The Molecular Effects of SGLT2i Empagliflozin on the Autophagy Pathway in Diabetes Mellitus Type 2 and Its Complications

Background Type 2 diabetes mellitus (T2DM), especially hyperglycemia, is associated with increased glucose cell toxicity and oxidative stress that can lead to irreversible damage in the kidney such as diabetic nephropathy (DN). Autophagy plays a key role in the degradation of damaged intracellular proteins in order to maintain intracellular homeostasis and cell integrity. The disturbance of autophagy is involved in the pathogenesis of diabetic nephropathy. We aim to investigate the molecular effect of sodium-glucose transporter 2 inhibitor (SGLT2i) on the expression of ATG5 and its downstream collaborator LC3-II in diabetic nice model. Material and Methods. We used eight weeks old male mice: twenty C57BL/6 wild type (C57BL/6), twenty BTBR ob/ob (DM), and twenty BTBR ob/ob that were treated with empagliflozin (DM+EMPA), FDA approved SGLT2i. Lysate from murine renal cortex was analyzed by Western blot and immunohistochemistry. ATG5, LC3B, and fibronectin expression were analyzed in murine kidney tissues. All mice were sacrificed 13 weeks after the beginning of the experiment. Results Histological and Western blot analyses reveal decrease ATG5, LC3-II, and fibronectin levels at renal specimens taken from DM mice. EMPA treatment reduced T2DM mice body weight and blood glucose and increased urine glucose. Further, it upregulated all of the abovementioned proteins. Conclusions Hyperglycemia reduces LC3-II and ATG5 protein levels which contribute to deficiencies in the autophagy process, with development and progression of DN. SGLT2i significantly reduces progression of DN and onset of end-stage renal disease in T2DM patients, probably through its effect on autophagy.


Introduction
Diabetes mellitus (DM) type 2 (T2DM) is a metabolic disease characterized by chronic hyperglycemia, resulting from insulin deficiency, insulin resistance, or both [1]. Chronic hyperglycemia is associated with disruption and alterations in carbohydrate, lipid, and protein metabolism [2][3][4], with cellular damage in different organs such as the kidney, heart, and retina [5,6]. Diabetic nephropathy (DN) is one of the common complications of diabetes. Above 50% of T2DM patients developed DN during their life. DN is characterized by renal functional and structural changes, such as glomerular mesangial expansion, accumulation of extracellular matrix (ECM), glomerular hypertrophy, and thickening of glomerular and tubular basement membranes with glomerular sclerosis, all lead to decrease in glomerular filtration rate [4]. The final stage of DN is end-stage renal disease and renal replacement therapy that lead to increased morbidity and mortality [3].
Numerous risk factors are associated with the development and progression of DN among them extended duration of DM, arterial hypertension, obesity, and hyperlipidemia, with most being modifiable by appropriate treatment. Other contributing factors, such as genetic factors including haptoglobin genotype, cannot be modified [5][6][7][8][9]. Nevertheless, hyperglycemia is of the greatest importance.
Autophagy is a catabolic mechanism that involves lysosomal-dependent degradation of unnecessary or dysfunctional intracellular components. Autophagy preserves cells and tissues maintenance by replacing damaged cellular components and promotes cell's metabolic homeostasis. The process starts with the formation of a cellular component named autophagosome, by autophagy-related genes (Atg) proteins such as ATG5 and LC3, and finalizes by its fusion with lysosome and intraphagosome lysosomal degradation of cellular organelles to their basal components [13][14][15][16][17][18][19]. It was shown that autophagy has an essential role in maintaining kidney's cellular homeostasis, especially in podocytes and proximal tubular cells [20][21][22][23][24]. Under pathological condition, intensified activity of autophagy is required to maintaining podocytes and other renal cells [21,22].
Studies suggest that both hyperinsulinemia and hyperglycemia inhibit autophagy activity in proximal tubular cells and podocytes, by inducing hyperactivation of mTORc1, an autophagy-regulated protein (20). Hyperglycemia suppresses the expression of LC3, Atg12-5, and Beclin-1, to inhibit the formation of the autophagosome membrane [25,26]. Thus hyperglycemia alters autophagy activity and contributes to diabetes-related podocyte and tubular cell damage.
Empagliflozin (EMPA), FDA approved for cardiovascular protection [27,28], is a renal sodium glucose cotransporter type 2 inhibitor (SGLT2i). It was seen that EMPA is highly effective in controlling blood glucose and HbA1c levels, increases glucose excretion, and slows the progression of renal injury [27][28][29][30]. Furthermore, it also improves blood pressure and decreases body weight (BW) and albumin secretion and slows the progression of renal failure [30][31][32][33][34]. Recent studies indicate that SGLT2is delay the progression of chronic kidney disease (CKD) in T2DM patients and cardiovascular morbidity and mortality by decreasing oxidative stress and apoptosis [32][33][34][35][36][37][38]. However, the precise mechanisms by which EMPA protect renal cells structure and function is not fully clear. In this study, we hypothesize that SGLT2i exerts its renal beneficial effects by affecting autophagy level. Thus, this study was designed to investigate the molecular effect of SGLT2i on the expression of ATG5/ LC3-II autophagy key proteins in diabetic nice model.

