The presence of sodium glucose co-transporter 2 in mesangial cells and pericytes and its roles in mesangial lesions and in capillaries under diabetic and ischemic conditions.

SGLT2 is expressed in mesangial cells and pericytes, and is upregulated byhigh glucose and ischemia. Upregulated SGLT2 in both cells might directly worsen ischemia in kidney interstitial legion, heart and brain. The overexpression of SGLT2 in these cells could induce various organ failures via damages or loss of capillaries and dysfunctions of mesangial cells, which are attenuated by SGLT2 inhibitors.

Low-dose sodium-glucose cotransporter 2 inhibitor ameliorates ischemic brain injury in mice through pericyte protection without glucose-lowering effects.

Antidiabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors have attracted attention for their cardiorenal-protective properties beyond their glucose-lowering effect. However, their benefits in ischemic stroke remain controversial. Here we show the effects of luseogliflozin, a selective SGLT2 inhibitor, in acute ischemic stroke, using a permanent middle cerebral artery occlusion (pMCAO) model in non-diabetic mice. Pretreatment with low-dose luseogliflozin, which does not affect blood glucose levels, significantly attenuated infarct volume, blood-brain barrier disruption, and motor dysfunction after pMCAO. SGLT2 was expressed predominantly in brain pericytes and was upregulated in peri- and intra-infarct areas. Notably, luseogliflozin pretreatment reduced pericyte loss in ischemic areas. In cultured pericytes, luseogliflozin activated AMP-activated protein kinase α and increased mitochondrial transcription factor A expression and number of mitochondria, conferring resistance to oxygen-glucose deprivation. Collectively, pre-stroke inhibition of SGLT2 induces ischemic tolerance in brain pericytes independent of the glucose-lowering effect, contributing to the attenuation of ischemic brain injury.

Roles of Sodium-Glucose Cotransporter 2 of Mesangial Cells in Diabetic Kidney Disease.

We have been studying the presence of sodium-glucose cotransporter 2 (SGLT2) in mesangial cells and pericytes since 1992. Recent large placebo-controlled studies of SGLT2 inhibitors in patients with type 2 diabetes mellitus have reported desirable effects of the inhibitors on the diabetic kidney and the diabetic heart. Most studies have indicated that these effects of SGLT2 inhibitors could be mediated by the tubuloglomerular feedback system. However, a recent study about urine sodium excretion in the presence of an SGLT2 inhibitor did not show any increases in urine sodium excretion. A very small dose of an SGLT2 inhibitor did not inhibit SGLT2 at the S1 segment of proximal tubules. Moreover, SGLT2 inhibition protects against progression in chronic kidney disease with and without type 2 diabetes. In these circumstances, the tubuloglomerular feedback hypothesis involves several theoretical concerns that must be clarified. The presence of SGLT2 in mesangial cells seems to be very important for diabetic nephropathy. We now propose a novel mechanism by which the desirable effects of SGLT2 inhibitors on diabetic nephropathy are derived from the direct effect on SGLT2 expressed in mesangial cells.

Lessons from the Trials for the Desirable Effects of Sodium Glucose Co-Transporter 2 Inhibitors on Diabetic Cardiovascular Events and Renal Dysfunction.

Recent large placebo-controlled trials of sodium glucose co-transporter 2 (SGLT2) inhibitors revealed desirable effects on heart failure (HF) and renal dysfunction; however, the mechanisms underlying these effects are unknown. The characteristic changes in the early stage of diabetic cardiomyopathy (DCM) are myocardial and interstitial fibrosis, resulting in diastolic and subsequent systolic dysfunction, which leads to clinical HF. Pericytes are considered to play crucial roles in myocardial and interstitial fibrosis. In both DCM and diabetic retinopathy (DR), microaneurysm formation and a decrease in capillaries occur, triggered by pericyte loss. Furthermore, tubulointerstitial fibrosis develops in early diabetic nephropathy (DN), in which pericytes and mesangial cells are thought to play important roles. Previous reports indicate that pericytes and mesangial cells play key roles in the pathogenesis of DCM, DR and DN. SGLT2 is reported to be functionally expressed in pericytes and mesangial cells, and excessive glucose and Na+ entry through SGLT2 causes cellular dysfunction in a diabetic state. Since SGLT2 inhibitors can attenuate the high glucose-induced dysfunction of pericytes and mesangial cells, the desirable effects of SGLT2 inhibitors on HF and renal dysfunction might be explained by their direct actions on these cells in the heart and kidney microvasculature.

