SGLT1 Expression Research Articles

Sodium-glucose co-transporter 1 and 2 expression in the mammary artery of patients with bypass surgery: role of the pro-inflammatory response and contribution to oxidative stress

Abstract Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection independently of glycemic control. Angiotensin II (Ang II) and H2O2 induced the expression of SGLT1 and 2 in cultured endothelial cells and isolated arteries to promote oxidative stress and endothelial dysfunction. However, the expression level and role of SGLT1 and 2 in human arteries remain poorly studied. Purpose This study examined the expression level of SGLT1 and 2 in the human internal mammary artery (IMA) obtained from bypass surgery patients, and, if so, determined the underlying mechanism and function. Methods IMAs were obtained from 40 bypass surgery patients (age 45 to 82). The expression level of target factors was assessed by Western blot analysis, immunofluorescence staining and RT-PCR, and the level of oxidative stress using dihydroethidium staining. Human kidney was used as a control tissue known to express SGLT1 and 2. Porcine coronary artery endothelial cells (CAEC) were cultured and studied at passage 1. Results Western blot analysis of 40 IMA samples indicated a high level of both SGLT1 and 2 in 16 and 17 IMAs, an intermediate level in 8 and 6 IMAs, and a low one in 16 and 17 IMAs, respectively. Immunofluorescence staining of IMA sections indicated that SGLT1 and 2 immunofluorescence signals were observed predominantly in the intima thickening and the media. The expression levels of SGLT1 and 2 were associated with p-p65 NF-kB signals but not angiotensin-converting enzyme (ACE), AT1R, MCP-1, VCAM-1. IMAs with a high expression level of SGLT1 and 2 had a high level of ROS throughout the arterial wall including the intima thickening and endothelium, which was inhibited by the antioxidant N-acetylcysteine, the ACE inhibitor perindoprilat, the AT1R antagonist losartan, and also by the dual SGLT1 and 2 inhibitor sotagliflozin and the selective SGLT2 inhibitor empagliflozin. Pro-inflammatory cytokines mRNA levels of IL-1β, TNF-α and IL-6 were detected in IMAs. Exposure of CAEC to either TNF-α, IL-1β or IL-6 caused a concentration-dependent upregulation of SGLT1 and 2. Conclusion The present findings indicate that SGLT1 and 2 expression is observed in some but not all IMAs of bypass surgery patients predominantly in the media, the intima thickening and the endothelium. High expression levels of SGLT1 and 2 are associated with NF-kB activation and oxidative stress that is prevented by a selective SGLT2 inhibitor and by a dual SGLT1/2 inhibitor. Since pro-inflammatory cytokines triggered SGLT1 and 2 expression in endothelial cells, the inflammatory burden of patients appears to be an important trigger regulating SGLT1/2 expression and the subsequent pro-oxidant response prompting pro-inflammatory and pro-thrombotic responses. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This work was supported by an unrestricted research grant from Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.

Litopenaeus vannamei BMAL1 Is a Critical Mediator Regulating the Expression of Glucose Transporters and Can Be Suppressed by Constant Darkness.

Simple SummaryGrowing evidence has indicated that glucose absorption exhibits profound circadian rhythmicity, mediated entirely by glucose transporters. We observed that the daily profile of BMAL1, GLUT1 and SGLT1 expression was also synchronized in the intestine and the hepatopancreas of Litopenaeus vannamei. Our result identified for the first time that BMAL1 is a critical mediator regulating the expression of glucose transporters, which could be suppressed by constant darkness in L. vannamei.Aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a core circadian transcription factor that controls the 24-h cycle of physiological processes. In shrimp, the role of BMAL1 in the regulating glucose metabolism remains unclear. Firstly, we observed that the daily profile of BMAL1, GLUT1 and SGLT1 expression were synchronized in the intestine and the hepatopancreas of Litopenaeus vannamei. Then we examined the effects of BMAL1 on the gene expression of glucose transporter type 1 (SGLT1) and sodium-glucose cotransporter 1 (GLUT1) in vivo and in vitro. BMAL1 in L. vannamei shares 70.91–96.35% of sequence identities with other shrimp species and possesses the conserved helix-loop-helix domain and polyadenylation site domain. The in vitro dual-luciferase reporter assay and in vivo RNA interference experiment demonstrated that BMAL1 exerted a positive regulation effect on the expression of glucose transporters in L. vannamei. Moreover, we conducted an eight-week treatment to investigate whether light/dark cycle change would influence growth performance, and gene expression of BMAL1, GLUT1 and SGLT1 in L. vannamei. Our result showed that compared with natural light treatment, constant darkness (24-h darkness) significantly decreased (p < 0.05) serum glucose concentration, and suppressed (p < 0.05) the gene expression of BMAL1, GLUT1 and SGLT1 in the hepatopancreas and the intestine. Growth performance and survival rate were also decreased (p < 0.05) by constant darkness treatment. Our result identified BMAL1 as a critical mediator regulating the expression of glucose transporters, which could be suppressed by constant darkness in L. vannamei. It would be quite interesting to explore the mechanism of dark/light cycles on glucose transport and metabolism in L. vannamei, which might provide a feeding strategy for improving carbohydrate utilization in the future.

