Sodium-glucose Cotransporter 2 Protein Research Articles

Breaking boundaries in diabetic nephropathy treatment: design and synthesis of novel steroidal SGLT2 inhibitors.

The activity of sodium glucose co-transporter 2 (SGLT2) has always been an important parameter influencing chronic kidney disease in type-2 diabetic patients. Herein, we have meticulously designed, synthesized, and evaluated several novel steroidal pyrimidine molecules that possess the capability to successfully bind to the SGLT2 protein and inhibit its activity, thereby remedying kidney-related ailments in diabetic patients. The lead steroidal pyrimidine compounds were selected after virtually screening from a library of probable N-heterocyclic steroidal scaffolds. A nano-catalyzed synthetic route was also explored for the synthesis of the steroidal pyrimidine analogs demonstrating an environmentally benign protocol. Extensive in vitro investigations encompassing SGLT2 screening assays and cell viability assessments were conducted on the synthesized compounds. Among the steroidal pyrimidine derivatives evaluated, compound 9a exhibited the highest SGLT2 inhibition activity and underwent further scrutiny. Western blot analysis was employed to determine the impact of 9a on inflammatory and fibrotic proteins, aiming to elucidate its mechanism of action. Additionally, in silico analyses were performed to illuminate the structural dynamics and molecular interaction mechanism of 9a. The overall investigation is crucial for advancing the development of the next generation of anti-diabetic drugs.

Isoquercitrin improves diabetes nephropathy by inhibiting the sodium-glucose co-transporter-2 pathway.

Diabetic nephropathy (DN) is one of the most severe kidney complications and the primary contributor to end-stage renal disease on a global scale. It exacerbates the morbidity, mortality, and financial burden for individuals with diabetes. Isoquercitrin, a natural compound found in various plants, has demonstrated potential as an antidiabetic agent. However, it remains uncertain whether isoquercitrin exerts a protective effect on DN. Therefore, the objective of this study was to explore whether isoquercitrin confers a protective effect on DN and its potential mechanism. In vivo, a mouse model of DN induced by streptozotocin was established in the study. The hypoglycemic effect of isoquercitrin was assessed by measuring fasting blood glucose levels, insulin tolerance tests, and glucose tolerance test in animals. Urinary albumin creatinine ratio, serum lipid levels, and pathological changes in renal tissues were measured to evaluate the protective effect of isoquercitrin against DN. The expression of Sodium glucose co-transporter-2(SGLT2) was analyzed using real-time quantitative PCR and immunohistochemistry. The studies suggest that isoquercitrin significantly reduces fasting blood glucose levels, enhances the body's capacity to regulate blood glucose and insulin resistance, and facilitates renal pathology and renal function. Simultaneously, it can lower blood lipids (total cholesterol and triglyceride) and improve the risk factors of DN. Meanwhile, isoquercitrin suppressed the expression of SGLT2 in renal tissues of DN mouse models. In vitro, real-time quantitative PCR and Western blot were used to detect the expression of SGLT2 in the human renal tubular epithelial (HK-2) cells. The effects of isoquercitrin on the survival rate and glucose uptake capacity of HK-2cells were determined by Cell-Counting-Kit-8 and glucose uptake methods. The results demonstrate that isoquercitrin suppressed the up-regulation of SGLT2 mRNA and protein in high-glucose-induced HK-2cells. Additionally, isoquercitrin inhibited glucose uptake in HK-2cells and mitigated high-sugar-induced damage. Thus, this study has concluded that isoquercitrin exhibits hypoglycemic and renal protective effects by inhibiting the SGLT2 pathway, indicating its potential as a promising anti-DN drug deserving further clinical investigation.

Myocardial SGLT2 expression and cardiac remodeling in patients with severe aortic stenosis: the BIO-AS Study

Abstract Background Cardiac remodeling plays a major role in the prognosis of patients with aortic stenosis (AS) and could impact the benefits of aortic valve replacement. Blood and tissue biomarkers have a differential pattern and expression level in patients with AS, which may retain a pathophysiological role in cardiac remodeling and metabolism. However, the data available are limited and contradictory. Aim Our study aimed to evaluate the expression of sodium-glucose cotransporter 2 (SGLT2) gene and protein in patients with severe AS stratified in high gradient (HG) and low flow-low gradient (LF-LG) AS and its association with cardiac functional impairments. Methods Gene expression and protein levels of main biomarkers of cardiac fibrosis (Galectin-3, sST2, Serpin-4, PINP, PICP, Collagen, TGF-b), inflammation (GDF-15, IL-6, NF-kB), oxidative stress (SOD1, SOD2), and cardiac metabolism (NHE, PPAR-a, PPAR-g, GLUT1, and GLUT4) were evaluated in blood samples and heart biopsies of 45 patients with AS. Results Our study showed SGLT2 gene and protein hyper-expression in patients with LF-LG AS, compared to controls and HG AS (p<0.05). These differences remained significant even after adjusting for age, gender, body mass index, history of diabetes mellitus, arterial hypertension, and coronary artery disease. SGLT2 gene expression was positively correlated with: i) TGF-b (r=0.72, p<0.001) and collagen (r=0.73, p<0.001) as markers of fibrosis; ii) NF-kB (r=0.36, p<0.01) and myocardial IL-6 (r=0.68, p<0.001) as markers of inflammation: iii) SOD2 (r=-0.38, p<0.006) as a marker of oxidative stress; iv) GLUT4 (r= 0.33, p<0.02) and PPAR-a (r=0.36, p<0.01) as markers of cardiac metabolism (Figure 1). Conclusion In patients with LF-LG AS, SGLT2 gene and protein were hyper-expressed in cardiomyocytes and associated with myocardial fibrosis, inflammation, and oxidative stress. The hyper-expression of the SGLT2 gene and protein in patients with LF-LG might pave the way for using SGLT2-Is in this subset population, with the potential to improve the outcomes of AVR and reduce the likelihood of re-hospitalization within one year.Figure 1

