1. Introduction
Chronic kidney disease (CKD) refers to a group of chronic progressive diseases that are characterized by increasing prevalence rates (currently about 14%) and high mortality that poses serious global threats to human life and health [
1]. CKD can progress to end stage renal disease (ESRD) that requires expensive renal transplantation or dialysis. There are currently no specific treatments that target the etiologies of CKD, and therefore there is still a pressing need to search for new agents for this disease, including novel and old repurposed drugs [
2,
3,
4].
The adenine-induced CKD model in rats and mice is a commonly used method for creating a metabolic abnormality that closely mimics the disease in humans. In this model, adenine is given to these animals in the feed at different concentrations for variable periods [
5,
6]. The excretion of nitrogenous compounds in the adenine-treated animals is diminished by renal tubular occlusion due to the formation of 2,8-dihydroxyadenine crystals, leading to accumulation of various guanidino compounds (such as methylguanidine and guanidinosuric acid) and urea nitrogen in blood [
7]. Adenine-induced CKD causes inflammation, oxidative, and nitrosative stress, and alters the composition of the microbiota [
8,
9,
10]. Several drugs and dietary supplements have been tested in this model for their efficacy against experimental CKD [
11,
12].
We have previously reported that streptozotocin (STZ)-induced diabetes worsens most of renal function tests in rats. Administration of adenine (to induce CKD) in STZ-diabetic rats aggravates further the renal damage induced by adenine alone [
13]. Diabetes is also known to worsen CKD in humans [
14].
The biguanide metformin is currently the first-line oral drug for treating type-2 diabetes mellitus [
15]. It produces its antidiabetic action through several mechanisms [
16,
17]. Metformin has also been reported to prevent fibrosis in several organs, including the kidneys [
18,
19]. In addition to treatment of diabetes, the drug has also been reported to be beneficial in treating several other diseases and conditions that include lung and breast cancers [
20,
21], inflammatory skin disorders [
22], and neurological diseases [
23].
In the present work, we have tested the possible ameliorative effects of metformin on adenine-induced CKD in diabetic and non-diabetic rats. While this work was being prepared for publication, a paper on the effect of metformin on the surgical (subtotal nephrectomy) model of CKD has been reported in non-diabetic rats [
24]. However, as far as we are aware, the effect of metformin has not been tested before in the adenine model of CKD in diabetic and non-diabetic rats.
2. Materials and Methods
2.1. Animals
Wistar rats (190–200 g) were obtained from the Small Animal House of the Sultan Qaboos University and were housed in a room with controlled environment (a temperature of 22 ± 2 °C, relative humidity of about 60%, with a 12 h light–dark cycle), and were provided ad libitium with additive-free standard diet (Oman Flour Mills, Muscat, Oman), and tap water.
2.2. Induction of Diabetes
Rats were rendered diabetic by an intraperitoneal (i.p.) injection of STZ (55 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5). Other animal groups were injected with citrate buffer. Seventy-two hours after STZ injection, a drop of blood was taken from the tail vein and fasting blood glucose level was measured using a blood glucose monitoring system (One Touch® UltraMini®, Life Scan, Inc., Milpitas, CA, USA). Rats with blood glucose level >20 mmol/L were considered diabetic. Treatments (for 35 days) were started three weeks after STZ injection.
2.3. Experimental Design
The animals (n = 48) were randomly distributed into eight equal groups and treated as follows for 35 consecutive days:
Control (CON) group continued to receive the same diet without treatment until the end of the study.
Adenine (A) group was switched to a powdered diet containing adenine (0.25% w/w in feed given daily).
Diabetes (STZ) group was induced by injecting the rats i.p. with STZ, as described above.
Adenine + Diabetes (A + STZ) group was treated with adenine and STZ, as mentioned in the second and third groups.
Metformin (MF) group was treated daily by oral gavage with metformin (200 mg/kg/day) dissolved in distilled water.
Adenine + Metformin (A + MF) group was treated with adenine and metformin as mentioned in the second and fifth groups.
Diabetes + Metformin (STZ + MF) group was treated with STZ and metformin, as mentioned in the third and fifth groups.
Adenine + Diabetes + Metformin (A + STZ + MF) group was treated with adenine, STZ and metformin, as mentioned in second, third and fifth groups, respectively.
One day before the rats were sacrificed, urine of each rat was collected over a 24-h period, and its volume measured. Immediately after the end of the treatment period, rats were anesthetized with a combination of ketamine (75 mg/kg) and xylazine (5 mg/kg) given i.p. injection. Blood was then collected from the inferior vena cava in heparinized tubes and centrifuged at 900× g for 15 min, at 4 °C to separate plasma. The plasma harvested was stored frozen at −80 °C pending biochemical analyses within 10 to 21 days. The rats were then sacrificed by an overdose of anesthesia. The kidneys were removed from the rats, washed with ice-cold saline, blotted with a piece of filter paper, and weighed. A small piece from the right kidney was fixed in 10% buffered formalin pending histological analysis. The remainder of the right and left kidneys were individually wrapped in aluminum foil and then dipped in liquid nitrogen and stored at −80 °C, pending analysis within about one to three weeks.