Abstract P237: Alterations In Renal Sodium Handling Proteins Determined By Urinary Extracellular Vesicle Analysis During Dietary Sodium Modulation And In Primary Aldosteronism

Sodium handling is altered during extremes in dietary sodium intake or in pathologic conditions such as primary aldosteronism. Urinary extracellular vesicles (uEVs) are excreted by all cell types along the nephron, are enriched for membrane proteins, and could provide insight into renal physiology in humans. We assessed uEV protein excretion of renal epithelial proteins relevant to sodium handling from 54 volunteers with pre-hypertension on high (HS, 200 mmol/d) and low (LS, 10 mmol/d) sodium diet for 5-7 days. To compare pathologic versus physiologic responses to aldosterone excess, samples were collected from 9 patients with primary aldosteronism (PA). uEVs were isolated by ultracentrifugation and trypsin digested proteins were quantified using multiple reaction monitoring mass spectrometry using isotopically labeled standards, normalized to the uEV-specific marker CD9. Wilcoxon signed rank and rank sum tests were used for within-subject and between-subject comparisons, respectively. LS diet altered uEV excretion of pendrin (4.0, expressed as fold-change vs HS), aquaporin-2 (-2.03), TRPV5 (-1.91), NCC (1.51), OSR1 (1.42; P &lt;0.01 for all), and SGLT2 (1.20, P =0.042). In primary aldosteronism, expression of ENaC-α, ENaC-β, and ENaC-γ, NCC, SGLT2, and OSR-1 were increased to a greater extent compared to during LS diet ( P &lt;0.05 for each), whereas AQP2 and TRPV5 were similar to HS diet ( Figure ). Expression of uEV proteins relevant to renal tubular sodium handling are dynamically altered by dietary sodium restriction and aldosterone excess. Further studies could identify patterns specific to pathologic versus physiologic aldosterone responses.

The effects of dietary Lactococcus spp. on growth performance, glucose absorption and metabolism of common carp, Cyprinus carpio L.

Based on beneficial effects of Lactococcus spp. and their extracellular polysaccharides on common carp (Cyprinus carpio L.) in previous studies, the main purpose of this paper was to investigate the functions of single or multi-strains of Lactococcus spp. on the growth performance, glucose absorption and metabolism of common carp. The results showed that when the fish consumed diets containing single (Lactococcus lactis Q-8, L. lactis Q-9, L. lactis Z-2) or multi-strains of Lactococcus spp. (5 × 108 CFU g−1 of feed) for 60 days, the final weight, weight gain (WG), specific growth rate (SGR) and condition factor (CF) had significantly (P < .05) increased, especially in the multi-strains addition groups. While the feed conversion ratio (FCR) of all Lactococcus spp. addition groups was significantly lower than that in the control group (P < .05). Viscera-somatic index (VSI) and survival rate in each experimental group was no significant difference during the feeding period (P > .05). For the glucose absorption, Lactococcus spp. addition groups significantly increased the blood glucose concentration and up-regulated the expression of Sglt1 and Gult2 in intestine compared with the control group (P < .05). In addition, the contents of hepatopancreas glycogen were also significantly increased in the Lactococcus spp. addition groups (P < .05). For the glucose metabolism in hepatopancreatic, the Lactococcus spp. addition groups significantly decreased the expression of GK and PK in glycolysis (P < .05), while increased the G6Pase and PEPCK expression in gluconeogenesis (P < .05). Furthermore, with the prolongation of feeding time, the synergistic effects on the expression levels of absorption and metabolism related genes, and the glycogen content were better in the multi-strains addition groups. Overall, these results suggested that the Lactococcus spp. improved the growth performance, and exerted glucose absorption and metabolism effects in common carp, which provide a theoretical basis for the rational use of potential probiotics and carbohydrate in aquaculture.