Abstract P138: Diet Influences the Expression of Renal Sodium-Glucose Cotransporters in Juvenile Male Rats

Poor nutrition and metabolic dysfunction are becoming more prevalent among children, as evident by increasing childhood obesity in recent years. The kidney sodium-glucose cotransporter 2 (SGLT2) is a contributor to overall metabolic homeostasis, with inhibition of SGLT2 significantly reducing circulating glucose and lipids. The study objective was to determine if diet influences the expression of renal glucose transporters in young rats. Weanling male Wistar Kyoto rats received 1) standard laboratory chow, 2) AIN-93G diet, or 3) a high-fat diet (60% kcal, HFD). Each dietary group was then further subdivided to receive either filtered water or 5% (w/v) sucrose solution ad libitum . Four weeks later, animals were euthanized and kidneys were collected and processed. mRNA expression of Sglt1 , Sglt2 , Sqstm1 , and Nlrp3 was determined using gene specific primers and RT-PCR. Renal SGLT2 protein expression was visualized using immunohistochemistry and quantified using Imagej software. Diet significantly influenced the renal expression of both Sglt1 ( P =0.005) and Sglt2 (P=0.046), while beverage did not have a significant effect on either ( P =0.7 and P =0.4, respectively). Rats fed the HFD had more than 30% reduction in Sglt1 mRNA expression as compared to rats fed chow, and ~20% reduction in Sglt2 as compared to rats fed AIN. Preliminary data suggest that diet and beverage significantly altered renal protein expression of SGLT2, with rats fed chow having the lowest renal expression that was slightly increased with sugar consumption (1.5% versus 2.7%, respectively). Rats fed AIN had the greatest expression, but was drastically reduced in rats consuming sugar (4% versus 1.2%, respectively). To determine if autophagy and inflammasome activation is involved, the expression of Sqstm1 and Nlrp3 was measured by RT-PCR. Similarly, rats fed HFD had the lowest expression of both Sqstm1 ( P<0.001) and Nlrp3 ( P=0.03). Sqstm1 and Nlrp3 mRNA expression was decreased with sugar consumption in rats fed chow and AIN, whereas Sqstm1 ( P =0.01) and Nlrp3 ( P <0.001) mRNA expression was increased in rats fed HFD with sugar beverage. Our data suggest that diet significantly influences renal glucose transporter expression in juvenile rats in a way that may be mediated by autophagic impairment. The modulatory effect of sugar consumption on SGLT2 protein, Sqstm1, and Nlrp3 was diet-dependent, highlighting the importance of nutrition to renal physiology within weeks after weaning.

Key results from observational studies and real-world evidence of sodium-glucose cotransporter-2 inhibitor effectiveness and safety in reducing cardio-renal risk.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, originally designed to manage blood sugar levels in individuals with type 2 diabetes (T2D), have emerged as a crucial class of drugs for managing cardio-renal diseases. These drugs work by targeting the SGLT2 protein in the kidneys, promoting the excretion of glucose and influencing metabolic pathways beyond glucose control. The relationship between cardio-renal diseases and SGLT2 inhibitors has been explored through landmark trials and real-world evidence (RWE) studies, demonstrating significant reductions in cardio-renal complications. This review discusses the importance of RWE studies alongside randomized controlled trials in understanding the real-world effectiveness and safety of SGLT2 inhibitors. It outlines the advantages and disadvantages of RWE compared to RCTs, highlighting their complementary roles in providing comprehensive insights into treatment outcomes. By examining a range of RWE studies, the review underscores the cardio-renal benefits of SGLT2 inhibitors across various patient populations. Safety assessments indicate that SGLT2 inhibitors are generally well tolerated, with severe adverse events being rare. Common issues, such as genital mycotic infections and urinary tract infections, are acknowledged, alongside less frequent but significant adverse events including diabetic ketoacidosis, lower-limb amputations, and bone fractures. In summary, SGLT2 inhibitors show promising cardio-renal protective effects in real-world scenarios across diverse populations in T2D, indicating their potential as early intervention measures. Continued research is essential for gaining a thorough understanding of their long-term effects and safety profiles.

