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Prevention of progression of diabetic nephropathy by the SGLT2 inhibitor ipragliflozin in uninephrectomized type 2 diabetic mice
European Journal of Pharmacology 830 (2018) 68–75
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Prevention of progression of diabetic nephropathy by the SGLT2 inhibitor ipragliflozin in uninephrectomized type 2 diabetic mice
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Atsuo Tahara , Toshiyuki Takasu Candidate Discovery Science Laboratories, Astellas Pharma Inc., Ibaraki, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: SGLT2 inhibitor Ipragliflozin Hyperglycemia Type 2 diabetes Nephropathy
Diabetic nephropathy is the leading cause of end-stage renal disease in the world. Although recent development of sodium-glucose cotransporter (SGLT) 2 inhibitors offers a new antidiabetic therapeutic strategy, it remains unclear whether such treatments are beneficial for limiting the progression of type 2 diabetic overt nephropathy. This study examined the effect of the SGLT2 inhibitor ipragliflozin on the progression of nephropathy in uninephrectomized KK/Ay type 2 diabetic mice, which exhibit not only typical diabetic symptoms such as hyperglycemia, hyperinsuemia, glucose intolerance, insulin resistance, hyperlipidemia, inflammation, and obesity, but also moderate hypertension and overt nephropathy with decline in renal function. Four-week repeated administration of ipragliflozin improved various diabetic symptoms, including hyperglycemia, insulin resistance, and inflammation by increasing urinary glucose excretion. In addition, ipragliflozin ameliorated albuminuria/ proteinuria; decline in renal function, as measured by creatinine clearance; hypertension; and renal injury, including glomerulosclerosis and interstitial fibrosis. These effects were significant at doses of 1 mg/kg or higher and were similar to those observed following administration of losartan (30 mg/kg). These results suggest that the SGLT2 inhibitor ipragliflozin prevents progression to diabetic overt nephropathy in uninephrectomized type 2 diabetic mice. SGLT2 inhibitors may therefore represent a promising therapeutic option for the management of type 2 diabetes to slow the progression of diabetic nephropathy.
1. Introduction Diabetic nephropathy is one of the most common complications of diabetes and the leading cause of end-stage renal disease. It is also associated with high cardiovascular risk and significant morbidity and mortality worldwide (Pálsson and Patel, 2014). In the past, several mechanisms have been implicated in the initiation and deterioration of diabetic nephropathy, including hyperglycemia, hypertension, dyslipidemia, and obesity, as well as ethnic and genetic factors (Tavafi, 2013). Mounting evidence suggests that proper control of blood glucose and blood pressure can reduce the risk of developing this complication. While current antidiabetic therapies to control blood glucose and administration of renin-angiotensin-aldosterone system (RAAS) inhibitors, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor antagonists (ARBs) to control blood pressure can slow the progression of diabetic nephropathy, they do not sufficiently prevent it (Fioretto et al., 2010). Therefore, there is a strong need to explore additional therapies for limiting the progression of diabetic nephropathy. In recent years, sodium-glucose cotransporter (SGLT) 2 inhibitors, which improve hyperglycemia via the stimulation of glucose excretion
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into the urine, have been proposed as novel drugs for treating type 2 diabetes (Chao, 2014). As chronic hyperglycemia is thought to contribute to the progression of diabetic nephropathy, the potent antihyperglycemic effects of SGLT2 inhibition may be promising for treating diabetic nephropathy. Several SGLT2 inhibitors including ipragliflozin have been shown to reduce urinary albumin excretion in type 2 diabetic animals with incipient nephropathy, which, at least in part, was attributed to inflammation, oxidative stress, and insulin resistance (Škrtić and Cherney, 2015; Tahara et al., 2013). However, the potential effects of SGLT2 inhibitors on the progression of overt nephropathy in type 2 diabetic animal models have not been determined in detail. Here, we investigated the effects of the SGLT2 inhibitor ipragliflozin on the progression of nephropathy in uninephrectomized type 2 diabetic mice, which exhibit not only typical diabetic symptoms such as hyperglycemia, insulin resistance, hyperlipidemia, hepatic steatosis, inflammation and obesity, but also moderate hypertension and overt nephropathy with decline in renal function. In addition, we compared the effects of ipragliflozin with those of the ARB losartan.
