Dapagliflozin Restores Impaired Autophagy and Suppresses Inflammation in High Glucose-Treated HK-2 Cells

Sodium-glucose cotransporter2 (SGLT2) inhibitors have a reno-protective effect in diabetic kidney disease. However, the detailed mechanism remains unclear. In this study, human proximal tubular cells (HK-2) were cultured in 5 mM glucose and 25 mM mannitol (control), 30 mM glucose (high glucose: HG), or HG and SGLT2 inhibitor, dapagliflozin-containing medium for 48 h. The autophagic flux was decreased, accompanied by the increased phosphorylation of S6 kinase ribosomal protein (p-S6RP) and the reduced phosphorylation of AMP-activated kinase (p-AMPK) expression in a HG condition. Compared to those of the control, dapagliflozin and SGLT2 knockdown ameliorated the HG-induced alterations of p-S6RP, p-AMPK, and autophagic flux. In addition, HG increased the nuclear translocation of nuclear factor-κB p65 (NF-κB) p65 and the cytoplasmic nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), mature interleukin-1β (IL-1β), IL-6, and tumor necrosis factorα (TNFα) expression. Dapagliflozin, SGLT2 knockdown, and NF-κB p65 knockdown reduced the extent of these HG-induced inflammatory alterations. The inhibitory effect of dapagliflozin on the increase in the HG-induced nuclear translocation of NF-κB p65 was abrogated by knocking down AMPK. These data indicated that in diabetic renal proximal tubular cells, dapagliflozin ameliorates: (1) HG-induced autophagic flux reduction, via increased AMPK activity and mTOR suppression; and (2) inflammatory alterations due to NF-κB pathway suppression.


Introduction
Diabetic kidney disease (DKD) is one of the main chronic complications of diabetes and the leading cause of end-stage renal disease (ESRD) [1]. Despite various therapeutic options for glucose control, many diabetic patients remain at high risk for development and progression of DKD. Therefore, there is an urgent need for more effective interventions for preventing the development and progression of DKD.
Sodium-glucose cotransporter 2 (SGLT2) is localized in the proximal tubule and is critical for 80-90% of the glucose filtered under physiological conditions [2]. SGLT2 inhibitors are effective antidiabetic drugs that attenuate hyperglycemia by suppressing renal glucose reabsorption. The results of large-scale clinical studies have shown that SGLT2 inhibitors improve hyperglycemia independently of insulin [3,4] and present renoprotective and cardioprotective effects in diabetic patients [5][6][7]. Although dapagliflozin treatment has led to remarkable renal outcomes [5,8], the relevant mechanisms have still not been fully elucidated.
Alterations in nutrient-sensing signaling pathways, including the AMP-activated kinase (AMPK) pathway, are involved in the pathogenesis of DKD through autophagy impairment and inflammation [9,10]. AMP-activated protein kinase (AMPK) is an energy Table 1. Sequences of the small interfering RNAs used in this study. SGLT2  GUCAUUGCUGCAUAUUUCCTT  GGAAAUAUGCAGCAAUGACTA  AMPK  GGUAGAUAUAUGGAGCAGUTT  ACUGCUCCAUAUAUCUACCTC  RELA  GGCUAUAACUCGCCUAGUGTT  CACUAGGCGAGUUAUAGCCTC  NLRP3 GGUGUUGGAAUUAGACAACTT GUUGUCUAAUUCCAACACCTG

Glucose Uptake
Glucose uptake was defined as the uptake of 2-deoxyglucose (2-DG) by HK-2 cells using a glucose uptake fluorometric assay kit (K666-100, BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. Generally, HK-2 cells were cultured in 96-well plates in keratinocyte SFM. When 50-60% confluent, the cells were treated with or without dapagliflozin (DAPA, 20 µM) for 48 h or cells were transfected with control siCON or siSGLT2 (200 nM) for 6 h. The cells were incubated in 0.5% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) with or without 2-DG for 1 h. The relative fluorescence units (RFU) were measured at Ex/Em = 544/590 nm using a Fluoroskan Microplate Fluorometer (#5210470, Thermo Fisher Scientific, San Jose, CA, USA). The uptake of 2-DG in all samples was calculated by the 2-DG-6-phosphate (2-DG6P) standard curve and the RFU of samples.