Amelioration of diabetic nephropathy by SGLT2 inhibitors independent of its glucose-lowering effect: A possible role of SGLT2 in mesangial cells.

Several clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on diabetic nephropathy. The underlying mechanisms are not fully understood. We found that administration of canagliflozin at a low dose (0.01 mg/kg/day) did not affect either blood glucose levels or glycosuria, but it improved albuminuria and mesangial expansion in db/db mice to a similar extent as at a high dose (3.0 mg/kg/day) that lowered blood glucose levels. This indicated the existence of a tubular SGLT2-independent reno-protective mechanism. Here we focused on the potential role of SGLT2 in mesangial cells (MCs). Western blot analysis revealed the expression of SGLT2 in cultured mouse MCs. Exposure of MCs to high glucose levels for 72 h significantly increased the expression of SGLT2. Canagliflozin or ipragliflozin (both 100 nM) treatment inhibited glucose consumption in the medium under high-glucose conditions but not under normal-glucose conditions. Furthermore, canagliflozin inhibited high-glucose-induced activation of the protein kinase C (PKC)-NAD(P)H oxidase pathway and increases in reactive oxygen species (ROS) production. Thus, the inhibition of mesangial SGLT2 may cause an inhibition of PKC activation and ROS overproduction in diabetic nephropathy, and this may at least in part account for the reno-protective effect of SGLT2 inhibitors.

Recovery from Diabetic Macular Edema in a Diabetic Patient After Minimal Dose of a Sodium Glucose Co-Transporter 2 Inhibitor.

BACKGROUND Diabetic macular edema (DME) causes serious visual impairments in diabetic patients. The standard treatments of DME are intra-vitreous injections of corticosteroids or anti-vascular endothelial growth factor antibodies and pan-photocoagulation. These treatments are unsatisfactory in their effects and impose considerable physical and economic burdens on the patients. CASE REPORT A 63-year-old woman was diagnosed as type 2 diabetes with retinopathy 7 years ago. Before the initiation of an SGLT2 inhibitor, the dipeptidyl peptidase-4 inhibitor, sitagliptin (50 mg daily), and metformin (250 mg dai- ly) were used for her glycemic control. The level of her hemoglobin A1c had been controlled around 7%. She began to feel decreased visual acuity and blurred vision of her left eye 8 months before the visit to our clin- ic. She was diagnosed as DME, which turned out to be corticosteroid-resistant. Her visual acuity further de- creased to 20/50. Metformin was changed to ipraglifl (25mg/day). Her left visual acuity started to improve after 4 weeks of treatment with ipragliflozin and improved to 20/22 after 24 weeks. The macular edema did not change until 12 weeks of the treatment, however, it decreased prominently after 16 weeks. CONCLUSIONS In our patient with steroid-resistant DME, her visual symptoms and macular edema recovered after the initiation of an SGLT2 inhibitor. SGLT2 inhibitors might be a potential candidate for the DME treatment.

Sodium Glucose Cotransporter 2 (SGLT2) Plays as a Physiological Glucose Sensor and Regulates Cellular Contractility in Rat Mesangial Cells.

PURPOSE: Mesangial cells play an important role in regulating glomerular filtration by altering their cellular tone. We report the presence of a sodium glucose cotransporter (SGLT) in rat mesangial cells. This study in rat mesangial cells aimed to evaluate the expression and role of SGLT2. METHODS: The SGLT2 expression in rat mesangial cells was assessed by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Changes in the mesangial cell surface area at different glucose concentrations and the effects of extracellular Na+ and Ca2+ and of SGLT and Na+/Ca2+ exchanger (NCX) inhibitors on cellular size were determined. The cellular sizes and the contractile response were examined during a 6-day incubation with high glucose with or without phlorizin, an SGLT inhibitor. RESULTS: Western blotting revealed an SGLT2 band, and RT-PCR analysis of SGLT2 revealed the predicted 422-bp band in both rat mesangial and renal proximal tubular epithelial cells. The cell surface area changed according to the extracellular glucose concentration. The glucose-induced contraction was abolished by the absence of either extracellular Na+ or Ca2+ and by SGLT and NCX inhibitors. Under the high glucose condition, the cell size decreased for 2 days and increased afterwards; these cells did not contract in response to angiotensin II, and the SGLT inhibitor restored the abolished contraction. CONCLUSIONS: These data suggest that SGLT2 is expressed in rat mesangial cells, acts as a normal physiological glucose sensor and regulates cellular contractility in rat mesangial cells.