Correspondence to: Candidate Discovery Science Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. E-mail address: [email protected] (A. Tahara).
https://doi.org/10.1016/j.ejphar.2018.04.024 Received 10 January 2018; Received in revised form 19 April 2018; Accepted 20 April 2018 Available online 25 April 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 830 (2018) 68–75
A. Tahara, T. Takasu
2. Materials and methods 2.1. Materials Ipragliflozin was synthesized at Astellas Pharma Inc. (Ibaraki, Japan) and losartan was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). These drugs were suspended or dissolved in 0.5% methylcellulose solution and administered orally via a stomach tube. Doses of these drugs were expressed as the free base form. 2.2. Animals Male C57BL/6 (normal) and KK/Ay type 2 diabetic mice were purchased from CLEA Japan (Kanagawa, Japan) at age 6 weeks and used at age 14 weeks. The left kidney of diabetic mice was removed under isoflurane anesthesia. Two weeks after uninephrectomy, diabetic mice were grouped such as to attain uniform mean blood glucose levels among the groups. All animals were housed under standard conditions of controlled temperature, humidity, and light (12-h light-dark cycle), and were given free access to standard commercial chow and water. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. Astellas Pharma Inc., Tsukuba Research Center has been awarded Accreditation Status by the AAALAC International.
Fig. 1. Effects of 4-week intervention with ipragliflozin or losartan on (A) blood pressure and (B) heart rate in uninephrectomized type 2 diabetic mice. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. nonNx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $ P < 0.05 vs. Nx diabetic vehicle group.
2.3. Repeated administration study Groups comprised the following: (1) normal mice: normal (vehicle); (2) non-nephrectomized (non-Nx) diabetic mice: control (vehicle); (3) nephrectomized (Nx) diabetic mice: (i) vehicle, (ii) ipragliflozin (0.1 mg/kg), (iii) ipragliflozin (0.3 mg/kg), (iv) ipragliflozin (1 mg/kg), (v) ipragliflozin (3 mg/kg), (vi) losartan (30 mg/kg). Six mice were used in each group/subgroup. To measure pretreatment creatinine clearance values, mice were transferred to metabolic cages where spontaneously voided urine was collected for 24 h, and blood samples were obtained from the tail vein. Subsequently, each drug was orally administered to Nx diabetic mice once daily (at night) for 4 weeks. Body weight and food intake were measured weekly. An oral glucose tolerance test (OGTT) was performed at Week 3. Mice were fasted over the inactive period (from 7:00–19:00) prior to drug administration. Two hours later, blood was sampled from a tail vein for the evaluation of fasting blood glucose and plasma insulin levels. A glucose solution (2 g/kg) was then orally administered, and blood sampling was conducted for 2 h. After drug administration on day 25, spontaneously voided urine was collected for 24 h. The morning after the final drug administration on day 28, blood samples were collected from the abdominal vena cava and tissues (kidneys, pancreas, liver, and epididymal adipose tissue) were isolated under isoflurane anesthesia. To measure blood pressure, a second set of animals was subjected to the same group composition and drug administration schedule. The morning after the final drug administration on day 29, systolic blood pressure and heart rate were measured using a photoelectric tail cuff pulse detection system (BP-98A-L; Softron, Tokyo, Japan).
using the Triglyceride E-test Wako, NEFA C-test Wako, and Cholesterol E-test Wako kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Hepatic lipid (triglyceride and cholesterol) content levels were measured according to a previously reported method (Tahara et al., 2011). Levels of aminotransferases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the Transaminase CII test reagent (Wako Pure Chemical Industries, Ltd.). Plasma levels of leptin, fibroblast growth factor 21 (FGF-21), adiponectin, interleukin (IL)− 1β, IL-6, tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and C-reactive protein (CRP) were measured using commercial ELISA kits (R&D Systems Inc., Minneapolis, MN, USA). Urinary albumin and protein excretion were measured using mouse albumin ELISA and a protein assay reagent (BioRad, Hercules, CA, USA), respectively. Plasma and urinary creatinine levels were measured using Determiner L CRE (Kyowa Medex, Tokyo, Japan), and creatinine clearance (µl/g body weight/min) was calculated based on urinary and plasma creatinine levels, urine volume, and body weight. Urinary concentrations of nephrin and podocalyxin were measured using ELISA kits (Exocell Inc., PA, USA). Urinary concentrations of kidney injury molecule-1 (KIM-1) were measured using an ELISA kit (R&D Systems Inc.). Urinary N-acetyl-β-D-glucosaminidase (NAG) activity was measured using NAG Test Shionogi (Shionogi Co. Ltd, Osaka, Japan).
