Liraglutide Regulates the Kidney and Liver in Diabetic Nephropathy Rats through the miR-34a/SIRT1 Pathway

Purpose To explore the regulatory effects of liraglutide on the kidney and liver through the miR-34a/SIRT1 pathway with related factors in diabetic nephropathy (DN) rats. Methods DN rats were randomly divided into two groups (n = 10) and were injected with liraglutide or normal saline twice a day. The 24-hour urine microalbumin content and biochemical index levels were measured. qRT-PCR was performed to detect the expression of miR-34a in the kidney and liver tissues. The levels of SIRT1, HIF-1a, Egr-1, and TGF-β1 in kidney and liver tissues were determined using qRT-PCR, western blot, and immunohistochemistry. Electron microscopy and HE staining were used to observe the ultrastructure and pathological changes. Results Liraglutide treatment in DN rats decreased blood glucose, 24-hour urine microalbumin, TC, TG, LDL-C, UA, Cr, UREA, ALT, and AST levels and increased the level of HDL-C (P < 0.05). Compared with the control group, the miR-34a levels were significantly decreased in kidney and liver tissues followed by liraglutide treatment (P < 0.05). The levels of SIRT1 in the liraglutide group are significantly higher than those in the control group with the kidney and liver tissues (P < 0.05). Conversely, the contents of HIF-1a, Egr-1, and TGF-β1 were significantly lower in the liraglutide group than in the control group (P < 0.05). Electron microscopy showed that the kidney of the liraglutide-treated group exhibited minor broadening of the mesangial areas, fewer deposits, and a well-organized foot process. HE staining revealed that the kidney of the liraglutide-treated rats had a more regular morphology of the glomerulus and Bowman sac cavity and lighter tubular edema. Additionally, the liraglutide-treated DN rats had a clear hepatic structure, a lower degree of steatosis, and mild inflammatory cell infiltration. Conclusion Liraglutide, through its effect on the miR-34a/SIRT1 pathway, may have a protective role in the kidney and liver of DN rats.

2 Journal of Diabetes Research the expression of SIRT1 but increases the expression of the hypoxia-inducible factor-1 α (HIF-1a) and early growth response-1 (Egr-1); this causes inflammation of the glomerular mesangial cells as well as an increase in the levels of profibrotic factors [23,24]. Hypoxia has been shown to be an important inducing factor of inflammation and fibrosis in chronic liver disease; HIF-1a is a key transcription factor for liver fibrosis and is involved in the biliary ligation (BDL) and carbon tetrachloride-(CCL4-) induced liver fibrosis in rats [25][26][27]. HIF-1a was present in elevated levels in the liver of T2DM rats with fatty liver disease. Egr-1 is a proinflammatory nuclear transcription factor; Egr-1 knockout mice were shown to exhibit more severe liver fibrosis, which was induced by treatment with acetaminophen (APAP) [28]. Obesity and T2DM are often accompanied by insulinemia; long-term stimulation with high insulin induces the activation of Egr-1 in hepatocytes and reduces the regulatory effect of insulin. Conversely, inhibiting the expression of Egr-1 in the liver significantly reduces glucose production, thereby resulting in an improvement in glucose tolerance and insulin sensitivity [29]. Therefore, we established a rat model of DN using streptozotocin (STZ) and explored the effects of liraglutide on the expression of miR-34a and SIRT1 in the kidney and liver tissues. This study was aimed at investigating whether liraglutide has a role in protecting the kidney and liver of DN rats through the miR-34a/SIRT1 pathway; hence, this study could provide a theoretical basis for the use of liraglutide in the prevention and treatment of DN.   This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University and performed in accordance with the rules and regulations of the Care and Use of Laboratory Animals.

Instruments.
The instruments used in our study included a portable blood glucose monitor and test paper (Accu-Chek, Germany), a PCR amplifier CFX96 (Bio-Rad, USA), a gel imager and an electrophoresis system (Bio-Rad, USA), and an automatic chemiluminescence analyzer (Tianneng Technology, China).

