiment. The temperature in the cages was 22 ± 2°C, and the relative humidity was 50-60%. The ethical approval for the study was obtained from Dicle University Institute of Animal Experiments of Ethics Committee (Diyarbakir, Turkey), which is engaged in animal protection (Approval no: 2017/18).
Diabetic rat model
STZ dissolved in citrate buffer was injected to induce experimental diabetes in a single dose of 60 mg/kg into the peritoneal cavity of rats that were fasted for 12 hours. In order to prevent fatal hypoglycemia, which occurred 6 hours later in STZ injected rats, the animals were fed orally with 10% glucose solution for one day. Tail blood samples were obtained 72 hours after streptozotocin injection and the blood glucose concentration was determined by the glucometer (Abbott Optium Xceed) to make sure the formation of diabetes. It was decided that animals with a blood glucose of 300 mg/dl or higher were diabetic and were included in the study.
Rats were placed in their cages as four different groups, each containing seven animals. Group 1 served as control rats. Group 2 served as diabetic rats. Group 3 served as diabetic rats with daily treatment of moclobmide (5 mg/kg/day) orally for 56 days. Group 4 served as diabetic rats with daily administration of rasagiline (10 mg/kg/day) orally for 56 days. Feed and water consumption, body weight, blood glucose, and physical conditions of the animals were monitored weekly from the beginning to the end of the experiment. The dose of moclobemide and rasagiline was adjusted throughout the experiment period according to body weight changes each week to maintain a similar dose per kg body weight of the animal. Animals were housed in metabolic cages a day before the end of the study to collect samples of 24-h urine for measuring protein, NAG and GGT levels. On the last day of the study, animals were anesthetized with ketamine (80 mg/kg; i.p.) after 12 hours fasting and sacrificed by cardiac puncture. Kidneys were removed, washed immediately with ice-cold saline, and stored at -80 °C until the parameters were measured.
The kidneys were homogenized in ice-cold Tris-HCL buffer (0,1 M; pH:7,4) using a teflon homogenizer and centrifuged at 4°C (12000g x 30 min.) to collect the supernatant. Supernatants were used to determine intrarenal dopamine levels, oxidant enzymes, MAO-A and MAO-B activity, and antioxidant enzymes, SOD and CAT activities. The protein content in renal homogenate samples was determined by relevant extra sensitive protein Assay Kit.
Antioxidant and oxidant enzyme activities and lipid peroxidation
CAT activity of the kidney was measured in the supernatants of homogenates by an enzymatic method as described by Aebi . Renal SOD activity was determined in the supernatant via a photometric method described by Sun et al. is based on the capacity of SOD to inhibit reduction of nitro blue tetrazolium . Lipid peroxidation was determined by measuring the level of MDA in kidney homogenate using an assay kit provided by Sigma–Aldrich. MAO-A and MAO-B activities in kidney homogenates as intracellular oxidant enzymes responsible for dopamine catabolism were measured using the rats-specific ELISA Kit provided by LifeSpan BioSciences, Inc. North America.
The levels of creatinine in serum and urine samples were determined by using an available colorimetric kit from Cayman Chemical Company (Michigan, USA). Serum and urine glucose levels were measured by the Rat Glucose Test Kit produced by the Crystal Chemical Company. Urinary pathologic enzymes activities in control and diabetic groups such as renal damage markers, GGT and NAG, were evaluated by Colorimetric Assay Kits and the results of the test were expressed as units.
The results obtained from seven rats in each group of this study were expressed as mean ± SEM. For statistical examination, SPSS version 20.0 for Windows statistical package program was used. The Kruskal Wallis Analysis of Variance test was used for statistical evaluation. The paired comparison of the differences between the parameters was evaluated with the nonparametric Mann-Whitney U test. In addition, Spearman correlation test was used to clarify possible relationships between parameters. Values were regarded as statistically significant when p< 0.05.
