Renoprotective Effects of Metformin and Its Relationship with Oxidative Stress in Type 2 Diabetic Mice

Background: Oxidative stress has previously been shown to play critical roles in the development of diabetes and its complications. The purpose of this research was to observe the reno-protective effect of metformin and its effect on oxidative stress in type 2 diabetic mice renal tissue. Methods: Type 2 diabetes mellitus mice model was established by High-fat feed combined with small-dose STZ and randomly divided into diabetes model group, Metformin [MET, 250mg/(kg.d)] group, Glibenclamide (GLIB) [GLIB, 2.5mg/(kg.d)] group, and normal control group (NC). After 8 weeks of intervention, blood and urine samples were collected for detection of FBG, HbA1c, urine albumin (Alb), retinol-binding protein (RBP), podocalyxin (PCX), 8-OHdG, 8-iso-PG, and creatinine (Cr). Renal tissue specimens were preserved for observing renal glomerular basement membrane thickness (GBMT) and foot process fusion rate (FPFR) under electron microscopy. Results: Compared with the NC group, FBG, HbA1c, urinary Alb/Cr (UACR), RBP/Cr (URCR), PCX/Cr (UPCR), 8-OHdG /Cr (UOHCR), and 8-iso-PG /Cr (UISOCR) signicantly increased in the T2DM group (P <0.05). Compared with the T2DM group, FBG, HbA1c, UACR, URCR, UPCR, UOHCR, and UISOCR were signicantly reduced in the GLIB group and MET group (P <0.05). Compared to the GLIB group, UACR, URCR, UPCR, UOHCR, and UISOCR decreased in the MET group (P <0.05), but FBG and HbA1c were not differenced statistically between the two groups. GBMT and FPFR increased in the T2DM group (P <0.05), which were reduced in the MET group and lighter than those in the GLIB group (P <0.05). Conclusion: Metformin intervention can play a reno-protective effect in type 2 diabetic mice, which may be related to its effect in inhibiting enhanced oxidative stress in vivo. for Alb, RBP, PCX, 8-OHdG, and 8-iso-PG by ELISA method and were tested for urine creatinine (UCR) by the picric acid method. Urinary Alb/UCR (UACR), urinary RBP /UCR (URCR), urinary PCX /UCR (UPCR), urinary 8-OHdG/UCR (UOHCR), and urinary 8-iso-PG/UCR (UISOCR) were calculated. . It was shown that can related to oxidative stress [27] and lipid peroxidation [28] . The study by Qi et al [29] showed that in the ICH model of rats, the levels of lipid peroxidation antioxidant enzymes and 8-iso-PGF2α were detected to assess oxidative stress. They found that metformin reduced oxidative stress and was maintained under ICH conditions [30] . The present study showed after 8 weeks of intervention with metformin and glibenclamide, urine UOHCR and UISOCR in type 2 diabetic mice were signicantly reduced, and the metformin group was lower than that of the glimepiride group, suggesting that metformin was more effective in reducing the risk of diabetic mice. Metformin reduces the oxidative stress mechanism is not yet clear. The study by Shambhoo S et al. [31] found that metformin can improve the redox imbalance of rat red blood cells induced by rotenone, increase the activity of AMP-activated protein kinase and increase the antioxidant effect [32] . Yang L et al. reported that metformin can exert an antioxidant effect through activation of the AMPK/Nrf2 pathway [33] . In addition, metformin can also reduce the production of ROS by activating the AMPK pathway and inhibiting NADPH oxidase [34] (cid:0) PCX: Podocalyxin; UPCR: PCX/creatinine; RBP: Retinol-binding protein; GBMT: Glomerular basement membrane thickness; FPFR: Foot process fusion rate; 8-OHdG: 8-Hydroxy-2'-Deoxyguanosine; 8-iso-PG: 8-iso-prostaglandin. DKD: Diabetic kidney disease.