Iridoids and polyphenols from chilean Gaultheria spp. berries decrease the glucose uptake in Caco-2 cells after simulated gastrointestinal digestion

Berries are rich food sources of potentially health-beneficial (poly)phenols. However, they may undergo chemical modifications during gastrointestinal digestion. The effect of simulated gastrointestinal digestion on the content and composition of secondary metabolites from Gaultheria phillyreifolia and G. poeppigii berries was studied. The influence of the digested extracts on the in vitro metabolism and absorption of carbohydrates was evaluated. After simulated digestion, 31 compounds were detected by UHPLC-DAD-MS. The total content of anthocyanins decreased by 98–100%, flavonols by 44–56%, phenylpropanoids by 49–75% and iridoids by 33–45%, the latter showing the highest stability during digestion. Digested extracts inhibited α-glucosidase (IC50 2.8–24.9 μg/mL) and decreased the glucose uptake in Caco-2 cells by 17–28%. Moreover, a decrease in the mRNA expression of glucose transporters SGLT1 (38–92%), GLUT2 (45–96%), GLUT5 (28–89%) and the enzyme sucrase-isomaltase (82–97%) was observed. These results show the effect of simulated gastrointestinal digestion on the content and composition of Gaultheria berries.

Intestinal Barrier Function and Performance of Broiler Chickens Fed Additional Arginine, Combination of Arginine and Glutamine or an Amino Acid-Based Solution.

Simple SummaryThere is growing evidence that amino acids can influence intestinal barrier function and inflammation in broiler chickens. Arginine, glutamine, and threonine are regarded as functional amino acids that can help restore gut integrity-related issues under enteric or stress-related conditions. The present study aimed to investigate different combination of arginine, glutamine, and threonine plus a grape extract specifically tailored to improve performance and intestinal functions of broilers. The results showed that tested amino acids were able to improve feed conversion ratio of broilers while alleviating the intestinal inflammation caused by administration of synthetic glucocorticoid through different pathways. There was also indication of changes in intestinal permeability by tested amino acids. The mechanistic understanding of different amino acids and their combinations presents opportunity to optimize intestinal barrier function particularly under stress related conditions.Two experiments were conducted to investigate the effect of arginine (Arg); the combination of Arg and glutamine (Gln); as well as an amino acid-based solution (MIX) containing Arg, Gln, threonine (Thr), and grape extract, on performance, intestinal permeability, and expression of selected mechanistic genes. Using 240 male Ross 308 off-sex broiler chickens, four experimental treatments were replicated six times with 10 birds per replicate. The experimental treatments included 5 g/kg Arg, 2.5 g/kg Arg and 2.5 g/kg Gln, and 1 g/kg MIX added to a basal diet as control. In the second study, the four dietary treatments were then given to 24 birds with or without a synthetic glucocorticoid, dexamethasone (DEX), as a gut dysfunction model. Feed conversion ratio was improved by all the supplemented treatments from day 7 to 35 of age (p < 0.001). DEX injections increased (p < 0.001) the intestinal permeability in all treatments, which tended to be reversed by Arg or MIX. Additional Arg, Arg-Gln, and MIX suppressed (p < 0.05) the overexpression of IL-1β generated by DEX. Feeding birds with MIX treatment increased (p < 0.05) expression of SGLT-1 and glutathione synthetase. In conclusion, tested amino acid supplements were effective in improving feed efficiency and restraining intestinal inflammation caused by DEX through IL-1β pathway.

Prognostic Impact of Membranous/Nuclear Epidermal Growth Factor Receptor Localization in Clear Cell Renal Cell Carcinoma.