Identification of phytocompounds as potent inhibitors of sodium/glucose cotransporter-2 leading to diabetes treatment

Type-II diabetes, a major metabolic disorder has threatened the very existence of a healthy life since long ago. Commercially available antidiabetic drugs are known for several adverse effects. The present study attempted to identify potential phytocompounds as inhibitors of sodium/glucose cotransporter-2 (SGLT2), a major protein that helps in glucose re-absorption from renal tubules. A total of 28 phytocompounds were collected based on the literature survey. 3D co-ordinates of phytocompounds were collected from PubChem database. Molecular docking was carried out with SGLT2 protein and the best 3 docking complexes were subjected to molecular dynamics simulation for 100 ns. Free energy changes were also analyzed using MM/PBSA analysis. Phytocompounds were also analyzed for their drug-likeness and ADMET properties. Docking study observed a strong binding affinity of phytocompounds (> −7.0 kcal/mol). More than 10 phytocompounds showed better binding affinity compared to reference drugs. Further analysis of three best docking complexes when analyzed by MD simulation showed better stability and compactness of the complexes compared to reference drug, empagliflozin. MM/PBSA analysis also revealed that van der Waals force and electrostatic energy are the major binding energy involved in the complex formation. Like docking energy, free energy analysis also observed stronger binding energies (ΔGGAS) in SGLT2-phytocompound complexes compared to empagliflozin complex. All the phytocompounds showed drug-likeness and considerable ADMET properties. The study, therefore, suggests that Trifolirhizin-6′-monoacetate, Aspalathin, and Quercetin-3-glucoside could be a possible inhibitor of SGLT2 protein. However, further studies need to be carried out to reveal the exact mode of activity. Communicated by Ramaswamy H. Sarma

Myocardial sodium-glucose cotransporter 2 expression and cardiac remodelling in patients with severe aortic stenosis: The BIO-AS study.

Cardiac remodelling plays a major role in the prognosis of patients with aortic stenosis (AS) and could impact the benefits of aortic valve replacement. Our study aimed to evaluate the expression of sodium-glucose cotransporter 2 (SGLT2) gene and protein in patients with severe AS stratified in high gradient (HG) and low flow-low gradient (LF-LG) AS and its association with cardiac functional impairments. Gene expression and protein levels of main biomarkers of cardiac fibrosis (galectin-3, sST2, serpin-4, procollagen type I amino-terminal peptide, procollagen type I carboxy-terminal propeptide, collagen, transforming growth factor [TGF]-β), inflammation (growth differentiation factor-15, interleukin-6, nuclear factor-κB [NF-κB]), oxidative stress (superoxide dismutase 1 [SOD1] and 2 [SOD2]), and cardiac metabolism (sodium-hydrogen exchanger, peroxisome proliferator-activated receptor [PPAR]-α, PPAR-γ, glucose transporter 1 [GLUT1] and 4 [GLUT4]) were evaluated in blood samples and heart biopsies of 45 patients with AS. Our study showed SGLT2 gene and protein hyper-expression in patients with LF-LG AS, compared to controls and HG AS (p < 0.05). These differences remained significant even after adjusting for age, gender, body mass index, history of diabetes mellitus, arterial hypertension, and coronary artery disease. SGLT2 gene expression was positively correlated with: (i) TGF-β (r = 0.72, p < 0.001) and collagen (r = 0.73, p < 0.001) as markers of fibrosis; (ii) NF-κB (r = 0.36, p < 0.01) and myocardial interleukin-6 (r = 0.68, p < 0.001) as markers of inflammation: (iii) SOD2 (r = -0.38, p < 0.006) as a marker of oxidative stress; (iv) GLUT4 (r = 0.33, p < 0.02) and PPAR-α (r = 0.36, p < 0.01) as markers of cardiac metabolism. In patients with LF-LG AS, SGLT2 gene and protein were hyper-expressed in cardiomyocytes and associated with myocardial fibrosis, inflammation, and oxidative stress.

Sodium-Glucose Cotransporter 2 (SGLT2) Inhibition Induces DNA Damage and Radiosensitization in Glioblastoma Cell Lines

Glioblastomas (GBM) are highly dependent on glucose for energy to promote cell proliferation and tumor growth. Preclinical studies have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors, drugs that are FDA approved for diabetes, reduce the viability and invasiveness of GBM cells. We hypothesize that SGLT2 inhibitors decrease GBM cell growth and viability by promoting DNA damage. The SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin were used and cell studies were performed under low glucose (0.6 mM) media conditions. In the cell proliferation assays, cells were counted in triplicate after 24-, 48- and 72-hour treatment with SGLT2 inhibitors. Glucose transport assays were performed using a 6-fluorescent iodinated glucose analog (6FIGA) which is selectively transported by SGLTs and not by glucose transporters (GLUTs). Immunoblots were used to measure protein expression of SGLT2 and markers of DNA damage (phospho-ATM and gamma-H2AX). Cellular radiosensitization was evaluated by clonogenic survival assays. SGLT2 inhibition with canagliflozin, dapagliflozin and empagliflozin decreased cell proliferation in LN229, U118MG and LN18 GBM cells lines. In LN229 cells, 72-hour treatment with canagliflozin (30 µM) decreased cell proliferation by 60% compared to vehicle control cells (p<0.0001) whereas dapagliflozin (30 µM) and empagliflozin (30 µM) decreased cell proliferation compared to vehicle control cells by 17% (p<0.0001) and 19% (p<0.001), respectively. Glucose transport assays show that 6FIGA uptake is reduced by canagliflozin and dapagliflozin in LN229 and U87 GBM cells. Canagliflozin treatment (50 µM) for 24 hours in LN229 cells results in increased phosphorylated ATM protein and increased gamma-H2AX protein, markers of DNA damage, by immunoblot. SGLT2 protein expression was not affected by canagliflozin. Treatment with canagliflozin (50 µM) 48 hours prior to 2, 4 and 6 Gy of ionizing radiation reduced clonogenic survival of LN229, LN18 and U118MG GBM cells. SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin decrease GBM cell proliferation, with the highest inhibitory effect seen with canagliflozin. Canagliflozin treatment alone increases DNA damage signaling in GBM cells. Canagliflozin radiosensitizes GBM cells. This work provides preliminary preclinical rationale for canagliflozin as a radiosensitizer in GBM.