2.4. Biochemical measurements Blood and urinary glucose concentrations were measured using Glucose CII test reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Hemoglobin A1c (HbA1c) levels were measured using a DCA2000 System (Bayer Medical, Tokyo, Japan). Plasma insulin levels were measured using an ultra-high-sensitivity mouse insulin enzymelinked immunosorbent assay (ELISA) kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). Pancreatic insulin content was measured using an insulin ELISA kit according to a previously reported method (Tahara et al., 2013). Plasma lipid [triglycerides, nonesterified fatty acids (NEFAs), and cholesterol] levels were measured
2.5. Histopathology Specimen preparation and histopathological examination were performed at CMIC Bioresearch Center Co., Ltd. (Yamanashi, Japan). Sagittal slices of renal tissue were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 2-μm thick sections for morphological study. These sections were stained with hematoxylin and eosin, and periodic acid Schiff. All tissue samples were evaluated by an independent investigator blinded to group information. All glomeruli and the entire microscopic area in each specimen were examined. 69
European Journal of Pharmacology 830 (2018) 68–75
A. Tahara, T. Takasu
Fig. 2. Effects of 4-week intervention with ipragliflozin or losartan on glucose tolerance and insulin resistance in uninephrectomized type 2 diabetic mice. Time course of changes in (A) blood glucose and (C) plasma insulin levels, and area under the (B) blood glucose and (D) plasma insulin concentration-time curves (AUCs) during the oral glucose tolerance test (OGTT). The (E) Matsuda and (F) disposition indexes were used as parameters of insulin resistance and secretion, respectively. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. OGTT was performed at Week 3. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group. Table 1 Effects of 4-week intervention with ipragliflozin or losartan on various diabetic parameters in uninephrectomized type 2 diabetic mice. Parameter
Body weight (g) Food intake (g/day) HbA1c (%) Blood glucose (mg/dL) Plasma insulin (ng/mL) Pancreatic insulin content (ng/mg tissue) Urine volume (mL/day) Urinary glucose excretion (mg/day) Epididymal adipose tissue weight (g) Plasma leptin (ng/mL) Plasma FGF-21 (ng/mL) Plasma adiponectin (µg/mL)
Normal
Diabetic (Control)
Nx diabetic
Vehicle
Vehicle
Vehicle
41.7 ± 0.6 3.52 ± 0.14 3.15 ± 0.11 125 ± 5 1.6 ± 0.2 99 ± 11 1.09 ± 0.15 0.9 ± 0.3 0.41 ± 0.02 6.8 ± 1.7 1.19 ± 0.13 9.53 ± 0.93
48.5 ± 0.6a 6.09 ± 0.11a 8.07 ± 0.40a 497 ± 24a 68.5 ± 8.5a 63 ± 9a 4.57 ± 0.44a 425 ± 52a 1.61 ± 0.09a 41.0 ± 5.6a 2.61 ± 0.12a 4.48 ± 0.50a
Ipragliflozin
47.5 ± 0.3a 6.21 ± 0.15a 7.98 ± 0.36a 472 ± 28a 74.2 ± 6.9a 65 ± 6a 4.32 ± 0.44a 478 ± 41a 1.77 ± 0.10a 38.8 ± 3.9a 2.80 ± 0.29a 4.31 ± 0.23a
Losartan
0.1 mg/kg
0.3 mg/kg
1 mg/kg
3 mg/kg
30 mg/kg
