The Impact of Tofogliflozin on Physiological and Hormonal Function, Serum Electrolytes, and Cardiac Diastolic Function in Elderly Japanese Patients with Type 2 Diabetes Mellitus

The sodium glucose transporter 2 (SGLT2) inhibitor tofogliflozin is a glucose-lowering drug that causes the excretion of surplus glucose by inhibiting SGLT2. Because of tofogliflozin’s osmotic diuresis mechanism, patients’ serum electrolytes, body fluid levels, and cardiac function must be monitored. We retrospectively analyzed the cases of 64 elderly Japanese patients with type 2 diabetes mellitus (T2DM) who received tofogliflozin for 3 months. Their HbA1c, serum electrolytes (sodium, potassium, chloride), hematocrit, brain natriuretic peptide (cardiac volume load marker) and renin and aldosterone (RAA; an index of regulatory hormones involved in body fluid retention) were continuously monitored during the investigation period. Renal function and cardiac function (by echocardiography) were assessed throughout the period. HbA1c significantly decreased (β1=−0.341, p<0.0001, linear regression analysis [LRA]). Most of the hormonal, electrolyte, and physiological parameters were maintained throughout the study period. In these circumstances, E/e’ tended to decrease (β1=−0.382, p=0.13, LRA). Compared to the baseline, E/e’ was significantly decreased at 1 and 3 months (p<0.01, p<0.05). In the higher E/e’ group (E/e’≥10, n=34), E/e’ decreased significantly (β1=−0.63, p<0.05, LRA). ΔE/e’ was correlated with body-weight change during treatment (r=0.64, p<0.01). The 3-month tofogliflozin treatment improved glycemic control and diastolic function represented by E/e’ in T2DM patients, without affecting serum electrolytes, renal function, or RAA. No negative impacts on the patients were observed. Three-month tofogliflozin treatment lowered glucose and improved cardiac diastolic function

Overexpression of the N-terminal part of p50 blocks the inhibitory effect of Ni²⁺ ions.

<p>(A) Stable transfectants expressing mock, WT, N- or C-terminal parts of p50 were established. Each transfectant was cultured in the presence of 1 mM Ni²⁺ ions for 24 h. After stimulation, the culture supernatants were harvested and subjected to IL-8 ELISA. The concentration of IL-8 in mock transfectant cultured in the absence of Ni²⁺ ions was set as 100%. The relative IL-8 secretion is shown. Data are means±SD of three independent experiments. *p<0.05. (B) Migration of HSC3 cells was determined with a scratch motility assay. At 18 h after scratching the cells, phase-contrast images (5×fields) of the scratch motility process were obtained. Representative images are shown. (C) Each scratched area was measured on the images, set at 100% for 0 h, and the mean percentage of the total closure of the scratched area was calculated.</p

Ni²⁺ ions inhibit the nuclear translocation of the NF-κB p50 subunit.

<p>(A) HSC3 cells were stimulated with 1 mM Ni²⁺ ions (or with medium as a control) for the indicated times. Cell lysates were prepared and subjected to Western blotting. The membranes were probed with anti-phospho-p65 Ab (top panel), anti-p65 Ab (middle panel) or anti-GAPDH Ab (lower panel), respectively. Representative data for three separate experiments are shown. (B) HSC3 cells were stimulated with 1 mM Ni²⁺ ions for the times indicated. The nuclear extracts were harvested and subjected to a Transfactor assay to measure p50. Data are means±SD of three independent experiments. (C) HSC3 cells were stimulated with 1 mM Ni²⁺ ions for 1 h. After stimulation, cells were immediately transferred to ice, washed with ice-cold PBS and fixed. The cells were subjected to immunofluorescence cell staining with anti-human p50 Ab followed by FITC-conjugated goat anti-rabbit IgG Ab. Green, p50; red, nuclei with monomeric cyanine nucleic acid stain. Bar, 10 µm.</p

NF-κB-dependent secretion of IL-8.

<p>(A) HSC3 cells were exposed to different concentrations of the NF-κB-specific inhibitors isohelenin (solid line) and TPCK (dashed line). After 24 h, secreted IL-8 concentration was measured. Data from at least three separate experiments are shown (mean±SD). (B) Total cell lysates were collected after treatment of HSC3 cells with or without TPCK or isohelenin and subjected to Western blotting. The membranes were probed with anti-phospho-p65 Ab (top panel), anti-p65 Ab (middle panel) or anti-GAPDH Ab (lower panel). Representative data for three separate experiments are shown. (C) HSC3 cells were transfected with pNF-κB-Luc plasmid (firefly) and pRL-CMV plasmid (renilla). After transfection, cells were stimulated with (+) or without (–) 1 mM Ni²⁺ ions for 1 h. Luciferase activity was measured and the firefly/renilla ratio was calculated. The ratio in the absence of Ni²⁺ ion stimulation was set as 100%. Data are means±SD of three independent experiments. *p<0.05. (D) HSC3 cells were stimulated as in (B). After stimulation, the expression of IL-8 mRNA was measured with real-time PCR. The IL-8/GAPDH ratio in the absence of Ni²⁺ ions was set as 1. *p<0.05.</p

Ni²⁺ ions bind directly to the p50 subunit of NF-κB.

