Tofogliflozin, a novel sodium-glucose co-transporter 2 inhibitor, improves renal and pancreatic function in db/db mice

Abstract

Background and purpose: Although inhibition of renal sodium-glucose co-transporter 2 (SGLT2) has a stable glucose-lowering effect in patients with type 2 diabetes, the effect of SGLT2 inhibition on renal dysfunction in type 2 diabetes remains to be determined. To evaluate the renoprotective effect of SGLT2 inhibition more precisely, we compared the effects of tofogliflozin (a specific SGLT2 inhibitor) with those of losartan (an angiotensin II receptor antagonist) on renal function and beta-cell function in db/db mice.

Experimental approach: The effects of 8-week tofogliflozin or losartan treatment on renal and beta-cell function were investigated in db/db mice by quantitative image analysis of glomerular size, mesangial matrix expansion and islet beta-cell mass. Blood glucose, glycated Hb and insulin levels, along with urinary albumin and creatinine were measured

Key results: Tofogliflozin suppressed plasma glucose and glycated Hb and preserved pancreatic beta-cell mass and plasma insulin levels. No improvement of glycaemic conditions or insulin level was observed with losartan treatment. Although the urinary albumin/creatinine ratio of untreated db/db mice gradually increased from baseline, tofogliflozin or losartan treatment prevented this increase (by 50-70%). Tofogliflozin, but not losartan, attenuated glomerular hypertrophy. Neither tofogliflozin nor losartan altered matrix expansion.

Conclusions and implications: Long-term inhibition of renal SGLT2 by tofogliflozin not only preserved pancreatic beta-cell function, but also prevented kidney dysfunction in a mouse model of type 2 diabetes. These findings suggest that long-term use of tofogliflozin in patients with type 2 diabetes may prevent progression of diabetic nephropathy.

Keywords: beta-cell loss; nephropathy; sodium-glucose co-transporter inhibitor; tofogliflozin.

Figure 1

Blood glucose and glycated Hb in db/db mice. (A–B) Tofogliflozin lowered blood glucose concentration (A) and suppressed glycated Hb (B) in db/db mice. (C–E) Long-term tofogliflozin administration lowered the urine volume (C), lowered UGE (D), and elevated glucose clearance (E) in db/db mice. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from control group by Dunnett's multiple comparison test. ^###P < 0.001 significantly different from control group by t-test.

Figure 2

Body weight and food consumption in db/db mice. (A–B) Tofogliflozin treatment increased body weight (A) and food consumption (B) in db/db mice. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from control group by Dunnett's multiple comparison test. ^###P < 0.001 significantly different from control group by t-test.

Figure 3

Urinary albumin excretion in db/db and db/ + m mice. (A–B) Values shown are the ratio of urinary albumin excretion to urinary creatinine excretion (ACR). Tofogliflozin treatment suppressed the 24 h urinary ACR in db/db mice. ACR was significantly lower in tofogliflozin-treated mice than in control db/db mice (A). Changes in ACR values from the start of study in the tofogliflozin-treated groups were comparable with changes in ACR values in the losartan-treated group at 4 and 8 weeks of treatment (B left, 0–4 weeks; B right, 0–8 weeks). Data shown are means± SEM *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from control group by Dunnett's multiple comparison test. ^##P < 0.01, ^###P < 0.001 significantly different from control group by t-test. (C–E) Scatter plot of plasma glucose and ACR values in db/db and db/ + m mice. (C) 0 weeks (Baseline value), (D) 4 weeks of treatment, (E) 8 weeks of treatment.

Figure 4

Creatinine clearance and kidney weight in db/db and db/ + m mice. (A) Creatinine clearance in db/db mice was not affected by 8 weeks’ treatment with tofogliflozin or losartan. Creatinine clearance was significantly higher in untreated db/db mice than in db/ + m mice. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). ^###P < 0.001 significantly different from control group by t-test. (B) Scatter plot of plasma glucose and creatinine clearance in db/db and db/ + m mice. (C) Kidney weight in db/db mice was not affected by 8 weeks’ treatment with tofogliflozin or losartan. Kidney weight was significantly greater in untreated db/db mice than in db/ + m mice. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). ^###P < 0.001 significantly different from control group by t-test. (D) Scatter plot of plasma glucose and kidney weight in db/db and db/ + m mice.

Figure 5

Histological analysis of glomerulus at 8 weeks of treatment (1). (A) Kidney sections were stained with anti-type IV collagen antibody. Representative images are shown (scale bar, 10 μm). (B–C) Kidney sections from each mouse were stained with anti-type IV collagen antibody. Glomerular size and mesangial matrix area were determined by imaging analysis. (B) Treatment with the diet containing tofogliflozin (0.015% ) significantly prevented glomerular expansion in db/db mice. (C) Mesangial matrix area was significantly lower in db/ + m mice than in untreated db/db mice. Tofogliflozin and losartan treatment had no effect on type IV collagen-positive area. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). **P < 0.01 significantly different from control group by Dunnett's multiple comparison test. ^###P < 0.001 significantly different from control group by t-test.

Figure 6

Histological analyses of glomeruli at 8 weeks of treatment (2). (A) Kidney sections were stained with PAS. Representative images are shown (scale bar, 10 μm). (B) Kidney sections from each mouse were stained with PAS. Mesangial matrix area was determined by scoring. Mesangial matrix area was significantly lower in db/ + m mice than in untreated db/db mice. Tofogliflozin and losartan treatment had no effect on the expansion of mesangial matrix area. Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). ^##P < 0.01 significantly different from control group by t-test.

Figure 7

Mean plasma insulin concentration and histological analysis of pancreatic beta-cell mass. (A) Tofogliflozin treatment increased plasma insulin levels (IRI) in db/db mice. (B–E) Total beta-cell mass was calculated from the insulin-positive area of pancreatic sections. Total beta-cell mass (% of total pancreatic area) was significantly smaller in db/ + m mice than in db/db mice at the start of the study (B, C). Total beta-cell mass was significantly higher in the tofogliflozin treatment groups than in the control group at the end of the study (D, E). Data shown are means± SEM (db/db mice, n = 8; db/ + m mice, n = 10). *P < 0.05, ***P < 0.001 significantly different from control group by Dunnett's multiple comparison test. ^#P < 0.05, ^###P < 0.001 significantly different from control group by t-test.