Autophagic Flux Assay
HK-2 cells were treated with control (Cont), HG (30 mM), or HG (30 mM) with dapagliflozin (DAPA, 20 µM). After 48 h, chloroquine (CQ, 50 µM, C6628, Sigma-Aldrich, St. Louis, MO, USA) or vehicle was added, and the cells were incubated for 1 h before the collection of samples. Autophagic flux was calculated by the ratio of LC3-II in the sample treated with CQ and the sample treated without CQ. The ratio of LC3-II in the sample cultured in HG (30 mM) and rapamycin (0.4 µg/mL, 48 h, R0395, Sigma Aldrich, St. Louis, MO, USA) was used as a positive control for mTOR inhibition and autophagy activation. The samples were collected and analyzed by western blot analysis.

Protein Extraction and Western Blot Analysis
Total proteins were harvested by radioimmunoprecipitation lysis buffer with phenylmethanesulfonylfluoride, sodium orthovanadate, and a protease inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX, USA). Nuclear and cytoplasmic proteins were harvested using a nuclear extraction kit (ab113474, Abcam, Cambridge, UK) according to the manufacturer's instructions. Concentrations of proteins were measured using a BCA protein assay kit (#23225, Pierce, Rockford, IL, USA) following the manufacturer's instructions. Protein lysates were denatured in 4× Laemmli sample buffer (#161-0747, Bio-Rad, Hercules, CA, USA) at 100 • C for 5 min.

Immunofluorescence
HK-2 cells were cultured in K-SFM serum-free medium in eight-well plates. When the cells were 70-80% confluent, the medium was changed and the following treatments were administered before 48 h of incubation: control (Cont), HG (30 mM), HG (30 mM) with dapagliflozin (DAPA, 20 µM), or HG (30 mM) with rapamycin (rapa 0.4 µg/mL). Alternatively, cells were transfected with siSGLT2, as explained above. The cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, blocked in 2% BSA/PBS for 30 min, and then incubated overnight with primary antibodies against IL-6 or p-S6RP (Table 2) at 4 • C. After being washed with PBS, the cells were incubated with secondary antibodies conjugated with Alexa Fluor 647 (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min. After the cells were washed with PBS, the nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI), and the cells were observed under a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

ELISA
HK-2 cells were cultured in K-SFM serum-free medium in six-well plates. When the cells were 70-80% confluent, cells were divided into 3 groups: control (Cont), HG (30 mM), and HG (30 mM) with dapagliflozin (DAPA, 20 µM). After 48 h, supernatants of cultured medium were collected. Levels of pro-inflammatory cytokines in the cultured medium were detected by using human ELISA kits targeting IL-1β (DLB50, R&D

Cytotoxicity Detection
To detect the cytotoxicity of dapagliflozin, HK-2 cells were treated with different concentrations of dapaliflozin (0-100 µM) under HG condition. After 48 h, the supernatants of the culture medium were collected. The activity of lactate dehydrogenase (LDH) in the culture medium that was released from damaged cells was determined using a Cytotoxicity Detection Kit (#04744926001, Roche, Mannheim, Germany) according to the manufacturer's instructions. The O.D. of the sample was measured and corrected by subtracting the readings at 650 nm and the reading of cell-free culture medium from the readings at 492 nm using an Emax Plus Microplate Reader (Molecular Devices, San Jose, CA, USA).

Statistical Analysis
GraphPad Prism software (ver. 7.0f; La Jolla, CA, USA) was used for statistical analysis. The experimental results represent the mean ± standard deviation (SD) values of three independent duplicate experiments, and the significance of the differences between three or more groups was evaluated using one-way ANOVA followed by Tukey's multiple comparison test. A value of p < 0.05 was considered statistically significant.