EGFR is overexpressed in the majority of clear cell renal cell carcinomas (CCRCCs). Although EGFR deregulation was found to be of great significance in CCRCC biology, the EGFR overexpression is not associated with EGFR-targeted therapy responsiveness. Moreover, the prognostic role of EGFR expression remains controversial. In the present study, we evaluated the role played by EGFR overexpression in CCRCC and its prognostic significance associated with different immunohistochemical localization patterns. In our study, the Total Score (TS) related to membranous-cytoplasmic EGFR expression showed a significant correlation with grade, pathologic stage (pT), and Stage, Size, Grade, and Necrosis (SSIGN) score, and a negative correlation with nuclear EGFR expression. No significant correlations were shown between nuclear EGFR and clinic-pathological features. Additionally, a correlation between SGLT1 expression levels and pT was described. Multivariate analysis identifies pT and SSIGN score as independent prognostic factors for CCRCC. A significantly increased survival rate was found in the case of positive expression of nuclear EGFR and SGLT1. Based on our findings, SGLT1 and nuclear EGFR overexpression defines a subgroup of CCRCC patients with good prognosis. Membranous-cytoplasmic EGFR expression was shown to be a poor prognostic factor and could define a CCRCC subgroup with poor prognosis that should be responsive to anti-EGFR therapies.

Deletion of heterogeneous nuclear ribonucleoprotein F in renal tubules downregulates SGLT2 expression and attenuates hyperfiltration and kidney injury in a mouse model of diabetes

We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in HnrnpfRT knockout (KO) mice. Non-diabetic HnrnpfRT KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. Akita HnrnpfRT KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita HnrnpfRT KO mice were studied up to the age of 24weeks (n = 8/group). Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita HnrnpfRT KO mice than Akita mice. Compared with Akita mice, Akita HnrnpfRT KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita HnrnpfRT KO mice. Treatment of Akita HnrnpfRT KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita HnrnpfRT KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita HnrnpfRT KO mice. The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

Heal Thyself: SGLT2 Inhibition Limits Regenerative Cell Exhaustion and Heals Damaged Vessels.

Chronic metabolic dysfunction as a result of type 2 diabetes (T2D) increases susceptibility to the development of ischemic cardiovascular disease (CVD) (1). The search for therapies to improve cardiovascular health in individuals afflicted with T2D has led to the discovery and utilization of a novel class of glucose-lowering drugs called sodium–glucose cotransporter 2 inhibitors (SGLT2i). Located in the proximal tubule of the kidney, the SGLT2 receptor directs the reuptake of glucose and sodium into the circulation (2,3). Inhibition of this pathway results in glucosuria/natriuresis and represents a novel metabolic mechanism to reduce hyperglycemia during T2D. Several randomized clinical trials in people with and without diabetes have now established a critical role for SGLT2i in the reduction of cardiovascular and cardiorenal events (4,5). While direct myocardial and indirect metabolic effects have been proposed to explain these benefits, recent data suggest that SGLT2 inhibition stimulates vascular repair via increased trafficking of bone marrow–derived provascular progenitor cells essential for vessel regeneration (6–9). In this issue of Diabetes , Albiero et al. (10) definitively demonstrate that despite low levels of expression of SGLT2 on hematopoietic cells, SGLT2 inhibition with dapagliflozin improved endothelial repair through enhanced recruitment of hematopoietic stem/progenitor cell (HSPC) progeny from the bone marrow (BM) to the site of vessel injury. In concept, circulating HSPC represent critical intermediates in blood vessel homeostasis via the coordination of angiogenic, arteriogenic, and vasculogenic processes (11). Seminal contributions by the Fadini group and others have previously established a significant depletion in circulating provascular HSPC during T2D, which contributes to …

Effects of canagliflozin on human myocardial redox signalling: clinical implications.

AimsRecent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans.Methods and resultsStudy 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide () sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM.ConclusionsWe demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin.

Potential impacts of diabetes mellitus and anti-diabetes agents on expressions of sodium-glucose transporters (SGLTs) in mice.

Sodium-glucose transporters (SGLTs) are important targets for therapeutic intervention of type 2 diabetes. This study aims to evaluate the physiological influences of diabetes mellitus and the potential impacts of metformin and fluoxetine on SGLTs expressions. Alterations of SGLT1 and SGLT2 were measured in organs involved in glucose homeostasis (kidney, intestine, liver and pancreas) of streptozotocin (STZ) and high-fat diet (HFD) induced diabetic mice by western blotting and real-time PCR (RT-PCR) respectively. In kidney, duodenal segments of intestine, liver, and pancreas of HFD diabetic mice, expressions of SGLT2 were all elevated compared to control mice. The level of SGLT1 was significantly increased in intestine, but was decreased in pancreas. SGLT1 expression in kidney was unaffected, and SGLT1 was undetectable in hepatocytes. Similar results were obtained in STZ diabetic mice. More importantly, here we noticed metformin decreased levels of SGLT2 in kidney, intestine, and pancreas of HFD mice markedly. Expressions of SGLT1 in intestine and pancreas were reduced by metformin as well. In contrast, fluoxetine increased abundances of SGLT2 and SGLT1 in kidney of HFD mice, but decreased SGLT1 expression in intestine. The present study provided evidence that expressions of SGLT1 and SGLT2 were significantly modulated by diabetes mellitus as well as by metformin and fluoxetine, which indicated the efficacy of SGLT2 inhibitors might be impacted by these factors.