Introduction
The prevalence of diabetes has increased signi cantly in the past few decades, and it has become one of the most important health problems in the world. According to the International Diabetes Federation, the number of people with diabetes worldwide has increased to 592 million by 2035 [1] . Diabetic kidney disease (DKD) is one of the most important complications of diabetes and can cause end-stage renal disease in severe cases [2] . Metformin is the most widely used anti-diabetic drug of choice for diabetes [3] . Recently, studies have shown that in addition to its hypoglycemic effect, it can reduce excessive endoplasmic reticulum stress, oxidative stress, and cell apoptosis, thereby exerting a protective effect on the kidneys [4][5][6] .
The 8-OHdG and 8-iso-PG are the biomarkers of DNA peroxidation [7] and lipid peroxidation [8] respectively. The excretion of 8-OHdG and 8-iso-PG in urine re ects the state of oxidative stress in the body index. However, the ability of metformin to improve 8-OHdG and 8-iso-PG in diabetic kidney disease has rarely been published. In this study, the renal protective effect of metformin on a model of type 2 diabetes mice and its effect on 8-OHdG and 8-iso-PG excretion in urine was observed, and the possible mechanism of reno-protective was explored.
Materials And Methods

animals
Eight-week-old SPF C57BL / 6J mice weighing 20-23 g, were purchased from Sandscobes Biotechnology Co., Ltd. All the mice were housed in 12h light/dark alternating regular illumination, ambient temperature (20 ± 1) ℃, the humidity was (48 ± 10) % and free access to food and water. During the experiment period, animals were treated strictly according to the rules in the Ethics Committee of the rst a liated hospital of the University of Science and Technology of China (Anhui Provincial Hospital). Every effort was made to minimize the number of animals used and their suffering.

experimental Grouping
After 7 days of acclimatization, mice were randomly divided into normal control group (n=8, fed with an ordinary diet until the end of the experiment) and high-fat diet (HFD) group (n=30, fed with HFD (containing 20 % protein, 20 % carbohydrate, 60 % fat until the end of the experiment)). After 16 weeks of feeding on a regular and high-fat diet, HFD mice had received a small dose of STZ (50 mg/kg was dissolved in 0.1mol/L citrate buffer, pH=4.2 and 3 days), and the NC group was injected with an equal amount of citrate buffer, and after a week, random blood glucose ≥16.7mmol/L for T2DM. Then the T2DM model mice (n=22) were randomly divided into 3 groups as follows (1) T2DM group (n=7), in which the mice were received by gavage of the equivalent volume of normal saline for 8 weeks; (2) MET group (n = 8), in which the mice were received by gavage of Metformin (Shanghai shimeigun Pharmaceutical Company) at a dose of 250 mg/kg/day for 8 weeks.
(3) GLIB group (n = 7), in which the mice were received by gavage of Glibenclamide (Tianjin Paci c Pharmaceutical Co., Ltd) at a dose of 2.5 mg/kg/day for 8 weeks. The mice in the normal control group were received a subcutaneous injection of the equivalent volume of normal saline for 8 weeks. After 8 weeks, the mice were weighed, fasting blood glucose was measured. Furthermore, blood, urine, and kidney tissue specimens were collected for further examination. The tissue specimens were stored at -80℃ before further use.

Electric microscope observation
The renal cortex was harvested and xed with 2.5% glutaraldehyde to make ultrathin sections. Five visual elds of sections were examined in a JEM-1230 transmission electron microscope. GBMT (glomerular basement membrane thickness) was measured at ve sites randomly. The total length of fused foot processes / total length of the basement membrane was measured and recorded as FPFR (foot process fusion rate). All parameters were measured by the Image J image analysis system.

Statistical analysis
Statistical analysis was performed using the Graphpad and spss 26.0 software. Data were expressed as mean ± standard deviation (means ± SD) and comparisons between two groups were performed using Student's t-test. Multigroup comparisons were analyzed using a one-way ANOVA test. If data were shown normally distributed, pairwise comparisons were conducted by LSD test. If not, pairwise comparisons were conducted by the Dunnett T3 test. All P values were two-sided, and a value of P less than 0.05 was considered statistically signi cant.

Comparison of body weight and hematuria in each group
The weight of mice and biochemical results were presented in Table 1. Elevated levels of FBG, HbA1c, UACR, UPCR, URCR, and reduced weight were detected in the T2DM groups compared with the NC group(p<0.05). Following eight weeks of treatment with MET and GLIB, weight levels increased and FBG, HbA1c, UACR, UPCR, URCR decreased signi cantly in the GLIB and MET groups(p<0.05). Furthermore, decreased levels of UACR, UPCR and URCR were detected in the MET group compared with the GLIB group(p<0.05), but the levels of FBG and HbA1c were not signi cant different.

Kidney pathology changes
Electron microscopy results showed that the thickness of the basement membrane in the NC group was uniform and clear, and the foot processes were basically not fused. The basement membrane in the T2DM group was thickened, the structure was fuzzy, and the foot processes were obviously fused and detached. The degree of GBMT and FPFR in the MET group and the GLIB group were improved compared with the DM group, and the difference between the two groups was statistically signi cant (p<0.05) These results are shown in Table 2 and Figure 1.