Hydrogen peroxide-induced Ca2+ responses in CNS pericytes.

OBJECTIVE: The aims of the present study were to elucidate the interaction of reactive oxygen species (ROS) and Ca(2+) response in central nervous system (CNS) pericytes. METHODS: The intracellular Ca(2+) concentration was measured using fluorescent Ca(2+) indicator, fura-2, in cultured CNS pericytes. RESULTS: Hydrogen peroxide evoked a dose-dependent increase in cytosolic Ca(2+), which was completely inhibited by catalase. Removal of external Ca(2+) or addition of nicardipine (1 microM) during application of hydrogen peroxide did not affect Ca(2+) response. Incubation of the cells in Ca(2+) free solution did not abolish but slightly reduced Ca(2+) response by hydrogen peroxide. Ca(2+) response to hydrogen peroxide was not altered by the depletion of intracellular Ca(2+) by thapsigargin (1 microM). Pretreatment of the cells with tyrosine kinase inhibitor genistein (100 microM) or tyrphostin A47 (30 microM) significantly reduced Ca(2+) increase by hydrogen peroxide. CONCLUSIONS: These results indicate that hydrogen peroxide evokes Ca(2+) increase predominantly by release from intracellular Ca(2+) store, which may be regulated by tyrosine kinases.

Hyperglycemia promotes microinflammation as evaluated by C-reactive protein in the very elderly.

OBJECTIVE: C-reactive protein (CRP) is not only an acute phase reactant but also a sensitive marker of subclinical inflammation associated with atherosclerosis. The aim of the present study was to determine whether glycemic control or blood pressure influences the vascular microinflammation as evaluated by CRP levels in the very elderly. METHODS: The study group consisted of 195 residents aged 85 years. The subjects were divided into three groups according to their CRP levels; a low (<1 mg/L), an average (1 to 3 mg/L), and a high (3 to 10 mg/L) CRP group. Hemoglobin A(1c) (HbA(1c)) levels were used as an index of glycemic control. RESULTS: The HbA(1c) level showed a significant positive correlation with the CRP level (r=0.289, p<0.0001). In contrast, systolic and diastolic blood pressures failed to correlate with the CRP level. The HbA(1c) was significantly greater in the high CRP group compared to the average and the low CRP groups (6.01+/-0.29%, 5.57+/-0.09% and 5.42+/-0.05%, respectively). Furthermore, the HbA(1c) adjusted by sex, body mass index, systolic blood pressure, serum triglyceride and current smoking status was significantly higher in the high CRP group than in the average and the low CRP groups. Multiple regression analysis also revealed that the HbA(1c) level was significantly and independently associated with the CRP level. CONCLUSIONS: These results suggest that tight good glycemic control, even in very elderly subjects, may be able to reduce the systemic inflammation that contributes to leads to atherosclerosis.

Decreased lipid peroxidation following periodontal therapy in type 2 diabetic patients.

BACKGROUND: Although diabetes mellitus and periodontal disease promote atherosclerosis, the relation of oxidative stress with these diseases remains unclear. To investigate the influence of periodontal disease on oxidative stress, we assessed the effects of initial periodontal therapy on lipid peroxide (LPO), an oxidative stress index, in type 2 diabetic and non-diabetic patients. METHODS: Seventeen subjects with or without type 2 diabetes were enrolled in this intervention study. No patient had a history of cardiovascular or peripheral vascular disease. Five type 2 diabetic and six non-diabetic patients, all with moderate to severe periodontal disease, received and completed the initial periodontal therapy and examination. Before and after the therapy, patients underwent medical examinations and blood determinations, including LPO. RESULTS: Before the therapy, the periodontal probing depth and bleeding on probing (BOP) were similar between groups. LPO, triglyceride, and white blood cell counts were significantly higher in diabetic than non-diabetic patients. Therapy improved the periodontal parameters in both groups and significantly decreased LPO in diabetic patients. Anti-malondialdehyde-modified low-density lipoprotein (MDA-LDL) antibody, a marker of oxidized LDL, significantly decreased with treatment in both groups. Overall, Spearman rank correlation showed no significance between periodontal parameters and LPO or anti-MDA-LDL antibody, but BOP tended to correlate with LPO in diabetic patients (r = 0.585; P = 0.0791). CONCLUSION: Although this is a small and preliminary study, and the changes of LPO and anti-MDA-LDL antibody were within the normal range, the initial periodontal therapy significantly decreased LPO, an oxidative stress index, in type 2 diabetic patients with periodontal disease.