Metabolic characteristics of the animals
Three days after STZ injection, rats with a significant increase in fasting blood glucose levels, water, and food consumption, and urine volume were considered to be diabetic animals and included in the study. In DN-group rats, body weight significantly reduced with respect to the initial body weight three weeks after STZ injection, and gradually decreased further in the following weeks till the end of study (Table 1). Treatment with moclobemide or rasagiline of diabetic rats prevented significant loss of weight compared to DN group.
Parameters related to kidney function
The level of serum creatinine and BUN in DN group rats showed a significant increase compared to the normal control because of reduced greatly excretion of urinary creatinine and urea. Treatment with moclobemide or rasagiline led to increased urinary creatinine and urea excretion in diabetic rats, thus resulting in normalization of serum creatinine and BUN concentration (Table 2). As shown in Table 2, urinary glucose loss and proteinuria showed a significant increase in diabetic rats with respect to the normal control group. The increase in blood glucose level, urine volum, urinary glucose loss and proteinuria due to diabetes, was significantly prevented by treatment with moclobemide or rasagiline, although the effect of rasagiline was less according to moclobemide. Urinary excretion of GGT and NAG has been used for a long time as a valid indicator of renal damage in nephropathy studies on human beings, laboratory rodents, and dogs. GGT and NAG levels were significantly higher in urine samples of untreated diabetic rats than in normal control values. MAO inhibitors, moclobemide, and rasagiline significantly reduced GGT and NAG urinary levels of diabetic rats. However, the effect of moclobemide was more than rasagiline (Table 2)
Renal antioxidant enzyme activities and lipid peroxidation
Renal SOD and CAT activity in DN rats was found significantly higher than normal control rats. Treatment with moclobemide or rasagiline for 8 weeks significantly reduced both SOD and CAT activity, thusboth antioxidant enzyme levels were close to normal control values with MAO inhibitors. MDA level increased significantly as an indicator of oxidative stress and lipid peroxidation in the kidney of diabetic rats. MAO-A or MAO-B inhibitors, moclobemide or rasagiline, significantly prevented the increase in lipid peroxidation in kidneys induced by diabetes (Table 3).
Renal MAO-A and MAO-B activity
Both MAO-A and MAO-B activity in diabetic rats were found to be significantly higher than the normal control group. The high MAO-A activity in diabetic rats kidney was reduced by moclobemide treatment, and similarly, high MAO-B activity decreased to normal levels with rasagiline treatment (Table 3).
Correlation Between Renal Function Parameters and Oxidant and Antioxidant Enzyme Activity and Lipid Peroxidation
As shown in Figures 1, 2, 3 and 4, there is a relationship between oxidant and antioxidant enzyme activities and renal function in diabetic rats. We found a positive correlation between SOD and urine GGT, CAT and urinary GGT, MAO-A and urinary GGT in kidneys of DN rats (Figure 1,2,3). In addition, renal MAO-A activity showed a linear correlation with proteinuria (Figure 4).
DN is the main cause of morbidity and mortality as one of the serious complications of diabetes leading to end-stage renal failure. Multiple pathophysiological mechanisms that contribute to the development of DN are quite complex. In recent years, experimental evidences have shown that reactive free oxygen radicals may be responsible for the pathogenesis of DN [1-7]. Free reactive oxygen species are produced continuously under physiological and pathological conditions as a result of metabolism and are balanced by the body’s antioxidant defense systems. If the amount of oxygen radicals produced exceeds the capacity of the defense system or the strength of antioxidant mechanisms decreases, oxidative stress can lead to tissue damage. It is believed that mitochondrial free oxygen radical production is increased in diabetics and this condition plays an important role in the formation of nephropathy [8-10]. In the present study, we determined that oxidative stress indicators such as MAO-A, MAO-B, SOD and CAT activities and MDA level were significantly increased in kidney tissues of DN rats. In addition, we found a positive correlation between nephropathy symptoms and renal MAO-A and antioxidant enzyme activities. Treatment with MAO inhibitors, moclobemide or rasagiline significantly prevented nephropathy symptoms in diabetic rats. Therefore, it was concluded that oxidative stress increased in kidney tissues of diabetic rats and this adverse condition may cause nephropathy. However, antioxidant agents were not effective sufficiently to meet the expectations for the prevention or treatment of nephropathy in diabetic patients.