Maternal dexamethasone exposure does not affect glucose tolerance but alters renal haemodynamics in F1 rats in a sex-dependent manner.

Prenatal programming with dexamethasone increases the risk of the development of hyperglycaemia and insulin resistance, leading to diabetes in adulthood. Dexamethasone also causes a decline in renal glomerular filtration in the adult offspring. Sodium-glucose cotransporter-2 (SGLT2) plays a significant role in regulating blood glucose and renal haemodynamics in diabetic patients. However, the role of SGLT2 in dexamethasone-induced programming and the putative sex-dependent effects on the changes named earlier is unknown. Therefore, this study aimed to investigate the impact of maternal dexamethasone treatment on glucose tolerance, insulin sensitivity, renal perfusion and renal function in adult male and female offspring and the possible contribution of SGLT2 to these changes. Pregnant Sprague Dawley rats (F0 ) were treated with either vehicle or dexamethasone (0.2 mg/kg ip) from gestation Day 15 to 20. F1 males and F1 females were randomly selected from each mother at 4 months of age. There was no change in serum Na+ , Na+ excretion rate, glucose tolerance or insulin sensitivity in F1 male or female rats. However, dexamethasone caused significant glomerular hypertrophy and decreases in CSinistrin and CPAH indicating decreased glomerular filtration rate and renal plasma flow, respectively, in dexamethasone-treated F1 male but not female rats. Dexamethasone did not affect SGLT2 mRNA or protein expression in F1 males or females. We conclude that dexamethasone-mediated prenatal programming of glomerular volume, renal function and haemodynamics is sex-dependent, occurring only in adult male offspring.

The Sympathetic Nervous System Regulates Sodium Glucose Co-Transporter 1 Expression in the Kidney

Hyperactivation of the sympathetic nervous system (SNS) has been demonstrated in various conditions including obesity, hypertension and type 2 diabetes. Elevated levels of the major neurotransmitter of the SNS, norepinephrine (NE), is a cardinal feature of these conditions. Increased levels of the sodium glucose cotransporter 1 (SGLT1) protein have been shown to occur in the parotid and submandibular glands of hypertensive rodents compared to normotensive controls. However, there was a need to examine SGLT1 expression in other tissues, such as the kidneys. Whether NE may directly affect SGLT1 protein expression has not yet been investigated, although such a link has been shown for sodium glucose cotransporter 2 (SGLT2). Hence, we aimed to determine (i) whether our murine model of neurogenic hypertension displays elevated renal SGLT1 expression and (ii) whether NE may directly promote elevations of SGLT1 in human proximal tubule (HK2) cells. We did indeed demonstrate that in vivo, in our mouse model of neurogenic hypertension, hyperactivation of the SNS promotes SGLT1 expression in the kidneys. In subsequent in vitro experiments in HK2 cells, we found that NE increased SGLT1 protein expression and translocation as assessed by both specific immunohistochemistry and/or a specific SGLT1 ELISA. Additionally, NE promoted a significant elevation in interleukin-6 (IL-6) levels which resulted in the promotion of SGLT1 expression and proliferation in HK2 cells. Our findings suggest that the SNS upregulates SGLT1 protein expression levels with potential adverse consequences for cardiometabolic control. SGLT1 inhibition may therefore provide a useful therapeutic target in conditions characterized by increased SNS activity, such as chronic kidney disease.

Obesity and overweight are linked to increased sodium-glucose cotransporter 1 and glucose transporter 5 levels in duodenum.