Animal Model.
Male BTBR ob/ob mice and C57BL/6 were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Animals were group-housed and maintained on a 12 h/12 h light/dark cycle with ad libitum access to food and water. The animals were treated in accordance with NIH Animal Welfare Guideline (ed. 2011), and the Azrieli Faculty of Medicine, Bar-Ilan University, Institutional Animal Care and Use Committee (IACUC) approved all procedures [Ethical #48-07-2019]. BTBR mice with the ob/ob leptin-deficiency mutation were used as T2DM model, as they develop severe DM, which is presented in hyperglycemia, and DN [47][48][49] C57BL/6 mice were used as healthy control. Eight-week old mice were randomly divided in three groups: Group one C57/BL/6 as control (n = 15) and group two BTBR ob/ob vehicle as (DM), and third group BTBR ob/ob that were treated with EMPA (DM+EMPA).
Mice body weight was monitored once a week on a calibrated scale with a sensitivity of 0.01 mg. Water intake (ml/day), food consumption (gr/day), blood glucose (Glucometer, Atcu-Check, Roch), and daily urine production (ml/day) were measured. Urine samples were analyzed for glucose, protein, and creatinine excretion per day. All mice were sacrificed 13 weeks after the beginning of the experiment by ketamine-xylazine overdose injection. At sacrifice, kidneys were harvested, one kidney was flashed frozen in liquid nitrogen and transferred to -80°C until analysis, and the second kidney was submerged in 4% paraformaldehyde for histological analysis.
2.2. EMPA Administration. EMPA was purchased from DA-TA Biotech, USA. EMPA powder was diluted in water (1 mg/kg mouse) and administrated to the mice, as was published in another study, via drinking water for a period of 12 consecutive weeks, and drug concentration in drinking water was adjusted weekly to the BW [32-36].

Proteins Extraction and Western Blot Analyses.
Mice kidneys were homogenized and incubated with radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling, USA) containing proteases and phosphatases inhibitors (Sigma, Israel) in ratio of 1 : 100 and 1 : 1000, respectively. Kidney's lysate was centrifuged, and the supernatant containing total proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% dry skim milk (Bio-Rad, CA, USA) in Tris-buffered saline with Tween 20 at room temperature and washed and incubated with primary antibodies (Anti-APG5L/ATG5 ab109490 [EPR4797] 1 : 1000; Anti-LC3-II ab48394 1 : 1000; Anti-GAPDH ab181602 1 : 10000) at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated to secondary antibody at room temperature. Bands were visualized using clarity enhanced chemiluminescence kit (Bio-Rad, CA, USA) and were analyzed using Bio-Rad image Lab software, are data presented in arbitrary units (AU).

Histology.
Fresh kidney from each mouse was fixed in 4% formaldehyde and embedded in paraffin. Then, 4-μm thick sections were transferred to positive charged glass slides and processed for Hematoxylin and Eosin (H&E) staining. Images were captured using an Axio Lab. A1 microscope. Images were captured with Axiocam 105 color  HRP-polymer antirabbit (Nichirei and Dako) was used as a secondary antibody, and proteins were visualized using the DAB Plus Substrate System (TA-125-HDX, Thermo Scientific, USA) and then counterstained with Hematoxylin. Images were taken at 20× and 10× by using an Axio Lab. By using an Axio Lab, a 1 microscope with the Axiocan 105 color digital camera and ZEIN software (Zeis, Oberkochen, Germany), photograph analysis presents data as % of stained tissue.
2.6. Statistics. All results are reported as mean ± SEM. After conforming data normality distribution and equal variation among groups, comparisons between the study groups were performed using ANOVA, in GraphPad prism version 5.00 for windows (GraphPad Software, La Jolla, CA, USA). P value < 0.05 was considered statistically significant.