Prediction of cognitive function by arterial stiffness in the very elderly.

BACKGROUND: Cognitive function is impaired in elderly subjects, so the aim of the present study was to determine the role of arterial stiffness on cognitive function. METHODS AND RESULTS: Cognitive function and arterial stiffness were assessed by the Mini-Mental State Examination (MMSE) and measurement of the brachial-ankle pulse wave velocity (PWV), respectively. The cross-sectional association of the MMSE score and PWV was studied in 203 subjects (87 men, 116 women), all of whom were 85 years old. Sex distribution, systolic and diastolic blood pressures did not differ between the normal (MMSE score >or=24, n=128) and impaired MMSE groups (MMSE score <24, n=75). In contrast, the PWV was significantly increased in the impaired MMSE group than in the normal MMSE group (25.0+/-0.8 vs 22.9+/-0.5 m/s, p<0.05). In multiple regression analysis, the PWV was also independently and significantly associated with the MMSE score. CONCLUSIONS: These results suggest that cognitive function could be predicted by arterial stiffness, as assessed by the PWV, in the very old. Preventing atherosclerosis may play an important role in preserving normal cognitive function until very old age.

NAD(P)H oxidases in rat basilar arterial endothelial cells.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries. METHODS: We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining. RESULTS: RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production. CONCLUSIONS: Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.

Calcium influx pathways in rat CNS pericytes.

In central nervous system (CNS), pericytes have been proposed to play a role in broad functional activities including blood-brain barrier, microcirculation, and macrophage activity. However, contractile responses and Ca2+ signaling in CNS pericytes have not been elucidated. The aim of the present study is to investigate contractility and Ca2+ influx pathway in CNS pericytes. CNS pericytes were cultured from rat brain. Contraction of the pericytes in response to various stimuli was evaluated by the change in surface area measured by a light microscope with a digital camera. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to examine the expression of mRNA of alpha-smooth muscle actin. Intracellular Ca2+ was measured using fura-2 fluorescence spectroscopy. A23187 (Ca2+ ionophore), high external K+ (4 x 10(-2) mol/l), endothelin-1, and serotonin induced contraction of CNS pericytes. RT-PCR analysis revealed the expression of alpha-smooth muscle actin in CNS pericytes. Cytosolic Ca2+ ([Ca2+]i) increased after application of high concentration of external K+, tetraethylammonium, and charybdotoxin, which was inhibited by nicardipine and removal of external Ca2+. Angiotensin-II, serotonin, acetylcholine, ATP, and endothelin-1 caused biphasic response in [Ca2+]i. In response to these agents, [Ca2+]i rapidly increased and then decayed to a relatively constant Ca2+ plateau. The Ca2+ plateau was partially inhibited by nicardipine and completely abolished by omission of external Ca2+. After intracellular Ca2+ store was depleted by the removal of external Ca2+ and addition of thapsigargin, reapplication of external Ca2+ evoked increases in [Ca2+]i. These results indicate that CNS pericytes express mRNA of alpha-smooth muscle actin and possess contractile ability. In CNS pericytes, resting membrane potential is regulated by large conductance Ca2+-activated K+ channels and Ca2+ enters into the cells via L-type voltage-dependent Ca2+ channels, agonist-activated Ca2+ permeable channels, and capacitative Ca2+ entry pathways.

PPARgamma ligands attenuate mesangial contractile dysfunction in high glucose.