Prior evidence indicates that individualswith obesity have an accelerated intestinal glucose absorption. This cross-sectional study evaluated whether those with overweight or obesity display higher duodenal protein levels of the glucose carriers sodium-glucose cotransporter 1 (SGLT-1), glucose transporter 2 (GLUT-2), and glucose transporter 5 (GLUT-5). SGLT-1, GLUT-2, and GLUT-5 protein levels were assessed on duodenal mucosa biopsies of 52 individuals without diabetes categorized on the basis of their BMI as lean, with overweight, or with obesity. Individualswith overweight and obesity exhibited progressively increased duodenal protein levels of SGLT-1 and GLUT-5 as compared with the lean group. Conversely, no differences in duodenal GLUT-2 abundance were found among the three groups. Univariate analysis showed that SGLT-1 and GLUT-5 protein levels were positively correlated with BMI, waist circumference, 1-hour post-load glucose, fasting and post-load insulin, and insulin secretion and resistance levels. Furthermore, a positive relationship was detected between intestinal GLUT-5 levels and serum uric acid concentrations, a product of fructose metabolism known to be involved in the pathogenesis of obesity and its complications. Individualswith overweight and obesity display enhanced duodenal SGLT-1 and GLUT-5 abundance, which correlates with increased postprandial glucose concentrations, insulin resistance, and hyperinsulinemia.

Effect of Dapagliflozin in Lowering Risk of Heart Failure Severity: Meta-Analysis

Background: Heart failure is a progressive health problem with high mortality and morbidity rates in developed as well as developing countries including Indonesia. Dapagliflozin is one of the oral antidiabetic medicines of the class of sodium-glucose cotransporter-2 (SGLT2) inhibitors, used in people with type 2 diabetes mellitus (DMT2). Unlike other oral anti-diabetes that work to stimulate insulin secretion or increase insulin sensitivity, dapagliflozin works in the kidneys by competitively inhibiting the SGLT2 protein reversibly which serves in glucose reabsorption in the glomerulus thereby lowering blood sugar levels in T2DM patients. This study aimed to determine the estimated effect of dapagliflozin on the severity of heart failure patients.Subjects and Method: This study was a systematic review and meta-analysis with PICO. Population= Heart failure patients over 18 years old. Intervention= administration of dapagliflozin. Comparison= Placebo. Outcome= severity of Heart Failure. The articles used PRISMA flowchart guidelines. The article search process was conducted from 2019 to 2022 using databases from PubMed, Google Scholar, and Scopus with the search keywords Dapagliflozin, Heart Failure, and Placebo. The analysis was performed using RevMan 5.4 software.Results: A total of 8 articles from across 2 continents, America and Asia, reviewed in the meta-analysis, showed that administering Dapagliflozin could reduce the risk of heart failure severity by 0.99 units compared to without dapagliflozin, however, it was statistically insignificant (OR= 0.99; 95% CI= 0.92 to 1.06; p= 0.710).Conclusion: The administration of Dapagliflozin lowers the risk of heart failure severity and is statistically significant. Keywords: dapagliflozin, heart failure, placebo Correspondence: Andreza. Universitas Hasanuddin. Jl. Perintis Kemerdekaan No.KM.10, Tamalanrea Indah, Kec. Tamalanrea, Makasar City, South Sulawesi. Email: andrezafauzialghifary@gmail.com. Mobile: +625299015320.

S-51-4: RENAL DENERVATION IMPROVES FLUID RETENTION BY MODULATING RENAL SGLT2 FUNCTION IN RATS WITH HEART FAILURE

Objective: Renal denervation (RDN) has been developed as a neuromodulation therapy for patients with treatment-resistant hypertension. RDN is expected to prevent not only hypertension but also heart failure (HF). Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to ameliorate HF and widely used for HF patients. The present study was conducted to assess the effect of RDN on HF and the interaction between renal sympathetic nerves and SGLT2 function in HF. Design and method: HF was induced by coronary artery ligation in rats. Four weeks after coronary artery ligation surgery, surgical bilateral RDN was performed. Western blot analysis was performed to assess the expression levels of SGLT2. One week after RDN surgery, as a functional test for SGLT2 activation, natriuretic response to intravenous injection of SGLT2 inhibitor dapagliflozin (0.5 mg/kg, i.v.) was investigated. Furthermore, the changes in expression and localization of SGLT2 were assessed in human embryonic kidney cells treated with norepinephrine directly, in vitro. Results: Western blot analysis indicated that renal SGLT2 expression is increased in rats with HF. RDN significantly reduced enhanced renal SGLT2 expression in HF. Natriuretic response to dapagliflozin were greater in HF than Sham (Cumulative sodium excretion at 120 min, 227 ± 35 vs. 133 ± 13 μEq/gkw, P &lt; 0.05, n = 6). RDN significantly attenuated natriuretic response to dapagliflozin in HF (Cumulative sodium excretion at 120 min, 138 ± 19 vs. 227 ± 35 μEq/gkw, P &lt; 0.05, n = 6). Direct application of norepinephrine on human embryonic kidney cells, in vitro, resulted in an increased expression of SGLT2 and specifically promoted the translocation of SGLT2 from the cytosol to the cell surface (Cytosolic SGLT2/GAPDH 0.51 ± 0.05 vs. 0.86 ± 0.08, Membranous SGLT2/Na-K-ATPase 1.06 ± 0.10 vs. 0.54 ± 0.03, P &lt; 0.01, n = 6). Moreover, RDN increased the cytosolic SGLT2 while decreasing membranous SGLT2 in the kidneys of rats with HF (Cytosolic SGLT2/GAPDH 1.30 ± 0.11 vs. 0.58 ± 0.09, Membranous SGLT2/Na-K-ATPase 0.51 ± 0.10 vs. 2.08 ± 0.08, P &lt; 0.01, n = 6). Conclusions: These findings suggest that the excessive activation of renal sympathetic nerves in HF increases the expression of SGLT2 protein trafficked by norepinephrine to cell membrane in proximal tubules resulting in greater sodium retention associated with HF condition. RDN mitigates the enhanced expression and activity of SGLT2 and ameliorates the subsequent sodium and water retention associated with HF, suggesting the potential therapeutic use of RDN for HF.