47.0 ± 0.9 6.17 ± 0.14 7.50 ± 0.35 446 ± 26 68.3 ± 7.7 72 ± 4 4.20 ± 0.50 531 ± 49 1.75 ± 0.09 33.3 ± 5.5 2.66 ± 0.28 4.84 ± 0.27
45.9 ± 0.7 6.29 ± 0.14 6.62 ± 0.37b 357 ± 15b 52.2 ± 5.1 81 ± 11 5.66 ± 0.38 745 ± 51b 1.64 ± 0.10 26.3 ± 4.1 2.44 ± 0.28 6.12 ± 0.61
44.7 ± 0.7b 6.32 ± 0.14 5.78 ± 0.38b 271 ± 15b 34.2 ± 5.2b 109 ± 3b 5.89 ± 0.56 946 ± 83b 1.35 ± 0.09b 20.6 ± 3.0b 1.85 ± 0.20b 7.28 ± 0.56b
43.4 ± 0.6b 6.25 ± 0.11 5.47 ± 0.33b 221 ± 14b 29.8 ± 4.5b 132 ± 13b 6.84 ± 0.50b 1109 ± 92b 1.31 ± 0.11b 18.8 ± 4.1b 1.59 ± 0.19b 8.13 ± 0.66b
47.8 ± 0.7 6.09 ± 0.15 7.57 ± 0.34 452 ± 23 64.9 ± 7.0 76 ± 6 3.96 ± 0.40 410 ± 42 1.86 ± 0.12 30.3 ± 5.0 2.40 ± 0.22 5.59 ± 0.60
HbA1c: hemoglobin A1c, FGF-21: fibroblast growth factor 21. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. a P < 0.05 vs. normal group (Tukey's test). b P < 0.05 vs. Nx diabetic vehicle group (Dunnett's test).
Histopathological changes (glomerulosclerosis, interstitial fibrosis, cell infiltration, and basophilic change) were evaluated using a semiquantitative scoring system according to the percentage of affected renal tissue: 0, no lesion; 1, very slight (< 10%); 2, slight (10% ≤ X < 25%); 3, moderate (25% ≤ X < 50%); 4, marked
(≥ 50%).
2.6. Statistical analysis The experimental results are expressed as the mean ± standard 70
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Table 2 Effects of 4-week intervention with ipragliflozin or losartan on various lipid and inflammatory parameters in uninephrectomized type 2 diabetic mice. Parameter
Plasma triglycerides (mg/dL) Plasma NEFAs (mEq/L) Plasma cholesterol (mg/dL) Liver weight (g) Hepatic triglyceride content (mg/g liver) Hepatic cholesterol content (mg/g liver) ALT (IU/L) AST (IU/L) Plasma IL-1β (pg/mL) Plasma IL-6 (pg/mL) Plasma TNF-α (pg/mL) Plasma MCP-1 (pg/mL) Plasma CRP (ng/mL)
Normal
Diabetic (Control)
Nx diabetic
Vehicle
Vehicle
Vehicle
106 ± 7 0.67 ± 0.05 77 ± 6 1.55 ± 0.07 10.7 ± 1.7 4.7 ± 0.8 14.9 ± 2.4 27.5 ± 4.8 71 ± 14 186 ± 21 55 ± 6 103 ± 27 150 ± 53
411 ± 39a 1.24 ± 0.07a 136 ± 7a 2.97 ± 0.09a 29.7 ± 3.8a 16.7 ± 2.4a 55.3 ± 7.2a 74.7 ± 6.7a 335 ± 52a 939 ± 128a 212 ± 25a 469 ± 68a 362 ± 29a
Ipragliflozin
395 ± 34a 1.21 ± 0.06a 139 ± 10a 3.09 ± 0.12a 30.9 ± 3.3a 16.9 ± 2.0a 64.4 ± 7.0a 79.1 ± 8.4a 397 ± 75a 959 ± 100a 220 ± 20a 534 ± 76a 426 ± 41a
Losartan
0.1 mg/kg
0.3 mg/kg
1 mg/kg
3 mg/kg
30 mg/kg
383 ± 30 1.21 ± 0.09 140 ± 7 2.94 ± 0.15 30.2 ± 3.2 16.9 ± 1.4 53.5 ± 10.5 69.8 ± 11.8 352 ± 49 837 ± 110 225 ± 31 455 ± 69 400 ± 33
302 ± 35 1.05 ± 0.07 141 ± 11 2.64 ± 0.13 24.2 ± 2.4 12.4 ± 1.5 40.5 ± 6.6 65.0 ± 5.8 282 ± 64 669 ± 100 175 ± 19 386 ± 76 325 ± 36
208 ± 29b 0.98 ± 0.07 115 ± 9 2.23 ± 0.13b 19.0 ± 2.8b 8.9 ± 1.5b 24.3 ± 6.3b 52.4 ± 7.3 187 ± 41b 564 ± 68b 134 ± 15b 267 ± 44b 212 ± 30b
220 ± 24b 0.87 ± 0.04b 96 ± 8b 2.22 ± 0.15b 15.7 ± 1.5b 6.6 ± 0.7b 21.5 ± 4.2b 40.0 ± 7.0b 150 ± 28b 510 ± 56b 117 ± 10b 245 ± 36b 197 ± 39b
388 ± 28 1.11 ± 0.08 147 ± 14 3.01 ± 0.10 27.5 ± 3.3 14.6 ± 1.8 50.9 ± 7.2 60.0 ± 7.8 313 ± 41 990 ± 93 200 ± 31 478 ± 44 359 ± 51