Dapagliflozin and SGLT2 Inhibition Suppressed Glucose Uptake by HK-2 Cells
SGLT2 is critical for glucose absorption in the human proximal tubule. In HK-2 cells, HG induced the expression of SGLT2, and both dapagliflozin and SGLT2 knockdown (efficiency 41.5%) significantly suppressed SGTL2 expression and glucose uptake ( Figure 1). of the culture medium were collected. The activity of lactate dehydrogenase (LDH) in the culture medium that was released from damaged cells was determined using a Cytotoxicity Detection Kit (#04744926001, Roche, Mannheim, Germany) according to the manufacturer's instructions. The O.D. of the sample was measured and corrected by subtracting the readings at 650 nm and the reading of cell-free culture medium from the readings at 492 nm using an Emax Plus Microplate Reader (Molecular Devices, San Jose, CA, USA).

Statistical Analysis
GraphPad Prism software (ver. 7.0f; La Jolla, CA, USA) was used for statistical analysis. The experimental results represent the mean ± standard deviation (SD) values of three independent duplicate experiments, and the significance of the differences between three or more groups was evaluated using one-way ANOVA followed by Tukey's multiple comparison test. A value of p < 0.05 was considered statistically significant.

Dapagliflozin Suppressed the HG-Induced Reduction in Autophagic Flux through AMPK Activation
To determine the optimal concentration of dapagliflozin, we examined the dose effect of dapagliflozin in HG treated-HK-2 cells on cytotoxicity and AMPK activation. Compared with the Cont, HG (30 mM) did not increase cell death, but significantly suppressed the expression of p-AMPK. Dapagliflozin (10-100 µM) restored p-AMPK expression in a dosedependent manner (Figure 2A,B). However, compared to the HG without dapagliflozin group, high concentrations of dapagliflozin (more than 50 µM) showed significant cytotox-Cells 2021, 10, 1457 6 of 16 icity in the high glucose condition ( Figure S5D). Then, we evaluated the AMPK-autophagy pathway in the treatment with dapagliflozin (20 µM) for 24 h and 48 h, respectively. The 24-h treatment with dapagliflozin (20 µM) ameliorated the decrease in p-AMPK and suppressed the increase in p-S6RP in high-glucose treated HK-2 cells. However, there were no significant changes in autophagic flux ( Figure S1). If the duration of dapagliflozin was extended to 48 h, HG-impaired autophagic flux could be restored by dapagliflozin treatment ( Figure 2C,D). On the other hand, dapagliflozin (20 µM) significantly ameliorated the decrease in p-AMPK, the increase in p-S6RP (a downstream protein of mTOR ( Figure 3A,B), and the decrease in autophagic flux due to HG in the 48-h treatment ( Figure 3E,F). To investigate the mechanism of dapagliflozin action on autophagic flux, AMPK siRNA was used to abrogate the activation of AMPK. The autophagic flux induced by dapagliflozin was abrogated by AMPK knockdown, which was accompanied by an increase in p-S6RP level ( Figure 3A,B,E,F). Rapamycin, an inhibitor of the mTOR pathway, also restored the increase in p-S6RP expression ( Figure 3C,D,G) and the reduction in autophagic flux caused by HG ( Figure 3E,F). These results indicate that the inhibitory effect of dapagliflozin on the reduction in autophagic flux by HG may be exerted through the activation of AMPK and the decrease in mTOR activity.

SGLT2 Inhibition Suppressed HG-Induced Inflammatory Alterations
Compared to those treated with Cont, the cells treated with HG showed increased SGLT2 expression and decreased p-AMPK expression, as well as inflammatory alterations, including increased protein levels of IL-6 ( Figure S2), TNFα, and mature IL-1β (Figure 4A,B,D,E) and secreted IL-6, TNFα, and IL-1β in cultured medium ( Figure S5A-C). Immunofluorescence staining also showed that, compared with those treated with Cont, the cells treated with HG induced the expression of IL-6. However, both dapagliflozin treatment ( Figure 4A-C, Figure S2A, Figure S5A-C) and SGLT2 knockdown ( Figure 4D-F, Figure S2B) attenuated all of the inflammatory alterations induced by HG.