Hedgehog interacting protein activates sodium-glucose cotransporter 2 expression and promotes renal tubular epithelial cell senescence in a mouse model of type 1 diabetes.

Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1β, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Sodium-glucose cotransporter-2 (SGLT2) expression in diabetic and non-diabetic failing human cardiomyocytes

This study aimed at investigating the SGLT2 expression in human cardiomyocytes. Human studies evaluating cardiomyocyte SGLT2s expression are limited. To better clarify this issue, SGLT2 protein expression was assessed in human hearts of diabetic and non-diabetic patients, and in AC16 human cardiomyocyte cell line. A prospective study with a follow-up of patients who underwent their first heart transplant (HTX) was performed. Explanted heart, basal (1 week after HTX), and final (48 weeks after HTX) endomyocardial biopsies (EMBs) from patients were evaluated for SGLT2 occurrence in cardiomyocyte with immunohistochemistry, immunofluorescence and SGLT2 quantization with both real-time reverse transcription-polymerase chain reaction and Western blot analysis. The immunofluorescence co-localization of SGLT2 in cardiomyocyte evidenced that an increased expression in the explanted heart from diabetic patients compared to non-diabetic (p < 0.001). In all final EMBs from diabetic patients, the expression of SGLT2 in cardiomyocyte was increased compared to non-diabetic (p < 0.01). This evidence was confirmed by Western blot analysis of SGLT2 protein. In addition, PCR analysis revealed very low mRNA levels in basal EMBs from diabetic and non-diabetic patients (p = NS), whereas final EMBs from diabetic patients showed higher SGLT2 mRNA levels in diabetic compared to non-diabetic patients (p < 0.05). Cultured human cardiomyocytes exposed to high-glucose showed increased expression of SGLT2 protein compared to cells exposed to normal glucose (p < 0.05). The presence of SGLT2 in cardiomyocytes supports the hypothesis of SGLT2i-mediated impact on metabolic pathways within cardiomyocytes. Moreover, metabolic disorders linked to diabetes may lead promptly to upregulation of SGLT2 levels in human cardiomyocytes.

Abstract 6402: Associations of sodium glucose cotransporter-2 (SGLT2), metabolic disorders, and survival following incident epithelial ovarian cancer

Abstract Background: Up to 75% of ovarian cancers will become platinum resistant and survival following the development of resistance is typically less than one year. New therapies with higher curative rates are needed to improve prognosis. There has been increasing interest in inhibitors of sodium glucose cotransporter-2 (SGLT2) in suppressing tumor growth. Use of SGLT2 inhibitors show promise not only due to their ability to inhibit glucose uptake, but also from the subsequent improvements in other metabolic disorders that may affect cancer prognosis. Purpose: We evaluated the associations of tumor SGLT2 protein expression with survival following diagnosis of incident epithelial ovarian cancer (EOC). Methods: Tumor samples were obtained from 492 patients undergoing debulking surgery for primary EOC at Roswell Park Comprehensive Cancer Center between 1995 and 2002. Only incident cases and patients who did not receive neo-adjuvant chemotherapy were included in analyses (n=396). Tissue samples were compiled into tissue microarray blocks that were sectioned, stained, imaged, and analyzed for SGLT2 expression. Data on patient, clinical, and tumor characteristics, along with available information on components of metabolic disorders, were linked to each patient. Multivariate Cox proportional hazards models were performed to determine hazards ratios (HR) and 95% confidence intervals (CI) for EOC-specific and all-cause mortality. Results: In our analysis of 396 EOC patients seen at Roswell Park, we observed significant associations between high SGLT2 expression and decreased risk of both EOC-specific (HR: 0.68; 95% CI: 0.49-0.94) and all-cause mortality (HR: 0.71; 95% CI: 0.53-0.95). In particular, among those with advanced-stage tumors, there was a significant association between high SGLT2 expression and lower risk of EOC-specific (HR: 0.61; 95% CI: 0.42-0.91) and all-cause mortality (HR: 0.64; 95% CI: 0.45-0.90). A lower risk of all-cause mortality was observed among those with high-grade tumors (HR: 0.69; 95% CI: 0.48-0.98). Trends of increasing SGLT2 associated with decreased risk of mortality varied by histological subtype. The inverse association between high SGLT2 and EOC-death was stronger among those with a history of hyperlipidemia (HR: 0.46; 95% CI: 0.19-0.99) and obesity (HR: 0.43; 95% CI: 0.23-0.82) compared to other metabolic syndrome components. These associations were less robust for all-cause mortality. Conclusion: Contrary to our initial hypothesis, higher SGLT2 expression among EOC patients may be associated with more favorable prognosis. This association may be stronger for high grade and advanced stage tumors as well as non-serous subtypes. Identifying other factors associated with levels of expression could aid in the development of potential screening interventions and more personalized treatment. Citation Format: Jennifer M. Mongiovi, Jo L. Freudenheim, Jeff C. Miecznikowski, Susan E. McCann, Ashley E. Stenzel, Wiam Bshara, Agnieszka Witkiewicz, Leighton Stein, Lisa D. Kellenberger, Ahmad Al Sulimani, Kirsten B. Moysich. Associations of sodium glucose cotransporter-2 (SGLT2), metabolic disorders, and survival following incident epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6402.