NEFAs: non-esterified fatty acids, ALT: alanine aminotransferase, AST: aspartate aminotransferase, IL: interleukin, TNF-αː tumor necrosis factor α, MCP-1: monocyte chemotactic protein-1, CRP: C-reactive protein. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. a P < 0.05 vs. normal group (Tukey's test). b P < 0.05 vs. Nx diabetic vehicle group (Dunnett's test).
improved obesity (body and fat weights), dyslipidemia, steatosis, and inflammatory symptoms without affecting food intake. Plasma levels of leptin, FGF-21, and adiponectin, which are highly correlated with insulin resistance, returned to normal levels, suggesting that ipragliflozin significantly and dose-dependently improved insulin resistance. Pancreatic insulin content was also significantly and dose-dependently increased to normal levels. In contrast, while losartan slightly improved these type 2 diabetes symptoms, the changes were not significant. Compared to normal mice, non-Nx diabetic mice exhibited increased urinary albumin excretion, creatinine clearance (hyperfiltration), tubular injury markers (urinary NAG activity and KIM-1 excretion) and glomerular podocyte injury markers (urinary excretion of nephrin and podocalyxin), as well as histopathological features of glomerulosclerosis, with little interstitial fibrosis (Figs. 3–6). Renal function was not reduced, as evidenced by the lack of a significant decrease in creatinine clearance (Fig. 4A) and changes in urinary protein excretion (Fig. 3B) and plasma creatinine levels (Fig. 4B). These results demonstrate that non-Nx diabetic mice exhibit symptoms associated with incipient diabetic nephropathy. In contrast, Nx diabetic mice exhibited reduced renal function at the end of the 4-week study, as evidenced by significantly increased urinary albumin and protein excretions, decreased creatinine clearance, and increased plasma creatinine levels. In addition, further increases were observed in tubular and glomerular podocyte injury markers and interstitial fibrosis, which was an additional pathological change to glomerulosclerosis. Since there was no evidence of reduced renal function in these mice at the start of the study, these findings indicate that Nx diabetic mice show rapid progression from incipient to overt nephropathy. Ipragliflozin significantly and dose-dependently decreased albumin and protein urinary excretions, alleviated renal hypertrophy (Fig. 3), reversed the decline in renal function (decreased creatinine clearance and increased plasma creatinine) (Fig. 4), decreased markers of renal tubular and glomerular podocyte injury (Fig. 5), and improved histopathological changes, including glomerulosclerosis and interstitial fibrosis (Fig. 6). Losartan also significantly improved nephropathy, almost as effectively as ipragliflozin. However, unlike with ipragliflozin, losartan significantly decreased creatinine clearance and slightly increased plasma creatinine.