Comparison of UOHCR and UISOCR in each group
As illustrated in Table III, compared with the NC group, UOHCR and UISOCR increased in each diabetes group(p<0.05). Compared with the T2DM group, UOHCR and UISOCR were signi cantly lower in the MET group and the GLIB group (p<0.05), and the MET group was lower than the GLIB group (p<0.05).

Discussion
Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, which occurs in approximately 30% of patients with type 1 diabetes mellitus (T1DM) and 50% of T2DM patients [9] . DKD is often accompanied by thickening of the glomerular basement membrane, tubular interstitial brosis, tubular atrophy, and altered podocyte morphology [10,11] . It has been shown that metformin treatment improves the renal structure and dysfunction associated with DKD [5,12,13] . Furthermore, it has been shown that metformin exerts its renal protective effect independently of hypoglycemic [14,15] . In the present study, after 8 weeks of intervention in type 2 diabetic mice, the excretion level of urinary albumin (an indicator of glomerular damage), retinol-binding protein (an indicator of proximal tubular function), and podocalyxin (an indicator of podocyte damage) were signi cantly increased in the T2DM groups compared with the NC group compared with the normal group. Moreover, compared with the normal group, GBMT thickened and FPFR increased in the T2DM group, suggesting that diabetic mice have obvious kidney damage in the state of hyperglycemia. After metformin and glibenclamide intervention, the above indicators were ameliorated at different degrees compared with T2DM mice. In addition, the metformin group was signi cantly better than the glibenclamide group under the condition of similar glycemic control, suggesting that metformin has relatively better renal protection, which may be partly independent of the hypoglycemic effect.
8-OHdG(a biomarker of DNA oxidative damage) [16] and 8-iso-PG(a biomarker of lipid peroxidation) [17] are that re ect the state of oxidative stress in the body [18] . Cellular interference caused by oxidative stress can lead to different changes in biomolecules (including DNA), with the nuclear base most sensitive to oxidative stress being guanine, whose damage results in a modi ed 8-OHdG [19] . 8-iso-PG is generated by binding to arachidonic acid through reactive oxygen species-mediated lipid peroxidation [20] . Urinary 8-OHdG and 8-iso-PG excretion can be used as indicators re ecting the oxidative stress status in vivo [16,21] . The observations in this study showed urinary 8-OHdG and 8-iso-PG were increased in type 2 diabetic mice. In addition, the excretion level of urinary 8-OHdG and 8-iso-PG were positively associated with UACR, UPCR, and URCR. This nding may partially suggest that oxidative stress is involved in the development of diabetic kidney disease.
Metformin is a basal antidiabetic drug for type 2 diabetes, and many basic and clinical studies have shown a better renal protective effect in recent years, and its mechanism may be multi-channel (AMPK/mTOR signaling pathway [22] , ER stress [23] , Autophagy [24] , Oxidative stress [25] et al), in which oxidative stress plays a key role in the development and progression of diabetic kidney disease (DKD) [26] . It was shown that metformin can reduce DNA damage related to oxidative stress [27] and lipid peroxidation [28] . The study by Qi et al [29] showed that in the ICH model of rats, the levels of lipid peroxidation antioxidant enzymes and 8-iso-PGF2α were detected to assess oxidative stress. They found that metformin reduced oxidative stress and was maintained under ICH conditions [30] . The present study showed after 8 weeks of intervention with metformin and glibenclamide, urine UOHCR and UISOCR in type 2 diabetic mice were signi cantly reduced, and the metformin group was lower than that of the glimepiride group, suggesting that metformin was more effective in reducing the risk of diabetic mice. Metformin reduces the oxidative stress mechanism is not yet clear. The study by Shambhoo S et al. [31] found that metformin can improve the redox imbalance of rat red blood cells induced by rotenone, increase the activity of AMP-activated protein kinase and increase the antioxidant effect [32] . Yang L et al. reported that metformin can exert an antioxidant effect through activation of the AMPK/Nrf2 pathway [33] . In addition, metformin can also reduce the production of ROS by activating the AMPK pathway and inhibiting NADPH oxidase [34] Conclusion In conclusion, metformin intervention has a clear protective effect on the kidney of type 2 diabetic mice, and the mechanism may be associated with its partial reduction of enhanced oxidative stress in the diabetic high glucose state in the body, and the complete mechanism needs to be further explored. The histopathological changes in mice under electron microscope among four groups(20000×