SGLT2 Inhibition Suppressed HG-Induced Inflammatory Alterations
Compared to those treated with Cont, the cells treated with HG showed increased SGLT2 expression and decreased p-AMPK expression, as well as inflammatory alterations, including increased protein levels of IL-6 ( Figure S2), TNFα, and mature IL-1β ( Figure 4A,B,D,E) and secreted IL-6, TNFα, and IL-1β in cultured medium ( Figure S5A-C). Immunofluorescence staining also showed that, compared with those treated with Cont, the cells treated with HG induced the expression of IL-6. However, both dapagliflozin treatment ( Figure 4A-C, Figures S2A and S5A-C) and SGLT2 knockdown ( Figure 4D-F, Figure S2B) attenuated all of the inflammatory alterations induced by HG.

Dapagliflozin Suppressed Inflammatory Alterations by Inhibiting HG-Induced NF-κB Pathway Activation through the Activation of AMPK
To explore the mechanisms underlying the anti-inflammatory mechanism of SGLT2 inhibition, nuclear and cytoplasmic protein levels were measured after dapagliflozin treatment or SGLT2 knockdown. Compared to the effect of the Cont treatment, HG increased the nuclear translocation of NF-κB (p65), which was accompanied by increases in cytoplasmic NLRP3, mature IL-1β, and TNFα expression in the cytoplasmic fraction (Figure 5A-D). However, both dapagliflozin and SGLT2 knockdown significantly reduced the extent of the inflammatory alterations ( Figure 5A-D). These results indicated that the antiinflammatory effects of SGLT2 inhibition may contribute to the suppression of NF-κB p65 translocation in HG-treated HK-2 cells. To confirm our hypothesis, RELA siRNA (a small interfering RNA that selectively knocks down the NF-κB p65 subunit) was transfected into cells to block the aggregation of nuclear NF-κB p65 induced by HG. NF-κB p65

Dapagliflozin Suppressed Inflammatory Alterations by Inhibiting HG-Induced NF-κB Pathway Activation through the Activation of AMPK
To explore the mechanisms underlying the anti-inflammatory mechanism of SGLT2 inhibition, nuclear and cytoplasmic protein levels were measured after dapagliflozin treatment or SGLT2 knockdown. Compared to the effect of the Cont treatment, HG increased the nuclear translocation of NF-κB (p65), which was accompanied by increases in cytoplasmic NLRP3, mature IL-1β, and TNFα expression in the cytoplasmic fraction ( Figure 5A-D). However, both dapagliflozin and SGLT2 knockdown significantly reduced the extent of the inflammatory alterations ( Figure 5A-D). These results indicated that the anti-inflammatory effects of SGLT2 inhibition may contribute to the suppression of NF-κB p65 translocation in HG-treated HK-2 cells. To confirm our hypothesis, RELA siRNA (a small interfering RNA that selectively knocks down the NF-κB p65 subunit) was transfected into cells to block the aggregation of nuclear NF-κB p65 induced by HG. NF-κB p65 (RELA) knockdown (efficiency 86.2%) significantly decreased NLRP3, mature IL-1β, and TNFα expression ( Figure 6A,B), and NLRP3 knockdown (efficiency 39.0%) suppressed the HG-induced increase in mature IL-1β expression ( Figure 6C,D, Figure S3). These results indicate that dapagliflozin may reduce HG-induced inflammasome activation and increase TNFα expression through inhibition of NF-κB p65 nuclear translocation.
sults indicate that dapagliflozin may reduce HG-induced inflammasome activation and increase TNFα expression through inhibition of NF-κB p65 nuclear translocation.
To investigate the effect of AMPK on inflammation, AMPK siRNA was transfected into cells to attenuate the effect of dapagliflozin. The inhibitory effect of dapagliflozin on the HG-induced increase in nuclear translocation of NF-κB p65 was abrogated by knocking down AMPK, which was accompanied by inflammatory alteration such as elevated NLRP3, mature IL-1β, IL-6, and TNFα expression ( Figure 7A,B, Figure S2E,F). These results indicated that the effect of dapagliflozin on inflammation may be dependent on the suppression of nuclear NF-κB p65 via AMPK activation.   To investigate the effect of AMPK on inflammation, AMPK siRNA was transfected into cells to attenuate the effect of dapagliflozin. The inhibitory effect of dapagliflozin on the HG-induced increase in nuclear translocation of NF-κB p65 was abrogated by knocking down AMPK, which was accompanied by inflammatory alteration such as elevated NLRP3, mature IL-1β, IL-6, and TNFα expression ( Figure 7A,B, Figure S2E,F). These results indicated that the effect of dapagliflozin on inflammation may be dependent on the suppression of nuclear NF-κB p65 via AMPK activation. . All data represent means ± standard deviation (SD). * p < 0.05 vs. the indicated group, ** p < 0.01 vs. the indicated group (n = 3). Cont, control; HG, high glucose; siCON, control siRNA; siAMPK, AMPK siRNA; NE, nucleoprotein extracts; CE, and cytoplasmic protein extracts.