Model Establishment.
Twenty SPF-grade male SD rats were fed with a high-fat and high-sugar diet for 8 weeks until their body weight increased to 400-450 g. The high-fat and high-sugar diet was resumed once again after the rats were injected intraperitoneally with 1 dose of STZ (30 mg/kg). After 3 days, the tail vein blood was collected; blood glucose levels equal to or greater than 16.7 mmol/L were used as an indicator for the successful establishment of a diabetic model. A DN model was considered to be successfully established if the urine albumin excretion rate exceeded 20 mg/24 h and the urine sample tested positive for glucose after 4 weeks. Thereafter, the DN rats were randomly divided into 2 groups (n = 10) and were subcutaneously injected with liraglutide (6 mg liraglutide dissolved in 11 mL normal saline, 0.6 mg/kg/d) or normal saline twice a day, then fed a high-fat and high-sugar diet.
The rats belonging to both the liraglutide and control groups were euthanized by an anesthetic overdose after 12 weeks.
2.5. Sample Collection. Following 12 weeks of treatment with liraglutide and normal saline, 24-hour urine samples were collected. Following an overnight fast, the rats were weighed and intraperitoneally injected with 3% pentobarbital sodium (40 mg/kg). Blood was collected from the abdominal aorta; a portion of each of the kidney and liver tissues was kept in RNAlater solution and stored at -80°C. Another portion was fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for use in subsequent experiments.
2.6. 24-Hour Urinary Microalbumin. The 24-hour urine samples were collected and centrifuged at 2500 × g for 20 min. The supernatants were collected, and the absorbance was measured at a wavelength of 450 nm using a microplate analyzer. The microalbumin content of the urine was calculated according to the regression equation.

Quantitative
Real-Time PCR (qRT-PCR). Total RNAs from the kidney and liver tissues were extracted using the TRIzol reagent and reverse transcribed to cDNA. Primers for SIRT1, HIF-1a, Egr-1, and TGF-β1 are shown in Table 1. The PCR reaction included 40 cycles of denaturation at 95°C for 10 min, annealing at 60°C for 1 min, and extension at 95°C for 15 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference. The expression levels of miR-34a were determined by performing a PCR reaction consisting of 40 cycles of denaturation at 94°C for 15 min, annealing at 55°C for 30 s, and extension at 70°C for 30 s. The has-U6 was used as the internal reference. Each experiment was repeated at least 3 times. The relative levels of SIRT1, HIF-1a, Egr-1, TGF-β1, and miR-34a were determined using the 2 -ΔΔCt (cycle threshold (CT)) formula.
2.9. Western Blot. The kidney and liver tissue samples were sonicated in the lysis buffer containing a phosphatase inhibitor and protease and were subsequently centrifuged at 14000 × g for 15 min at 4°C, The supernatants were collected, and the protein concentration was determined using a bicinchoninic acid (BCA) protein detection kit. Protein samples (20 μg/well) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to a polyvinylidene fluoride (PVDF) membrane, and thereafter blocked in 5% bovine serum at room temperature for 2 h. The membranes were incubated with the primary antibodies against SIRT1 (11kDa, 1 : 1000), HIF-1a (1 : 1000), Egr-1 (80kDa, 1 : 1000), TGF-β1 (1 : 1000), and GAPDH (1 : 5000) overnight at 4°C. After being washed with a Tris-buffered saline and Tween 20 (TBST) buffer three times, the membrane was incubated with horseradish peroxidase-(HRP-) labeled IgG (1 : 10000) at room temperature for 2 h. Following a three-time wash with TBST, the protein bands were detected using enhanced chemiluminescence (ECL) reagents to avoid light and color and take photos in gel electrophoresis apparatus. The gray value was measured by image processing software, and the relative expression level of the target protein was calculated by the gray value. GAPDH was used as an internal reference.
2.10. Immunohistochemistry. The kidney and liver tissue samples were fixed, embedded in paraffin, and dewaxed and rehydrated in graded alcohols. This was followed by quenching of endogenous peroxidases and antigen retrieval. Nonspecific protein binding was inhibited with the serum. The experiment was performed using a primary antibody followed by an appropriate secondary antibody. Phosphate- 2.11. Transmission Electron Microscopy. Kidney tissues were fixed with 2.5% glutaraldehyde for 2 h, rinsed overnight, and fixed with 1% osmic acid for 2 h. Thereafter, the tissues were dehydrated in gradient pyruvate acid and stained with lead and uranium. The ultrastructure was observed under a transmission electron microscope.
2.12. Hematoxylin/Eosin (HE) Staining. The kidney and liver tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and stained with hematoxylin/eosin (HE) for histopathological observation.  Journal of Diabetes Research 2.13. Statistical Analysis. Experimental data were analyzed using SPASS25.0 software. The quantitative data were expressed as mean ± standard deviation. Differences between the two groups were analyzed using independent t-tests. P values < 0.05 were considered to be statistically significant.