Dapagliflozin induces vasodilation in resistance-size mesenteric arteries by stimulating smooth muscle cell KV7 ion channels

Dapagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor that, in addition to glucose reduction, lowers systemic blood pressure. Here, we investigated if dapagliflozin could directly relax small mesenteric arteries that control peripheral vascular resistance and blood pressure, and the underlying molecular mechanism. We used pressurized arterial myography, pharmacological inhibition and Western blotting to investigate the direct effect of dapagliflozin on the contractility of freshly isolated, resistance-size rat mesenteric arteries. Our pressure myography data unveiled that dapagliflozin relaxed small mesenteric arteries in a concentration-dependent manner. Non-selective inhibition of KV channels and selective inhibition of smooth muscle cell voltage-gated K+ channels KV7 attenuated dapagliflozin-induced vasorelaxation. Inhibition of other major KV isoforms such as KV1.3, KV1.5 channels as well as large-conductance Ca2+-activated K+ (BKCa) channels, ATP-sensitive (KATP) channels did not abolish vasodilation. Dapagliflozin-evoked vasodilation remained unaltered by pharmacological inhibition of endothelium-derived nitric oxide (NO) signaling, prostacyclin (PGI2), as well as by endothelium denudation. Our Western blotting data revealed that SGLT2 protein is expressed in rat mesenteric arteries. However, non-selective inhibition of SGLTs did not induce vasodilation, demonstrating that the vasodilatory action is independent of SGLT2 inhibition. Overall, our data suggests that dapagliflozin directly and selectively stimulates arterial smooth muscle cells KV7 channels, leading to vasodilation in resistance-size mesenteric arteries. These findings are significant as it uncovers for the first time a direct vasodilatory action of dapagliflozin in resistance mesenteric arteries, which may lower systemic blood pressure.

Dapagliflozin Reduces Apoptosis of Diabetic Retina and Human Retinal Microvascular Endothelial Cells Through ERK1/2/cPLA2/AA/ROS Pathway Independent of Hypoglycemic.

Introduction: It is known that the metabolic disorder caused by high glucose is one of pathogenesis in diabetic retinopathy (DR), the leading cause of blindness, due to the main pathological change of apoptosis of endothelial cells (ECs). In previous studies, the potential impact of sodium glucose cotransporter-2 (SGLT-2), whose inhibitors slow the progression of DR, has not been elucidated. The purpose of the presented study was to explore the effect of SGLT-2 inhibitors dapagliflozin (DAPA) on apoptosis of diabetic mice retina and human retinal microvascular endothelial cells (HRMECs), examine the effects of dapagliflozin on HRMECs metabolism, and explore the molecular processes that affect DR. Methods and Results: The eyeballs of male streptozotocin (STZ)-induced diabetic C57BL/6N mice were evaluated. C57BL/6N mice were divided into control group (CON), diabetic untreated group (DM), diabetic dapagliflozin treatment group (DM + DAPA) and diabetic insulin treatment group (DM + INS). Hematoxylin-Eosin (HE) staining was performed to observe the pathological structure of the mice retina, and TUNEL staining to detect apoptosis of mice retinal cells. In vitro, DCFH-DA and western blot (WB) were used to evaluate ROS, Bcl-2, BAX, cleaved-caspase 3 in HRMECs and metabolomics detected the effect of dapagliflozin on the metabolism of HRMECs. And then, we performed correlation analysis and verification functions for significantly different metabolites. In vivo, dapagliflozin reduced the apoptosis of diabetic mice retina independently of hypoglycemic. In vitro, SGLT-2 protein was expressed on HRMECs. Dapagliflozin reduced the level of ROS caused by high glucose, decreased the expression of cleaved-caspase3 and the ratio of BAX/Bcl-2. Metabolomics results showed that dapagliflozin did not affect the intracellular glucose level. Compared with the high glucose group, dapagliflozin reduced the production of arachidonic acid (AA) and inhibited the phosphorylation of ERK1/2, therefore, reducing the phosphorylation of cPLA2, which is a key enzyme for arachidonic acid release. Conclusion: Collectively, results unearthed for the first time that dapagliflozin reduced apoptosis of retina induced by DM whether in vivo or in vitro. Dapagliflozin did not affect the glucose uptake while mitigated intracellular arachidonic acid in HRMECs. Dapagliflozin alleviated HRMECs apoptosis induced by high glucose through ERK/1/2/cPLA2/AA/ROS pathway.