Empagliflozin Inhibits Proximal Tubule NHE3 Activity, Preserves GFR, and Restores Euvolemia in Nondiabetic Rats with Induced Heart Failure.

SGLT2 inhibitors reduce the risk of heart failure (HF) mortality and morbidity, regardless of the presence or absence of diabetes, but the mechanisms underlying this benefit remain unclear. Experiments with nondiabetic HF rats tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) inhibits proximal tubule (PT) NHE3 activity and improves renal salt and water handling. Male Wistar rats were subjected to myocardial infarction or sham operation. After 4 weeks, rats that developed HF and sham rats were treated with EMPA or untreated for an additional 4 weeks. Immunoblotting and quantitative RT-PCR evaluated SGLT2 and NHE3 expression. Stationary in vivo microperfusion measured PT NHE3 activity. EMPA-treated HF rats displayed lower serum B-type natriuretic peptide levels and lower right ventricle and lung weight to tibia length than untreated HF rats. Upon saline challenge, the diuretic and natriuretic responses of EMPA-treated HF rats were similar to those of sham rats and were higher than those of untreated HF rats. Additionally, EMPA treatment prevented GFR decline and renal atrophy in HF rats. PT NHE3 activity was higher in HF rats than in sham rats, whereas treatment with EMPA markedly reduced NHE3 activity. Unexpectedly, SGLT2 protein and mRNA abundance were upregulated in the PT of HF rats. Prevention of HF progression by EMPA is associated with reduced PT NHE3 activity, restoration of euvolemia, and preservation of renal mass. Moreover, dysregulation of PT SGLT2 may be involved in the pathophysiology of nondiabetic HF.

Do sodium-glucose co-transporter-2 inhibitors increase plasma glucagon by direct actions on the alpha cell? And does the increase matter for the associated increase in endogenous glucose production?

Sodium-glucose co-transporter-2 inhibitors (SGLT2is) lower blood glucose and are used for treatment of type 2 diabetes. However, SGLT2is have been associated with increases in endogenous glucose production (EGP) by mechanisms that have been proposed to result from SGLT2i-mediated increases in circulating glucagon concentrations, but the relative importance of this effect is debated, and mechanisms possibly coupling SGLT2is to increased plasma glucagon are unclear. A direct effect on alpha-cell activity has been proposed, but data on alpha-cell SGLT2 expression are inconsistent, and studies investigating the direct effects of SGLT2 inhibition on glucagon secretion are conflicting. By contrast, alpha-cell sodium-glucose co-transporter-1 (SGLT1) expression has been found more consistently and appears to be more prominent, pointing to an underappreciated role for this transporter. Nevertheless, the selectivity of most SGLT2is does not support interference with SGLT1 during therapy. Paracrine effects mediated by secretion of glucagonotropic/static molecules from beta and/or delta cells have also been suggested to be involved in SGLT2i-induced increase in plasma glucagon, but studies are few and arrive at different conclusions. It is also possible that the effect on glucagon is secondary to drug-induced increases in urinary glucose excretion and lowering of blood glucose, as shown in experiments with glucose clamping where SGLT2i-associated increases in plasma glucagon are prevented. However, regardless of the mechanisms involved, the current balance of evidence does not support that SGLT2 plays a crucial role for alpha-cell physiology or that SGLT2i-induced glucagon secretion is important for the associated increased EGP, particularly because the increase in EGP occurs before any rise in plasma glucagon.

SGLT2 promotes cardiac fibrosis following myocardial infarction and is regulated by miR-141.