Fasting Blood Glucose and 24-Hour Urine Microalbumin
Changes. Following treatment with liraglutide, the blood glucose levels were significantly decreased in the DN rats (P < 0:05) (Figure 1(a)). Additionally, the 24-hour urine microalbumin content was also significantly reduced in the liraglutide-treated DN rats when compared to the control group (P < 0:05) (Figure 1(b)).

Changes in Biochemical
Indices. Treatment with liraglutide resulted in a significant decrease in the serum levels of TC, TG, and LDL-C (P < 0:05) (Figures 2(a) and 2(b)); conversely, the level of HDL-C was increased (P < 0:05) in DN rats when compared with the control group (Figure 2(b)). Furthermore, the levels of blood UA, Cr, and UREA were significantly reduced in the liraglutide-treated DN rats than in the control group (P < 0:05) (Figure 3). AST and ALT levels were also significantly decreased (Figures 4(a) and 4(b)), and the AST/ALT ratio tended to be within the normal range (Figure 4(c)) following liraglutide treatment.

Liraglutide Inhibited the Expression of miR-34a in the
Kidney of DN Rats. Through quantitative real-time PCR analyzing the miR-34a of extracted tissues, we found that compared to the control DN group, the expression of miR-34a was significantly decreased in the kidney tissues of the DN rats treated with liraglutide (P < 0:05) ( Figure 5). Immunohistochemical staining showed that the expression level of SIRT1 in the renal tubules and glomeruli was significantly increased following treatment with liraglutide (Figure 9(a)); conversely, the levels of HIF-1a, Egr-1, and TGF-β1 were decreased in the renal tubules and glomeruli of liraglutide-treated rats (Figures 9(b)-9(d)).

Ultrastructural and Pathological Changes in the Kidney of
Liraglutide-Treated DN Rats. In the liraglutide-treated rats, we observed a slight widening of the mesangial area, an occasional appearance of deposits, mild edema of endothelial cells, and well-arranged foot processes in the kidney tissues ( Figure 10). In the DN rats, electron microscopy revealed mesangial cell hyperplasia with lipid droplets, a widening of the mesangial area, low-density electron deposits, a shrunken basement membrane, a vascular cavity filled with a large number of red blood cells, and fusion foot processes ( Figure 10).
HE staining revealed that the kidney of liraglutide-treated rats adopts a more regular morphology of the glomerulus and Bowman sac cavity and lighter tubular edema ( Figure 11). Conversely, the DN rats exhibited obvious pathological changes including irregular glomerular morphology, adhesion of the wall and visceral layer of the Bowman sac, an increased number of cells, and renal tubular edema with partial necrosis (Figure 11).

Liraglutide Inhibited the Expression of miR-34a in the
Liver of DN Rats. Through quantitative real-time PCR analyzing the miR-34a of extracted tissues, we found that compared to the control group, liraglutide treatment significantly decreased the expression of miR-34a in the liver tissues (P < 0:05) (Figure 12).
Immunohistochemical staining showed that the expression level of SIRT1 was significantly increased in hepatocytes following the treatment of DN rats with liraglutide ( Figure 16(a)); conversely, decreased levels of HIF-1a, Egr-1, and TGF-β1 in hepatocytes were detected in liraglutidetreated rats when compared to the control group (Figures 16(b)-16(d)).