Endothelial NADPH oxidases protect against sympathetic nervous system hyperactivity-induced cardiac dysfunction and remodeling in mice: contribution of SGLT2

Abstract Background NADPH oxidase-derived reactive oxygen species (ROS) contribute to cardiac dysfunction, often characterized by coronary microvascular dysfunction, an inflammatory response and cardiomyocyte hypertrophy. Hyperactivity of the sympathetic nervous system (SNS) induces oxidative stress, promoting cardiac dysfunction and the development of heart failure. Selective inhibitors of sodium-glucose co-transporter 2 (SGLT2), have shown remarkable cardioprotective effects in clinical studies. Recently, SGLT2 inhibitors have been reported to prevent endothelial dysfunction and pro-inflammatory responses in endothelial cells in response to angiotensin II involving NADPH oxidases. Purpose Therefore, the aim of the study was to determine whether endothelial NADPH oxidases promote SNS-induced cardiac dysfunction and to clarify the role of SGLT2. Methods Male wild-type mice and mice lacking the NADPH oxidase subunit p22phox in the endothelium (p22phox ecKO, 11-week-old) were treated with isoproterenol (100 mg/kg) for five consecutive days and sacrificed at day 14. Hemodynamic measurements of left (LV) and right (RV) ventricles were performed by a transthoracic approach. Heart tissue sections were stained with Sirius red to evaluate fibrosis and wheat germ agglutinin to assess cardiomyocyte size. Cultured human microvascular endothelial cells (HMEC-1) were stimulated with 100 nM isoproterenol and ROS levels were assessed by dihydroethidium fluorescence. The expression level of target genes and proteins was assessed by quantitative real-time PCR and Western blot, respectively. siRNA approaches were used to down-regulate either the NADPH oxidase subunit p22phox or SGLT2. Results The isoproterenol treatment increased LV and RV systolic pressures in wild-type mice but not in p22phox ecKO mice. p22phox ecKO mice were protected against isoproterenol-induced fibrosis, cardiac remodeling characterized by upregulation of mRNA levels of ANP, BNP and β-MHC, and pulmonary congestion. LV remodeling was associated with upregulation of the NADPH oxidase subunits p22phox, Nox2, and Nox4 as well as of SGLT2 in wild-type mice, however no such effects were observed in p22phox ecKO mice. Exposure of HMEC-1 to isoproterenol stimulated the formation of ROS and caused an upregulation of p22phox and SGLT2 protein levels in a time- and concentration-dependent manner. No such effects were observed following silencing of either p22phox or SGLT2, or use of a selective SGLT2 inhibitor. Conclusion Deletion of the NADPH oxidase subunit p22phox in the endothelium protected against SNS hyperactivity induced LV cardiac dysfunction and remodeling, and prevented upregulation of SGLT2. Since depletion of SGLT2 prevented the pro-oxidant response to isoproterenol in endothelial cells, the endothelial NADPH oxidase/SGLT2 pathway seems to have a prominent role in promoting cardiac remodeling and dysfunction in SNS hyperactivity. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Deutsches Herzzentrum München

Overexpression of SGLT2 in the kidney of a P. gingivalis LPS-induced diabetic nephropathy mouse model

BackgroundThe overexpression of sodium-glucose cotransporter 2 (SGLT2) in diabetic kidneys has been reported. It has also been established that the diabetic glomerular endothelium expresses the toll-like receptors TLR2 and TLR4. The present study aims to examine the renal SGLT2 induction by the TLR2/4 ligand Porphyromonas (P.) gingivalis lipopolysaccharide (Pg-LPS) in mouse diabetic nephropathy.MethodsImmunohistochemical study and tissue RT-PCR analyses were performed on mouse kidneys in streptozotocin (STZ)-induced diabetic ICR mice (STZ-ICR), in healthy ICR mice administered Pg-LPS (LPS-ICR), and in diabetic ICR mouse kidneys with Pg-LPS-induced nephropathy (LPS-STZ).ResultsIn the quantitative analysis of blood sugar levels, the mean time to reach 600 mg/dl was shorter in the LPS-STZ than in the STZ-ICR kidneys. The rise in blood glucose levels was significantly steeper in the LPS-STZ than in the STZ-ICR kidneys. According to these data the LPS-STZ model suggests a marked glucose intolerance. The expression of SGLT2 was significantly stronger in the whole of the renal parenchyma of the LPS-STZ than in the LPS-ICR or in the STZ-ICR. The expression of SGLT2 was observed both in the renal tubules and around the renal tubules, and in the glomeruli of the LPS-STZ kidneys. In the analysis by tissue real-time PCR and cell ELISA, the expression of the SGLT2 gene and protein was significantly stronger in the LPS-STZ than in the LPS-ICR or in the STZ-ICR. There were no differences in the renal SGLT2 production in the LPS-ICR and the STZ-ICR kidneys.ConclusionsAbnormally high renal expression of SGLT2 occurs in diabetic kidneys with P. gingivalis LPS. Periodontitis may be an exacerbating factor in diabetic nephropathy as well as in diabetes.