Discussion
Tubular injury is one of the characteristics of DKD [10]. SGLT2 is critical for glucose reabsorption by proximal tubular cells [2]. HK-2 cells are well-differentiated proximal tubular cells which keep the basal functions of proximal tubular epithelium such as glucose reabsorption [29]. Previous studies showed that the expression of SGLT2 is high and stable from passage 6 to passage 12 and significantly decreased at passage 18 in HK-2 cells [30,31]. Moreover, although immune cells are the main source of pro-inflammatory cytokines in kidney, studies have confirmed that HG-induced pro-inflammatory cytokines in proximal tubular cells are also involved in tubular injury under a diabetic condition [20,32]. Therefore, HK-2 cells are suitable for exploring potential mechanisms of DKD.
Previous studies demonstrated that both SGLT2 knockdown [33] and SLGT2 inhibitors [34] significantly decreased glucose uptake by human proximal tubular cells. Our results ( Figure 1) were consistent with the previous studies. AMPK is extremely sensitive to changes in energy status in response to cellular ATP consumption and the ratio of AMP or ADP/ATP [11,35]. Therefore, activation of AMPK is a key regulator of multiple

Discussion
Tubular injury is one of the characteristics of DKD [10]. SGLT2 is critical for glucose reabsorption by proximal tubular cells [2]. HK-2 cells are well-differentiated proximal tubular cells which keep the basal functions of proximal tubular epithelium such as glucose reabsorption [29]. Previous studies showed that the expression of SGLT2 is high and stable from passage 6 to passage 12 and significantly decreased at passage 18 in HK-2 cells [30,31]. Moreover, although immune cells are the main source of pro-inflammatory cytokines in kidney, studies have confirmed that HG-induced pro-inflammatory cytokines in proximal tubular cells are also involved in tubular injury under a diabetic condition [20,32]. Therefore, HK-2 cells are suitable for exploring potential mechanisms of DKD.
Previous studies demonstrated that both SGLT2 knockdown [33] and SLGT2 inhibitors [34] significantly decreased glucose uptake by human proximal tubular cells. Our results (Figure 1) were consistent with the previous studies. AMPK is extremely sensitive to changes in energy status in response to cellular ATP consumption and the ratio of AMP or ADP/ATP [11,35]. Therefore, activation of AMPK is a key regulator of multiple nutritional and metabolic pathways, including gluconeogenesis, glycolysis, lipogenesis, protein synthesis, and autophagy pathways [11]. DKD is considered a state of overnutrition, with a marked inhibition of AMPK phosphorylation due to a decreased ratio of AMP/ATP or ADP/ATP [14,34,36]. Multiple studies have demonstrated that SGLT2 inhibitors including canagliflozin, empagliflozin, luseogliflozin, and ipragliflozin can activate p-AMPK [14,16,34,37,38]. A previous study showed that the SGLT2 inhibitor canagliflozin, not dapagliflozin or empagliflozin, had a dose-dependent effect (0-100 µM) on AMPK activation in HEK-293 cells [14]. In our study, dapagliflozin showed an effect equivalent to that of canagliflozin in inducing AMPK activity at concentrations from 10 to 100 µM in HK-2 cells. Monitoring the cytotoxicity of high concentrations of dapagliflozin is necessary in cell experiments. Our cytotoxicity detection showed that more than 50 µM of dapagliflozin may increase cell death ( Figure S5D), so the minimum effective concentration (20 µM) was safe and effective for further cell experiments in our study.