Discussion
DN presents a significant morbidity and mortality burden, and until recently, treatment options have been limited to risk factor control such as hypertension and hyperglycemia by RAAS inhibition and standard antidiabetic drugs [14][15][16][17]. Recently, different studies have concentrated in other pathways involved in DN prevention and progression such as autophagy, and other modalities of treatments that confer not only glycemic effect but also reno-protective effects were tested [14][15][16][17][18].
Autophagy pathway is crucial for cell survival, differentiation, development, and homeostasis under stress conditions such as hyperglycemia [19,20]. Various studies have provided evidences suggesting that autophagy-related key proteins ATG5 and LC3-II play a critical role in a variety of disease processes such as DM [19][20][21][22]. Yet only few have examined the possible role of ATG5/LC3-II proteins in the development and progression of DM and its vascular complication such DN [22,23,[38][39][40]. We analyzed the ATG5 protein that dissociates from the membrane next to the completion of autophagosome formation, and the LC3-II  Journal of Diabetes Research protein, one of the most frequently used biomarkers for autophagy. LC3-II is a gold standard marker for tracing changes in the autophagy process [24,[38][39][40][41]. ATG5 might play a crucial role in the production and secretion of insulin in pancreatic β cells, and deletion of ATG5 results impaired insulin secretion and glucose intolerance [42]. In recent years, autophagy has become a hot topic in the field of DN, suggesting that it is likely to be a key target for the prevention and treatment of DN. Yet, the activity of autophagy in the renal cells and changes in the autophagy-lysosomal system during DN is still under debate [41,[43][44][45]. Hyperglycemia was considered an autophagy suppressor in the glomerular, proximal convolute tubules, podocytes and mesangial cells [43].
The emergence of SGLT2i such as EMPA, a new class of glucose-lowering compounds, has shown not only blood glucose lowering effect, but also pleiotropic effects with renoprotective actions in preclinical and human studies [27]. In the present study, we provide novel mechanistic evidence showing that EMPA improves the autophagy process activities in the glomerular cells and renal proximal tubular cells, all of which led to reduced renal fibrosis caused by hyperglycemia. The acceleration of autophagy flux under EMPA treatment is associated with regression of glomerular sclerosis and preservation of glomerular (podocytes) morphology, as well as with a slowdown in the growth of albuminuria.
Various studies have assessed the impact of DM on the reno-protective autophagy processes on renal cells exposed to hyperglycemia [22,45]. Anton I. Korbut et al. demonstrate that EMPA as a monotherapy and in combination with other antidiabetic drugs; enhanced the areas of glomerular staining for beclin-1, LAMP-1, LC3-II, and BcL-2; and increased volume density of autophagosomes and autolysosomes in podocytes. All together indicate restoration of autophagy by EMPA. Further, the data provide explanation for the mechanism of the reno-protective effect of SGLT2i and possibly DPP4 inhibitors as well in diabetes.
Here, we report that DM mice exhibit significantly decreased levels of ATG5 and LC3-II protein levels compared to healthy control mice, which restored to near normal with EMPA treatment. We used immunohistochemistry on mouse tissue sections as well as Western blot analysis. Our data suggest that there is a link between ATG5-LC3-II dysregulation and DM. These results correlate with previous studies, which showed that alterations in ATG5/LC3 expression are implicated in many pathological conditions, including DM [26,43]. Restoration of the autophagy process, decreased glomerular hypertrophy, and regression of fibrosis in diabetic mice in this model identifies an unappreciated potential benefit of SGLT2i in patients with DN.

Conclusions
DN patients have increased risk of morbidity and mortality. Hyperglycemia is a modifiable risk factor for cardiovascular complications and progression of DN. There is now conclusive evidence and consensus that SGLT2i significantly reduce progression of DN and onset of end-stage renal disease, stroke, heart attack, and death in T2DM [46].
In our study, EMPA treatment decreases blood glucose levels in the T2DM mice with increased excretion of glucose in the urine. Both ATG5 and LC3-II protein levels decreased in those mice compared to control mice and increased following EMPA treatment. Our findings may be translated into clinical practice approach and may lead to further studies to address DM and its vascular complications by selective modulation of ATG5/LC3 expression with new agents such as SGLT2i and newer medications such as DPP4 inhibitors [46][47][48].

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

Ethical Approval
The study, involving diabetic mice model, was reviewed and approved by the animal committee ethics of Bar-Ilan University.