Cardiac fibrosis is a primary event during myocardial infarction (MI) progression, which impairs cardiac function. The present study aimed to investigate the effect of SGLT2 on cardiac fibrosis following MI. To validate the role of SGLT2 in the regulation of cardiac fibrosis in vivo, an MI rat model was established. Echocardiography was performed to determine cardiac function at 4 weeks post-MI. MI model rats were transfected with short hairpin RNA (sh)-SGLT2 or sh-negative control lentiviruses to investigate the effect of SGLT2 on rat heart function post-MI. Subsequently, the effects of SGLT2 on the cardiac fibrosis of infarcted hearts were assessed by performing Masson's trichrome staining. To further clarify the effect of SGLT2 on cardiac fibroblast proliferation, TGFβ was used to stimulate primary cardiac fibroblasts in vitro. The results demonstrated that SGLT2 served a key role in cardiac fibrosis. SGLT2 expression levels in infarct tissues were significantly increased at week 1 post-MI compared with the sham group. Compared with the control group, SGLT2 knockdown attenuated cardiac fibrosis by inhibiting the expression of collagen I and collagen III in cardiac fibroblasts in vitro and in vivo. Furthermore, the results indicated that SGLT2 expression was modulated by miR-141 in cardiac fibroblasts. In summary, the present study indicated that upregulated SGLT2 expression in cardiac fibrosis following MI was regulated by miR-141 and SGLT2 that knockdown reduced cardiac fibrosis and improved cardiac function after MI.

Nephron progenitor cell organization and nephrogenesis is dependent on Shroom3

The functional unit of the kidney, termed the nephron, develops from specialized mesenchyme cells known as the nephron progenitors. Nephron progenitors must undergo significant cell shape changes to regulate cell migration, cell movements, and cell projection formation during nephrogenesis. These dynamic cellular changes are essential for orientating and organizing the cells for progression through various stages of nephrogenesis to form a proper functioning nephron. The actin cytoskeleton regulates these dynamic morphological changes by interacting with intracellular proteins and their extracellular environment. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are strongly associated with poor nephron function and chronic kidney disease. Our previous work demonstrated unique Shroom3 expression patterns in nephron progenitor cells, yet the function of Shroom3 during nephrogenesis is not known. Here, we investigated functional roles for Shroom3 in nephron progenitor organization during nephrogenesis. Analysis of E13.5 and E18.5 kidney sections from Wildtype and Shroom3-/- null mutants demonstrated clustering abnormalities of nephron progenitor cells characterized by large abnormal gaps between cells in Shroom3 mutants. Analysis of Wildtype and Shroom3-/- nephron progenitor cell orientation by GM130 and Integrin-α8 immunofluorescence, markers of cell polarization, displayed altered cell orientation in some but not all cells. Transmission electron microscopy (TEM) confirmed that nephron progenitors of Shroom3-/- were irregularly shaped, abnormally clustered, and found at large distances from the neighboring epithelium. TEM analysis also revealed several filopodia-like long and thin actin-based membrane protrusions from both Wildtype and Shroom3-/- nephron progenitors. However, the broad, thick lamellipodia-like protrusions, found at the leading edge of the cells, were virtually absent from Shroom3-/- nephron progenitors. As nephrogenesis proceeds, Wildtype nephron progenitors develop into renal vesicles that consist of a central lumen surrounded by polarized wedge-shaped epithelial cells, which was not observed in Shroom3-/- renal vesicles. These alterations in the early stages of nephrogenesis in Shroom3-/- kidneys were also associated with a disruption in the epithelial polarization of mature nephrons demonstrated by irregular Sglt2 and Slc12a3 expression, key ion channels localized to the apical aspect in Wildtype epithelia. Our results demonstrate that Shroom3 is an important regulator of nephron progenitor organization and cell morphology in the earliest stages of nephron formation. It also suggests these abnormalities translate to a poorly polarized nephron epithelium and likely poor nephron function. These studies could explain why mutations in Shroom3 are so highly associated with poor nephron function and chronic kidney disease.