BACKGROUND: To elucidate the regulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and its roles in mesangial cells, we examined the expression of PPARgamma1 and effects of its ligands on cell phenotypes and angiotensin II-induced contractile response in cultured rat mesangial cells under a high (20 mmol/L) glucose condition. METHODS: The effects of tumor necrosis factor alpha (TNFalpha), protein kinase C (PKC) activation, antisense DNA for PPARgamma1, PPARgamma ligands and PD98059 were examined in mesangial cells cultured in either 5 mmol/L or 20 mmol/L glucose. The expressions of PPARgamma1 protein and alpha-smooth muscle actin (alphaSMA) as a marker of phenotype of cells were determined by Western blot. The expression of PPARgamma1 mRNA was determined by a reverse transcription-polymerase chain reaction method. The reduction of cell surface area in response to angiotensin II was measured by microscope to determine cellular contraction. RESULTS: PKC activation, TNFalpha, and 20 mmol/L glucose decreased PPARgamma1 at both protein and mRNA levels, which was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase (MAPK). Decreases of PPARgamma1 protein and contractile response and an increase of alphaSMA occurred simultaneously in the cells treated with 20 mmol/L glucose after 5 days, which were attenuated to the normal levels by PPARgamma ligands. The antisense DNA also induced the decrease of PPARgamma1 protein, contractile dysfunction, and increase of alphaSMA. CONCLUSION: MAPK suppresses PPARgamma1 at the transcriptional level, and the reduction of PPARgamma1 in cultured rat mesangial cells under the high glucose condition induces phenotypic change and loss of contractile function. PPARgamma ligands recover both reductions of PPARgamma 1 protein and contractile response.

Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase.

BACKGROUND: Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. METHODS AND RESULTS: Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. CONCLUSIONS: These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.

Prevalence of hepatitis viral infection in dental patients with impacted teeth or jaw deformities.

OBJECTIVE: The prevalence of infection with hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis G virus (HGV), and transfusion-transmitted viruses (TTV) was evaluated in patients with impacted teeth or jaw deformities. STUDY DESIGN: Of 486 patients, 268 had serum samples available for retrospective TTV DNA and HGV RNA assays. In addition, the sera of 404 patients were assayed for HB surface antigen and the sera of 340 were assayed for HCV antibody. RESULTS: HGV RNA was detected in 3 of 268 patients (1.1%), and TTV DNA was detected in 60 of 268 (22.4%). Of 404 patients, 3 had HB surface antigens (0.7%). Furthermore, 13 of 340 were HCV-seropositive (3.8%). The rate of infection was similar between patients with impacted teeth and those with jaw deformities, respectively, as follows: 1.1% versus 0%, respectively, for HBV prevalence; 4.1% versus 3.2% for HCV prevalence; 1.8% versus 0% for HGV prevalence; and 22.9% versus 21.4% for TTV prevalence. CONCLUSIONS: Universal precautions to prevent hepatitis and TTV infection during oral surgical procedures are important.

ATP-sensitive potassium channels mediate dilatation of basilar artery in response to intracellular acidification in vivo.

BACKGROUND AND PURPOSE: During cerebral ischemia, both hypoxia and hypercapnia appear to produce marked dilatation of the cerebral arteries. Hypercapnia and hypoxia may be accompanied by extracellular and intracellular acidosis, which is another potent dilator of cerebral arteries. However, the precise mechanism by which acidosis produces dilatation of the cerebral arteries is not fully understood. The objective of the present study was to examine the mechanisms by which intracellular acidosis produces dilatation of the basilar artery in vivo. METHODS: Using a cranial window in anesthetized rats, we examined responses of the basilar artery to sodium propionate, which was used to cause intracellular acidosis specifically. Expression of subunits of potassium channels was determined by reverse transcription and polymerase chain reaction (RT-PCR). RESULTS: Topical application of propionate increased diameter of the basilar artery in a concentration-related manner. Propionate-induced dilatation of the artery was attenuated by glibenclamide, an inhibitor of ATP-sensitive potassium channels. However, inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine), large-conductance calcium-activated potassium channels (iberiotoxin), and cyclooxygenase (indomethacin) did not affect the vasodilatation. Expression of mRNA for SUR2B and Kir6.1 was detected, with the use of RT-PCR, in the cultured basilar arterial muscle cells. CONCLUSIONS: The findings suggest that intracellular acidification may produce dilatation of the basilar artery through activation of ATP-sensitive potassium channels in vivo. Kir6.1/SUR2B may be the major potassium channels that mediate propionate-induced dilatation of the artery.