2.4. Drugs, Chemicals, and Biochemical Analysis
Adenine and STZ were obtained from Sigma (St. Louis, MO, USA). Metformin was a gift from the National Pharmaceutical Industries (Muscat, Oman). The rest of the chemicals were of the highest purity grade available. Urea, uric acid, calcium, phosphorus, and albumin were measured using an automated biochemical analyzer, Mindray BS-120 chemistry analyzer, from Shenzhen Mindray Bio-Medical Electronics Co. (Shenzhen, China). Creatinine, Superoxide dismutase (SOD), glutathione reductase (GR), total antioxidant capacity (TAC), and N-acetyl-β-D-glucosaminidase (NAG) were measured by colorimetric method, using BioVision kit (Milpitas, CA, USA). Indoxyl sulfate, 8- isoprostane and nuclear factor erythroid 2-related factor 2 (Nrf2) were measured using ELISA kits from MyBioSource, Inc. (San Diego, CA, USA). NF-κB (Nuclear Factor-kappa B), 8-hydroxy-2-deoxy guanosine (8-OHdG) and transforming growth factor (TGF-β1) were measured using ELISA kit from Cusabio Biotech Co. Ltd. (Wuhan, Hubei, China). The ELISA kits for measuring cystatin C, interleukin-1β (IL-1β), adiponectin, interleukin-6 (IL-6), and neutrophil gelatinase-associated lipocalin (NGAL) were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Interleukin-10 (IL-10) was measured using ELISA kits from Abcam (Cambridge, UK). Osmolality was measured by the freezing point depression method using the Osmomat 3000 osmometer (Gonotec GmbH, Berlin, Germany).
2.5. Histopathological Analysis
Formalin-fixed renal tissues were dehydrated, cleared in xylene and paraffin, and embedded using standard technique. Sections were cut by a rotary microtome at 4 μm thickness and were stained by hematoxylin and eosin (to evaluate acute tubular necrosis), Periodic Acid–Schiff (to assess glomerular integrity) and Picro-Sirius red (to assess interstitial fibrosis). Details of the techniques used were reported before (Ali et al., 2018). Sections were examined blindly under a light microscope by a histopathologist unaware of the treatments given. The percentage of renal tubular necrosis was scored by semi-quantitative method, as previously described by Ali et al., [
5], on a scale 0–4 as following; 0 = normal, no necrosis; 1 < 10%; 2 = 10–25%; 3 = 26–75%; 4 > 75%. Three 40X fields were evaluated from each kidney section of each animal of the 8 groups and the mean percentage was converted to the score value. Sirius red stained slides were analyzed following the procedure described by Manni et al. [
25]. The slides were examined by Olympus B51X microscope attached to Olympus DP70 camera, and images were acquired using the x40 objective lens. Three random images of the renal cortex were acquired from each kidney of each animal of the eight groups and stored as TIFF 24 bit RGB color image files. Image analysis was performed on the stored images using ImageJ
® image analysis software. Briefly, the images were converted into grey scale and the red stained collagen was isolated using the hue histogram filter available in “Threshold Color” followed by measuring the isolated area as a percentage. Fibrosis index (%) assessed the collagen content of the tissues [
26,
27], by calculating the ratio of the mean Sirius red-stained positive area to the mean whole area of each section for each animal.
2.6. Western Blotting
The activation of the signaling pathway of intracellular mitogen-activated protein kinase (MAPK) was assessed by western blotting as described before [
28].
2.7. Statistical Analysis
Data were given as mean ± SEM and were analyzed by one-way analysis of variance followed by Bonferroni’s multiple comparison test (GraphPad Prism version 5.03, San Diego, CA, USA); p < 0.05 was considered statistically significant.
4. Discussion
The present results showed that the rats that have been fed a diet mixed with adenine exhibited all the previously reported physiological, biochemical, histopathological, and molecular effects that occur in adenine-induced CKD [
5,
10]. The results also confirmed that STZ-induced diabetes aggravated these actions in rats with adenine-induced CKD [
13]. We have experimentally shown here that metformin treatment significantly mitigates the actions of CKD in diabetic and non-diabetic rats.
In the present work we have used metformin at a dose of 200 mg/kg/day, which is known to be a safe dose for rats and does not elevate lactate concentration in the blood [
29,
30]. Metformin at this dose did not cause any significant alterations in renal function or structure. Similar findings have been found by others before [
31].