Interaction between Renal Sodium‐Glucose Cotransporter 2 and Renin‐Angiotensin System in a PCOS Rat Model

Introduction : Polycystic Ovary Syndrome (PCOS) is characterized by androgen excess, oligo/anovulation, and polycystic ovaries. PCOS is co-prevalent with insulin resistance (IR), increased blood pressure (BP), and a dysfunctional renin-angiotensin system (RAS). The RAS has both a vasoconstrictive arm with angiotensin-converting enzyme (ACE) and a vasodilatory arm with ACE2. While PCOS patients require multiple medications to control their comorbidities, it is unknown how these medications interact with each other. ACE inhibitors (ACEi) and sodium-glucose cotransporter 2 inhibitors (SGLT2i) seem to influence each other in male rodents. However, how androgens influence these interactions in females is unknown. We have a well-established rat model that mimics PCOS, including IR, increased BP, and overactive RAS. Using this model, we tested the hypothesis that SGLT2i would attenuate the RAS and that ACEi would decrease renal SGLT2 expression in a rat model of PCOS. Methods : In Experiment 1, four-week old female Sprague Dawley (SD) rats implanted with DHT pellets (7.5mg/90 days) or control were randomized to drinking water with and without the SGLT2i empagliflozin at a dose of 10 mg/kg/day during the last 3 weeks of the study (n=10/group). At 3 months of DHT, fasting plasma was collected for the homeostatic model assessment of IR (HOMA-IR). BP was measured by radiotelemetry. Renal cortex and medulla were collected for quantitative real-time polymerase chain reaction (qRT-PCR) and western blot for ACE and ACE2. In Experiment 2, four-week old female SD rats implanted with DHT pellets (7.5mg/90 days) or control were randomized to drinking water with and without the ACEi enalapril at a dose of 250 mg/mL throughout the study (n=10/group). At 3 months of DHT, kidneys were collected for qRT-PCR for SGLT2. Results : PCOS rats had increased IR, BP, and renal expression of ACE and ACE2 compared to controls. SGLT2i had no impact on IR while causing a small reduction in BP in both groups. SGLT2i decreased cortical ACE mRNA and protein (1.03 ± 0.08 vs 1.37 ± 0.04, P<0.05) in PCOS, and had no impact in controls. SGLT2i also decreased cortical ACE2 protein (1.02 ± 0.05 vs 1.18 ± 0.03, P<0.05) in PCOS, but not in controls. SGLT2i decreased mRNA and protein of medullar ACE and ACE2 in both groups. In Experiment 2, cortical SGLT2 mRNA was increased in PCOS compared to controls. ACEi increased SGLT2 mRNA in controls (30.32 ± 8.03 vs 1.00 ± 0.32, P<0.01) with no effect in PCOS. Conclusions In summary, PCOS rats had increased IR, BP, and intrarenal ACE. Without impacting IR, SGLT2i attenuated both intrarenal ACE and ACE2 expression in PCOS. This suggests ACEi could work synergistically with SGLT2i to reduce BP. PCOS rats have increased SGLT2 mRNA, suggesting a differential response to SGLT2i between PCOS rats and controls, such as shown with cortical ACE. ACEi also increased cortical SGLT2 mRNA in controls, but androgen excess appeared to blunt this effect. Overall, this suggests that there could be a differential response in hyperandrogenemic women taking both SGLT2i and ACEi compared to a woman with normal androgen levels.

Angiotensin II up-regulates sodium-glucose co-transporter 2 expression and SGLT2 inhibitor attenuates Ang II-induced hypertensive renal injury in mice.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P<0.001). In vitro, angiotensin II (Ang II) dose-dependently stimulated SGLT2 expression in HK-2, human immortalized renal proximal tubular cells (RPTCs); losartan and antioxidants inhibited it. Sglt2 expression was increased in transgenic (Tg) mice specifically overexpressing Agt in their RPTCs, as well as in WT mice with a single subcutaneous injection of Ang II (1.44 mg/kg). Moreover, Ang II (1000 ng/kg/min) infusion via osmotic mini-pump in WT mice for 4 weeks increased systolic blood pressure (SBP), glomerulosclerosis, tubulointerstitial fibrosis, and albuminuria; canaglifozin (Cana, 15 mg/kg/day) reversed these changes, with the exception of SBP. Fractional glucose excretion (FeGlu) was higher in Ang II+Cana than WT+Cana, whereas Sglt2 expression was similar. Our data demonstrate a link between intrarenal RAS and SGLT2 expression and that SGLT2i ameliorates Ang II-induced renal injury independent of SBP.