Pathological
Changes in the Liver of Liraglutide-Treated DN Rats. In the liraglutide-treated DN rats, liver sinusoids were partially dilated ( Figure 17). Conversely, disordered hepatic sinusoids with obvious dilation were detected in the liver of the control group ( Figure 17).

Discussion
This study sought to investigate if liraglutide has a protective effect on the kidney and liver of DN rats that is mediated by  Figure 14: Expression of SIRT1, HIF-1a, Egr-1, and TGF-β1 proteins in the liver. Control: diabetic nephropathy group; DN+LRG: diabetic nephropathy+liraglutide group; Egr-1: early growth response-1; HIF-1a: hypoxia-inducible factor-1 alpha; TGF-β1: transforming growth factor-β1. * P < 0:05 vs. the control. 8 Journal of Diabetes Research inhibit the release of glucagon; GLP-1 also has glucose concentration-dependent hypoglycemic effect [30,31]. Liraglutide, a GLP-1 analog, is reported to delay gastric emptying, reduce food intake, and maintain stable blood glucose levels by promoting a glucose-dependent secretion of insulin and inhibiting the release of glucagon. Meanwhile, liraglutide also alleviates blood lipid levels in obese patients with T2DM and controls weight gain [32,33], and compared with exenatide, liraglutide has higher safety and tolerance and more significant effect in improving beta cell function and insulin sensitivity [34]. Moreover, liraglutide could decrease the incidence of proteinuria in diabetic patients, reduce urine protein through anti-inflammatory and antioxidative stress activities, improve kidney function, and prevent the occurrence of DN and acute kidney injury [35]. Multiple studies have shown that miR-34a is closely associated with the occurrence of diabetes; miR-34a is significantly increased in the peripheral blood mononuclear cells in patients with T2DM and is correlated positively with LDL-C/HDL-C and negatively with TG/HDL-C [36]. TGF-β1 stimulation promotes the resynthesis of miR-34a in fibroblasts and increases miR-34a expression in microvessels; hence, TGF-β1 has a role in renal tubule-interstitial fibrosis [37]. Exogenous injection of miR-34a-containing microvesicles in mice promotes apoptosis of renal tubular cells, which in turn is closely related to renal fibrosis. In the presence of high glucose, downregulation of miR-34a inhibits the proliferation of mesangial cells; miR-34a regulates early mesangial proliferation and glomerular hypertrophy in DN by targeting the growth arrest-specific protein 1 (GAS1) [38]. Silent information regulator 1 (SIRT1), a nicotinamide adenine dinucleotide-(NAD + -) dependent deacetylase, plays an important role in the pathophysiology of kidney disease [39]. High glucose could induce HIF-1a expression, leading to the apoptosis of renal tubular epithelial cells; the polymorphism of HIF-1a Pro582Ser has differential effects on DN [40,41]. High levels of Egr-1 result in an increase in the expression of profibrotic protein and influence DN development [42]. In glomerular mesangial cells cultured with high glucose, activation of the miR-34a/SIRT1 pathway upregulates the expression of HIF-1a and Egr-1, resulting in the overexpression of profibrotic factors, such as TGF-β1 and type IV collagen, in glomerular mesangial cells [23,24].
In the present study, we detected changes in the biochemical indices and levels of miR-34a, SIRT1, HIF-1a, Egr-1, and TGF-β and explored whether liraglutide plays a protective role in DN rats through the regulation of the miR-34a/SIRT1 pathway. Our findings indicate a significant decrease in the levels of blood glucose, 24-hour urine microalbumin, TC, TG, LDL-C, UA, Cr, and UREA, while the HDL-C content was observed to be increased following liraglutide treatment. The data showed miR-34a expression to be significantly lower while SIRT1 levels were shown to be higher in the kidney of liraglutide-treated rats than control rats (P < 0:05); the mRNA and protein levels of HIF-1a, Egr-1, and TGF-β1 were significantly