Metformin significantly elevated urinary albumin/creatinine ratio in rats with adenine-induced CKD and STZ-induced rats. The mechanism of this action was not certain, but it is possibly related to change in glomerular filtration, decreased blood glucose levels, blood pressure, and degree of insulin resistance and/or renal proximal tubular impairment [
32]. NAG is a hydrolytic lysosomal enzyme, found predominantly in the proximal tubules [
33] and is considered one of the most important and commonly used indices of tubular damage, mainly because NAG assays are sensitive enough to allow dilution of the urine, thus overcoming any enzyme inhibition [
34,
35]. NAG activity in urine was significantly increased by adenine and STZ when given singly or together. In rats given adenine and STZ (singly or together), metformin treatment was effective in significantly mitigating the increased NAG activity. Urine osmolality was markedly and significantly decreased in rats with adenine-induced CKD, and this was mitigated by metformin. It has been shown that sustained high urine volume and low urinary osmolality are independent risk factors for quicker decline in glomerular filtration rate in patients with CKD [
36,
37]. Metformin significantly mitigated the actions of adenine and STZ, which is in line of the report of Efe et al. [
38].
Increased production of reactive oxygen species (ROS) and/or reduced antioxidant defense capacity), apoptosis, and inflammation are established to be involved in the pathogenesis of CKD in humans and experimental animals [
5,
39,
40,
41]. Imbalances in the ROS are known to be important drivers in the inflammatory process [
42]. These actions may be the underlying basis of the consequent cardiovascular and other health outcomes of CKD [
43,
44]. Treatment with adenine in this work caused the expected and previously reported actions on the biomarkers of oxidative stress and inflammation in plasma, urine, and kidney homogenates [
8,
45]. Our present results indicated that metformin treatment significantly increased the plasma concentration of the indices of the oxidative stress and reduced the pro-inflammatory mediators. These actions support the reports that metformin is a strong antioxidant and anti-inflammatory agent [
22,
45].
Notable among the biochemical findings in the present work is that adenine feeding significantly increased the key profibrotic growth factor TGF-β1 level in the kidneys. In a previous work, we have shown that TGF-β1 is also increased in other tissues, such as gastrointestinal mucosa [
9,
46]. This has also been reported by others [
47]. TGF-β1 signaling pathway is established to be activated in CKD and promote renal fibrosis [
48]. In this work, metformin significantly antagonized the increase in TGF-β1. This property of metformin was reported before [
49]. This action of metformin may explain the decreased level of renal fibrosis seen histopathologically in this work. Metformin direct renoprotective action, independent of its glucose lowering effect, has been reported before in other models of renal disease such as peri-renal adipose inflammation-induced renal dysfunction [
50], and unilateral ureteral obstruction [
51].
The transcription factor Nrf2, is known to regulate the expression of more than 200 cytoprotective genes encoding antioxidant proteins, induce antioxidant enzymes to respond to oxidative stress, and antagonize oxidative and inflammatory damage [
42,
52]. NF-
κB is a nuclear transcription factor that has a significant role in many pathophysiological processes that includes inflammation, oxidative stress, immune reaction, and apoptosis [
53]. In this work, renal Nrf2, measured by an ELISA method, was found to be significantly decreased in adenine-treated rats, and in rats with STZ-induced diabetes. Concomitant treatment with metformin in these latter groups was significantly effective in ameliorating the decrease in Nrf2 level. The opposite was found with NF-
κB level in all the experimental groups. This finding is in line with the results of indices of oxidative stress and inflammation in these groups.
In the present adenine model of CKD, metformin produced a similar ameliorative action as in the subtotal nephrectomy model of CKD [
24]. However, in this investigation we extended our study by including diabetic and non-diabetic rats. Further, we used here more traditional and novel biomarkers to assess the effect of metformin on adenine-induced CKD. The latter included indoxyl sulfate, which is a reliable uremic toxin, especially for early-stage CKD [
54], and 8-OHdG [
55].
It has recently been reported that in non-diabetic mice with partial nephrectomy-induced CKD, metformin protects against experimental acute kidney disease (AKI) but not against AKI-CKD progression [
56]. It was found in a retrospective analysis that type-2 diabetic patients with CKD stage 5 who took metformin were less likely to develop ESRD than those who do not use it [
57]. More recently, a systematic and meta-analysis concluded that taking metformin is associated with significantly less risks of all-cause mortality and cardiovascular events in type-2 diabetic patients with mild to moderate CKD [
12].
It is well known that inflammation and apoptosis are regulated by MAPK pathway. We assessed the role of metformin on MAPK activation by determining the phosphorylation of ERK1/2 at p44 and p42 sites. Adenine-induced CKD resulted in activation of MAPK treatment, and when it was given to animals with metformin, the phosphorylation of MAPK p44 in renal tissue was significantly mitigated. Thus, there was attenuation of apoptosis and inflammation in renal tissue of animals that were treated with metformin. Therefore, we concluded that metformin alleviated kidney inflammation and apoptosis protecting against adenine induced-CKD via inhibiting NF-κB p65 and MAPK signaling pathways, and that their increased levels are associated with injured kidneys [
58].
A limitation of this study is that it used one dose of metformin (based on previously-published literature). Using three or more doses would have provided useful dose response data for all the analytes measured here. However, this was not feasible due to technical and financial limitations. Notwithstanding this limitation, the study has shown, using various biochemical, histopathological, histochemical, and molecular techniques, that metformin, at the dose used, was effective in mitigating the actions of adenine-induced CKD in diabetic and non-diabetic rats.