error of the mean (S.E.M.) or standard deviation (S.D.). Differences between normal, diabetic (non-Nx) control, and Nx diabetic vehicle groups were assessed using Tukey's multiple comparisons test. Differences between the Nx diabetic vehicle and ipragliflozin-treated groups were assessed using a Dunnett's multiple comparisons test, while those between the Nx diabetic vehicle and losartan-treated groups were assessed using a Student's t-test. Histopathological scores were compared using a Mann-Whitney test to analyze differences between two groups and Dunn's multiple comparisons test among multiple groups. A value of P < 0.05 was considered to be significant. Statistical and data analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
3. Results Compared to normal mice, non-Nx diabetic mice exhibited a slight but non-significant increase in blood pressure (Fig. 1). In contrast, Nxdiabetic mice exhibited a significant increase in blood pressure in the absence of changes in heart rate. Ipragliflozin slightly but significantly and dose-dependently decreased blood pressure, and losartan markedly decreased blood pressure. Nx diabetic mice exhibited glucose intolerance and insulin resistance during the OGTT (Fig. 2). In addition, these mice exhibited hyperglycemia, hyperinsulinemia, glycosuria and polyuria, obesity with overeating, dyslipidemia (increased plasma levels of triglycerides, NEFAs, and cholesterol), steatosis (hepatic hypertrophy, increased hepatic contents of triglycerides and cholesterol, and increased plasma ALT/AST levels), and inflammatory symptoms (increased plasma levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1, and the inflammatory marker CRP) (Tables 1 and 2). Nx diabetic mice also showed increased plasma leptin and FGF-21 and decreased plasma adiponectin levels, suggesting the development of insulin resistance. In addition, the mice showed decreased pancreatic insulin content (pancreatic exhaustion), probably due to insulin resistance and hyperinsulinemia. This pathology was not markedly different from that in non-Nx diabetic (control) mice. Ipragliflozin improved hyperglycemia and hyperinsulinemia by significantly and dose-dependently stimulating urinary glucose excretion. In addition, ipragliflozin attenuated glucose intolerance and insulin resistance. Ipragliflozin also significantly and dose-dependently 71
European Journal of Pharmacology 830 (2018) 68–75
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albuminuria, glomerular hyperfiltration, and glomerulosclerotic lesions in addition to symptoms of type 2 diabetes such as obesity, hyperglycemia, and insulin resistance. However, nephropathy in these mice does not progress over time, unlike that in diabetic patients. A model of more severe nephropathy with reduced renal function was successfully created in uninephrectomized type 2 diabetic mice to increase the burden and contribution of various factors/mechanisms to exacerbate nephropathy in the kidney (O'Brien et al., 2013; Zheng et al., 2011). Based on these reports, we attempted to create a model of overt nephropathy in uninephrectomized KK/Ay type 2 diabetic mice. Compared with nonNx diabetic mice, Nx diabetic mice not only exhibited increased urinary protein excretion, decreased creatinine clearance, increased plasma creatinine levels and glomerulosclerosis, but also an additional pathological change in the form of interstitial fibrosis as well as increased blood pressure. These mice showed notable renal function decline and exacerbated renal pathological changes that appeared to correspond to those observed in overt diabetic nephropathy. Since there was no clear evidence of reduced renal function in these mice at the start of the study, these findings suggest that this model may represent a rapid form of diabetic nephropathy, progressing from incipient to overt nephropathy in a relatively short period of 4 weeks. In this animal model, ipragliflozin improved various symptoms of type 2 diabetes, including hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, obesity, dyslipidemia, steatosis, and inflammation. In addition, ipragliflozin significantly increased pancreatic insulin content. We hypothesize that this was mainly induced by preventing the exhaustion of pancreatic insulin stores through alleviation of hyperglycemia-induced excess insulin secretion. In addition, this effect might also be correlated with the ipragliflozin-induced improvement in glucose tolerance and insulin resistance caused by glucose toxicity. These improvements by ipragliflozin are similar to those shown previously in other type 2 diabetes models (Tahara et al., 2013), demonstrating that ipragliflozin is also effective for treating the symptoms of type 2 diabetes in this model. In addition, ipragliflozin significantly decreased the urinary excretion of albumin and protein and reversed the decline in renal function, including increasing plasma creatinine levels and decreasing creatinine clearance. Ipragliflozin also improved the characteristic pathological changes of diabetic nephropathy, including glomerulosclerosis, interstitial