One of the most investigated subjects related to the pathophysiology of DN is the renin-angiotensin system. Regardless of their hemodynamic effects, it has been reported that Ang II contributes to the development and progression of renal damage through the AT1 receptor in diabetic patients [17-20]. The activity of monoamine oxidases, especially MAO-A, is increased by Angiotensin II through the AT1 receptor in kidneys . On the other hand, MAO activity also reduces the availability of dopamine. Therefore, the reduction of intrarenal dopamine activity by the renin-angiotensin system suggests that it may be one of the pathophysiological mechanisms of DN in diabetics. In fact, increased renal MAO-A activity and nephropathy findings in diabetic rats treated with AT1 receptor antagonist losartan have returned to normal .
Dopamine is mainly produced by proximal tubule cells in the kidneys independently of the nerves and regulates renal functions such as sodium excretion and glomerular filtration . Another important effect of dopamine is activation of antioxidant enzymes while inhibiting pro-oxidant enzymes and thus to reduce oxidative stress . Studies based on the renoprotective effects of intrarenal dopamine have shown that when dopamine levels in the kidneys are raised, nephropathy can be prevented very effectively. According to Mikusic et al., the renal dopaminergic system prevents the development of hypertension and renal inflammation in the experimental metabolic syndrome . However, the studies have shown that intrarenal dopamine activity is low and this state may be an important physiopathological mechanism responsible for abnormal renal functions in STZ-induced diabetic animals [27,28,29]. We found renal dopamine levels in diabetic rats were significantly lower than the control values. In diabetic rats treated with MAO inhibitors, intrarenal dopamine levels were elevated, nephropathy findings were significantly eliminated and renal functions improved significantly. In addition, treatment with MOA inhibitors diminished lipid peroxidation in kidney tissue of diabetic rats. However, we have found that to prevent the development or progression of nephropathy in diabetic rats by MAO-A inhibition is more effective than MAO-B inhibition.
The mitochondrial MAOs responsible for the catabolism of monamines produce reactive oxygen species in the kidneys. Experimental evidences have suggested that MAOs may be one of the important factors in many chronic pathological conditions such as DN. In this study, we found that the activity of MAO-A and MAO-B was significantly increased in the kidney of DN rats and there was a correlation between MAO-A and nephropathy findings such as high urinary GGT and proteinuria. We also demonstrated that nephropathy findings reduced significantly in diabetic rats treated with MAO-inhibitors, moclobemide or rasagiline. These results support data from studies that directly or indirectly show that long-term high levels of renal monoamine oxidases are associated with the pathogenesis of DN.
In conclusion, antioxidant enzyme activities are increased as a compensatory mechanism, against to lipid peroxidation and increasing MAOs activity in kidney tissue of DN rats. However, increased antioxidant enzymes SOD and CAT in the kidney of diabetic rats were not sufficient to prevent the development and progression of DN. MAO inhibitors such as moclobemide and rasagiline can significantly reduce the severity of nephropathy symptoms in diabetic rats. Inhibition of MAO-A in diabetic rats shows more renoprotective effect than MAO-B inhibition. Therefore, MAO-A inhibition may be added to new treatment protocols to prevent nephropathy in diabetic patients.
Scientiﬁc Responsibility Statement
The authors declare that they are responsible for the article’s scientiﬁc content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientiﬁc review of the contents and approval of the ﬁnal version of the article.
Animal and human rights statement
All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.
Conflict of interest
None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.
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