Autophagy impairment in renal cells is related to the pathogenesis of DKD [39]. Inhibition of AMPK caused by HG leads to the activation of the mTOR pathway, which contributes to renal hypertrophy and injury [40,41]. Our previous studies demonstrated that the suppression of AMPK and activation of the mTOR pathway contributed to HGinduced autophagy impairment under DKD conditions [42][43][44]. However, a recent study indicated that autophagy activity has the opposite manifestation in type 1 and type 2 diabetes mellitus (T1DM and T2DM) [45]. In a previous in vitro study, HG (treatment for 6-12 h, imitating T1DM) directly induced autophagic flux due to decreased lysosomal activity, while HG suppressed autophagic flux only in the presence of insulin (imitating T2DM) via mTOR activation [45]. In our study, HG induced p-S6RP increases with or without insulin (100 nM), indicating HG can directly activate the mTOR pathway, which was consistent with results from previous research [40][41][42][43]. Although compared with HG, the activation of the mTOR pathway in the presence of insulin was more obvious, there was no difference in the suppression of p-AMPK and autophagic flux ( Figure S4). The difference between our study and the previous study may be based on the incubation time of cells with HG medium (24-48 h vs. 6-12 h). Previous studies demonstrated that SGLT2 inhibitors enhance autophagy, including increases in the microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and decreased p62 via the AMPK/mTOR pathway [15,16,46]. In our study, with the suppression of p-AMPK via HG or HG plus insulin, autophagic flux was significantly suppressed. Dapagliflozin treatment can restore HG-impaired p-AMPK and autophgic flux in the presence and absence of insulin ( Figure S4). These data indicated that dapagliflozin can restore impaired autophagic flux in both a T1DM and T2DM condition in vitro. In addition, AMPK knockdown via siRNA induced p-S6RP expression and suppressed autophagic flux. Moreover, the mTOR inhibitor rapamycin activated autophagic flux by suppressing p-S6RP expression. All this evidence indicated that dapagliflozin restored the HG-induced autophagy impairment previously induced through via the AMPK/mTOR pathway, which is consistent with the reports of previous research [15,16]. There was an interesting phenomenon in our experiment. Compared with other groups, basal LC3-II was significantly increased in the AMPK-knockdown group. However, this change did not indicate that autophagy was activated, as chloroquine did not increase the expression of LC3-II in the AMPK knockdown group. We speculated that the increase in basal LC3-II induced by AMPK knockdown may be due to the failure of autophagosome clearance.
Inflammation is another significant feature of DKD [47]. Hyperglycemia activates the production of multiple proinflammatory cytokines (e.g., IL-1, IL-6, and TNFα) and leads to the recruitment of inflammatory cells to the kidney [48,49]. Previous studies demonstrated that SGLT2 inhibitors decreased the accumulation of nuclear NF-κB p65 in db/db mice [50], high-fat diet-fed Wistar rats [51], and HG-treated HK-2 cells [52] and then suppressed downstream proteins, including TNFα and IL-1β [51]. All these studies indicated that the translocation of the NF-κB p65 subunit is crucial for activating inflammation in DKD. On the other hand, the release of IL-1β is controlled by NLRP3 inflammasome activation [22,23,53]. NLRP3 activation may be involved in multiple inflammatory or stress pathways, including NF-κB signaling pathways [23]. Lipopolysaccharide (LPS)-induced NF-κB can bind to the NLRP3 promoter to directly regulate its expression in human macrophages [21], which indicates that NLRP3 is a downstream gene of NF-κB. Multiple studies have shown that SGLT2 inhibition suppresses the NLRP3 inflammasome in different diabetic animal models [26,27,[54][55][56]. Dapagliflozin suppressed the activation of the NLRP3 inflammasome in diabetic myocardial tissues [26] and