decreased following liraglutide treatment (P < 0:05). Immunohistochemical staining showed an obvious increase in SIRT1 levels while the levels of HIF-1a, Egr-1, and TGF-β1 were decreased in the renal tubules and glomeruli of DN rats (P < 0:05). Electron microscopy detected a slight widening of the mesangial area, occasional deposits, mild edema of endothelial cells, and well-arranged foot processes in the kidney tissues. In the DN rats, electron microscopy revealed mesangial cell hyperplasia with lipid droplets, a widening of the mesangial area, low-density electron deposits, a shrunken basement membrane, a large number of red blood cells filling the vascular cavity, and fusion foot processes. HE staining revealed that the kidney in liraglutide-treated rats had a more regular morphology of the glomerulus and Bowman sac cavity and lighter tubular edema. Conversely, the DN rats exhibited obvious pathological changes including irregular glomerular morphology, adhesion of the Bowman sac wall and visceral layer, increased number of cells, and renal tubular edema with partial necrosis. These findings suggest that liraglutide plays a protective role in the kidney of DN rats by decreasing the blood glucose/lipid levels and expression of miR-34, HIF-1a, Egr-1, and TGF-β1 while increasing the SIRT1 levels. Additionally, liraglutide treatment alleviates oxidative stress-induced injury in ZDF-induced DN rats [14]. During the process of epithelialmesenchymal transition (EMT), inhibition of the miR-34a/SIRT1 pathway downregulates TGF-β1 expression in hepatocytes, thus playing an antifibrotic role [18]. HIF-1a is not only involved in kidney injury in DN but also closely related to inflammatory reactions and glucagon sensitivity in the liver [43]. Similarly, Egr-1 is not only involved in high glucose-induced mesangial cell injury but also associated with liver injury, which is mediated by various pathways [44,45]. Therefore, our study further explored if liraglutide had a protective role in the liver by targeting the miR-34a/SIRT1 pathway. Our results showed that liraglutide treatment significantly decreased AST and ALT levels; moreover, the AST/ALT ratio also tended to be within the normal range. Data showed a significant decrease in the miR-34a and SIRT1 levels and suppressed HIF-1a, Egr-1, and TGF-β1 in the liver in DN rats (P < 0:05). Immunohistochemical staining showed that liraglutide treatment resulted in a significant increase in SIRT1 and a decrease in the protein levels of HIF-1a, Egr-1, and TGF-β1 in the hepatocytes of DN rats. From the staining of the liver with HE, a partial dilation of the liver sinusoids was observed in the liraglutide-treated DN rats. Conversely, a disordered hepatic sinusoid, which presented DN+LRG Control SIRT1 HIF-1a Egr-1 TGF-1 GAPDH Figure 15: Western blot of SIRT1, HIF-1a, Egr-1, and TGF-β1 protein expression in the liver. Control: diabetic nephropathy group; DN+LRG: diabetic nephropathy+liraglutide group; Egr-1: early growth response-1; HIF-1a: hypoxia-inducible factor-1 alpha; TGF-β1: transforming growth factor-β1. 9 Journal of Diabetes Research with obvious dilation, was detected in the control group, which had not received the liraglutide treatment. These findings suggest that liraglutide plays a protective role in the liver of DN rats by decreasing AST and ALT levels and miR-34a expression while increasing SIRT1 levels and inhibiting HIF-1a, Egr-1, and TGF-β1. Notably, the precise mechanism is unclear and requires further study.

Conclusion
In short, liraglutide plays a protective role in the kidney and liver of DN rats by decreasing miR-34a expression, increasing SIRT1 levels, and inhibiting HIF-1a, Egr-1, and TGF-β1. These findings provide a theoretical basis for the use of liraglutide in the prevention and treatment of DN.

Data Availability
The data analyzed during the study are not publicly available. Rigorous analysis of the data in order to ensure the objective authenticity of the results.