<p>(A) HSC3 cells were transfected with p50 expression vector (pcDNA-p50 WT). Cell lysates were harvested and subjected to Western blotting. (B) The cell lysates of pcDNA-p50 WT or mock-transfected cells were collected and incubated with either Ni²⁺-column (Ni) or protein G-sepharose beads (G) for 1 h. The samples were washed with ice-cold cell lysis buffer five times and subjected to Western blotting. (C) The cell lysates from p50-transfected HSC3 cells were incubated with Ni-beads for 1 h in the presence or absence of graded concentrations of imidazol. The samples were subjected to Western blotting.</p

Ni²⁺ ions bind to the N-terminal part of the p50 subunit.

<p>(A) Schematic illustration of the structure of the p105 molecule. NTD; N-terminal domain, DimD; dimerization domain, NLS; nuclear localizing signal, GRR; glycine-rich region, ARD; ankyrin repeat domain, DD; death domain. The lower panel shows the His clusters at position 108, 110 and 112. (B) HSC3 cells were transfected with mock, pcDNA-p50 WT, pcDNA-p50 N or pcDNA-p50 C plasmids. The cell lysates were harvested and subjected to Ni<b>²⁺</b>-column precipitation (upper) or Western blotting (lower). (C) The His cluster was deleted and transfected to HSC3 cells. The cell lysate was harvested and subjected to a Ni²⁺-column precipitation assay followed by Western blotting.</p

Ni²⁺ ions exerts an inhibitory effect through a TLR4-independent pathway.

<p>(A) Expression of TLR4 and MD2 mRNA in HSC2, HSC3 and Ca9–22 cells was examined using RT-PCR. Expression levels varied significantly among the cells. (B) HSC3 cells were pre-incubated with 2 µg anti-TLR4 Ab or class-matched control Ab for 2 h. After pre-incubation, the cells were stimulated with or without 1 mM Ni²⁺ ions for 24 h. The culture supernatants were harvested and subjected to IL-8 ELISA. Data for the three separate experiments are shown (mean±SD). *p<0.05. HUVECs (C) and HSC3 cells (D) were transfected with TLR4 siRNA or control siRNA. The transfectants were cultured in the presence or absence of 1 mM Ni²⁺ ions for 24 h. IL-8 concentration was measured by ELISA. The IL-8 concentration of control siRNA-transfected HUVECs cultured without Ni²⁺ was set as 1 and the fold increase was shown (C). The IL-8 concentration of non-transfected HSC3 cells cultured without Ni²⁺ was set as 100% (D). Data are means±SD of three independent experiments. *p<0.05. (E) HSC3 cells were transfected with TLR4 siRNA or control siRNA. After transfection, TLR4 expression was examined by RT-PCR. Representative values for three independent experiments are shown.</p

Effects of a High-Protein Diet on Kidney Injury under Conditions of Non-CKD or CKD in Mice

Considering the prevalence of obesity and global aging, the consumption of a high-protein diet (HPD) may be advantageous. However, an HPD aggravates kidney dysfunction in patients with chronic kidney disease (CKD). Moreover, the effects of an HPD on kidney function in healthy individuals are controversial. In this study, we employed a remnant kidney mouse model as a CKD model and aimed to evaluate the effects of an HPD on kidney injury under conditions of non-CKD and CKD. Mice were divided into four groups: a sham surgery (sham) + normal diet (ND) group, a sham + HPD group, a 5/6 nephrectomy (Nx) + ND group and a 5/6 Nx + HPD group. Blood pressure, kidney function and kidney tissue injury were compared after 12 weeks of diet loading among the four groups. The 5/6 Nx groups displayed blood pressure elevation, kidney function decline, glomerular injury and tubular injury compared with the sham groups. Furthermore, an HPD exacerbated glomerular injury only in the 5/6 Nx group; however, an HPD did not cause kidney injury in the sham group. Clinical application of these results suggests that patients with CKD should follow a protein-restricted diet to prevent the exacerbation of kidney injury, while healthy individuals can maintain an HPD without worrying about the adverse effects