reduced the production of IL-1β in aortic and liver tissues [54,55]. AMPK can suppress inflammatory signaling [25] and NLRP3 activation [57,58]. Other studies demonstrated that AMPK activation may inhibit the protein modification of NF-κB p65, including its phosphorylation [58][59][60]. As a transcriptional regulator of a variety of proinflammatory cytokines, the nuclear translocation of NF-κB p65 is an important step for the activation of proinflammatory cytokines. In the present study, we found that the increased translocation of NF-κB p65 from the cytoplasm to the nucleus was critical for the inflammation caused by HG and that AMPK activation suppresses these changes. Our findings provide another potential mechanism of dapagliflozin in the inflammation induced via AMPK/NF-κB p65/NLRP3 signaling in human kidney proximal tubular cells. Our study illustrated the possible mechanisms of the renoprotection of dapagliflozin in the treatment of DKD and suggested other potential benefits of dapagliflozin beyond glucose control. However, as this was only an in vitro study, our results need to be further verified in rodent models of T1DM and T2DM in the future.
activation [22,23,53]. NLRP3 activation may be involved in multiple inflammatory or stress pathways, including NF-κB signaling pathways [23]. Lipopolysaccharide (LPS)-induced NF-κB can bind to the NLRP3 promoter to directly regulate its expression in human macrophages [21], which indicates that NLRP3 is a downstream gene of NF-κB. Multiple studies have shown that SGLT2 inhibition suppresses the NLRP3 inflammasome in different diabetic animal models [26,27,[54][55][56]. Dapagliflozin suppressed the activation of the NLRP3 inflammasome in diabetic myocardial tissues [26] and reduced the production of IL-1β in aortic and liver tissues [54,55]. AMPK can suppress inflammatory signaling [25] and NLRP3 activation [57,58]. Other studies demonstrated that AMPK activation may inhibit the protein modification of NF-κB p65, including its phosphorylation [58][59][60]. As a transcriptional regulator of a variety of proinflammatory cytokines, the nuclear translocation of NF-κB p65 is an important step for the activation of proinflammatory cytokines. In the present study, we found that the increased translocation of NF-κB p65 from the cytoplasm to the nucleus was critical for the inflammation caused by HG and that AMPK activation suppresses these changes. Our findings provide another potential mechanism of dapagliflozin in the inflammation induced via AMPK/NF-κB p65/NLRP3 signaling in human kidney proximal tubular cells. Our study illustrated the possible mechanisms of the renoprotection of dapagliflozin in the treatment of DKD and suggested other potential benefits of dapagliflozin beyond glucose control. However, as this was only an in vitro study, our results need to be further verified in rodent models of T1DM and T2DM in the future.

Conclusions
In diabetic renal proximal tubular cells, HG-induced decreased AMPK activity is involved in (1) mTOR activation and autophagic flux reduction and (2) inflammatory alterations (inflammasome activation and increased TNFα expression) due to NF-κB (p65) pathway activation (Figure 8). Dapagliflozin ameliorates the alterations caused by HG. Figure 8. The mechanism of SGLT2 inhibition in diabetic renal proximal tubular cells. High glucose-induced decrease in AMPK activity is involved in (1) mTOR activation and autophagic flux reduction and (2) inflammatory alterations (inflammasome activation and increased TNFα and IL-6 expression) due to NF-κB (p65) pathway activation. Dapagliflozin attenuates the alterations caused by high glucose.

Data Availability Statement:
The data presented in this study are available on reasonable request from the corresponding author.