ROLE OF ALDOSTERONE AND P66SHC PROTEIN ON EXPRESSION OF THE SODIUM-GLUCOSE TRANSPORTER SGLT2 IN THE VASCULAR SYSTEM

Objective: SGLT2 is a transporter involved in sodium and glucose reabsorption in the kidney. SGLT2 inhibitors are a novel class of antidiabetic drugs which inhibit sodium and glucose reabsorption in the tubule, and they have shown a significant protective effect on cardiovascular outcomes in clinical trials. However the role of SGLT2 in the cardiovascular system is not fully elucidated. The aim of this study was to investigate the modulation of SGLT2 expression in the vascular system and kidney in a mouse model of aldosterone-induced hypertension and to explore the role of p66shc, through which aldosterone/MR signals. Design and method: Wild type mice (WT) and p66shc mice -/- (14 weeks, 6 per group) were treated or not with aldosterone (300 μg/kg/day) for 4 weeks. Systolic blood pressure (SBP) was measured by the tail-cuff method. The expression of SGLT2 and the mineralocorticoid receptor (MR) in the kidney and aorta was evaluated by immuno-blotting. Results: At the beginning of study SBP was similar in WT and p66shc-/-. Aldosterone increased SBP in both WT (192.3 ± 4.522 vs 103.3 ± 2.996; p &lt; 0.05) and p66shc-/- mice (176.8 ± 5.313 vs 105.3 ± 1.542; p &lt; 0.05), although at significant less extent in p66shc-/-. SGLT2 expression was significantly lower in p66shc -/- compared to WT in both kidney and aorta (-50%, for both tissues, p &lt; 0.05). Aldosterone increased SGLT2 expression by 3-fold only in WT (p &lt; 0.05) in aorta but not in the kidney. MR expression was similar in all groups. Conclusions: SGLT2 is expressed in vascular tissue as well as in the kidney. Aldosterone modulates its expression only in the vascular tissue, suggesting a tissue specific effect. The p66shc protein may be involved in the modulation of SGLT2 expression in both tissues. These findings suggest that aldosterone and p66shc adaptor protein may contribute to the modulation of SGLT2 expression in the vascular system. Thus SGLT2 may play a role in the vascular homeostatic functions of aldosterone-induced hypertension.

Perturbations of cancer cell metabolism by the antidiabetic drug canagliflozin

Notwithstanding that high rates of glucose uptake and glycolysis are common in neoplasia, pharmacological efforts to inhibit glucose utilization for cancer treatment have not been successful. Recent evidence suggests that in addition to classical glucose transporters, sodium-glucose transporters (SGLTs) are expressed by cancers. We therefore investigated the possibility that SGLT inhibitors, which are used in treatment of type 2 diabetes, may exert antineoplastic activity by limiting glucose uptake. We show that the SGLT2 inhibitor canagliflozin inhibits proliferation of breast cancer cells. Surprisingly, the antiproliferative effects of canagliflozin are not affected by glucose availability nor by the level of expression of SGLT2. Canagliflozin reduces oxygen consumption and glutamine metabolism through the citric acid cycle. The antiproliferative effects of canagliflozin are linked to inhibition of glutamine metabolism that fuels respiration, which represents a previously unanticipated mechanism of its potential antineoplastic action.

Anti-hyperglycaemic effects of dioxidovanadium complex cis-[VO2(obz)py] avert kidney dysfunction in streptozotocin-induced diabetic male Sprague-Dawley rats.

Despite the success of antidiabetic drugs in alleviation of hyperglycaemia, diabetic complications, including renal dysfunction, continue to be a burden. This raises the need to seek alternative therapies that will alleviate these complications. Accordingly, the aim of this study was to investigate the effects of dioxidovanadium(V) complex cis-[VO2(obz)py] on renal function in diabetic rats. Streptozotocin-induced diabetic rats were treated with cis-[VO2(obz)py] (40 mg·kg-1) twice every third day for five weeks. Diabetic untreated and insulin-treated rats served as the diabetic control and positive control, respectively. Blood glucose concentrations, water intake, urinary output, and mean arterial pressure (MAP) were monitored weekly for five weeks. Rats were then euthanized, and blood and kidney tissues were collected for biochemical analysis. Significant decreases in blood glucose concentrations, MAP, glomerular filtration rate (GFR), and SGLT2 expression, as well as plasma angiotensin and aldosterone concentrations, were observed in the treated groups compared with the diabetic control. The complex also increased urinary glucose concentrations, antioxidant enzymes GPx and SOD concentrations, and decreased MDA concentrations and kidney injury molecule (KIM-1) concentrations. These findings suggest that the anti-hyperglycaemic effects of this vanadium complex may ameliorate kidney dysfunction in diabetes.