fibrosis, inflammatory cell infiltration, and basophilic change (Fioretto and Mauer, 2007). Therefore, ipragliflozin improves the histopathological changes associated with diabetic nephropathy throughout the whole kidney, including the glomeruli, tubules, and interstitium. It also showed efficacy for improving tubular disorder, as indicated by significant decreases in the tubular injury markers, urinary NAG activity and KIM-1 excretion, following ipragliflozin administration. In addition, ipragliflozin significantly decreased urinary nephrin and podocalyxin excretions, glomerular podocyte injury markers that are highly correlated with the exacerbation of nephropathy (Lioudaki et al., 2015). This suggests that the actions of ipragliflozin in the glomeruli are not only to improve glomerulosclerosis, which arises from extracellular matrix accumulation in the mesangial region, but also to restore podocyte function, which plays an important role in the barrier mechanism of the basement membrane. Ipragliflozin may improve diabetic nephropathy by primarily ameliorating hyperglycemia, and thereby glomerular hyperfiltration, to improve glycemic control and reduce complications, such as nephropathy, as observed in a large clinical study (ADVANCE Collaborative Group, 2008; Neal et al., 2017; Wanner et al., 2016). Compared with normal mice, non-Nx and Nx diabetic mice showed little change in and significantly increased blood pressure, respectively. Since nephrectomy-induced reduction in renal function is widely known to result in hypertension (Gava et al., 2012), our model may exhibit hypertension-induced chronic renal failure. Ipragliflozin significantly and dose-dependently improved hypertension. In clinical settings, SGLT2 inhibitors have been shown to mildly decrease blood
Fig. 3. Effects of 4-week intervention with ipragliflozin or losartan on urinary (A) albumin and (B) protein excretions and (C) kidney weight in uninephrectomized type 2 diabetic mice. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, # P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
4. Discussion Many studies have reported that various SGLT2 inhibitors, including ipragliflozin, are effective for improving nephropathy in type 2 diabetic models (Tahara et al., 2013; Gembardt et al., 2014; Terami et al., 2014; Kojima et al., 2013; Nagata et al., 2013; Abdel-Wahab et al., 2016). Most of these studies have used animals with genetic type 2 diabetes, such as ob/ob mice, which exhibit characteristics of incipient nephropathy, characterized by increased urinary albumin excretion and creatinine clearance (glomerular hyperfiltration). Therefore, SGLT2 inhibitors are effective for improving the characteristics of incipient nephropathy, such as decreasing urinary albumin excretion and glomerulosclerotic lesions, by improving hyperglycemia and glomerular hyperfiltration. While this efficacy of SGLT2 inhibitors for incipient diabetic nephropathy is very important, it is also necessary to evaluate their efficacy for overt nephropathy, which is characterized by reduced renal function, and may therefore be more clinically significant. Nevertheless, few studies have evaluated the efficacy of SGLT2 inhibitors for overt diabetic nephropathy due to the scarcity of suitable pathological models (Kojima et al., 2013). Mice with genetic type 2 diabetes, including ob/ob, db/db, and KK/ Ay mice, exhibit typical symptoms of incipient nephropathy such as 72
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Fig. 4. Effects of 4-week intervention with ipragliflozin or losartan on (A) creatinine clearance and (B) plasma creatinine levels in uninephrectomized type 2 diabetic mice. Effects before (pre, left) and after (4 w, right) drug administration. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
albumin and protein excretion and renal tissue lesions, and a more potent antihypertensive effect compared with a high dose of ipragliflozin. These effects of losartan are consistent with the results of previous studies (Weil et al., 2013; Bakris et al., 2008), and may primarily be attributed to the alleviation of glomerular hyperfiltration resulting from stimulated RAAS-mediated efferent arteriolar contraction and partially to its antihypertensive actions. In addition, the RAAS is reportedly correlated with the initiation and progression of diabetic nephropathy not only through hemodynamic mechanisms such as systemic and glomerular hypertension, but also various non-hemodynamic mechanisms, including fibrosis factors such as transforming growth factor-β1 (TGF-β1), proinflammatory cytokines such as TNF-α, and oxidative stress (Wiecek et al., 2003). Unlike ipragliflozin, losartan decreased creatinine clearance and slightly elevated plasma creatinine levels. This may be the result of a sustained and potent reduction in renal blood flow and glomerular filtration resulting from RAAS inhibition, suggesting that ipragliflozin and losartan alleviate glomerular hyperfiltration via different mechanisms of action. Long-term clinical studies have shown that ARB and ACE inhibitors, which inhibit the RAAS, reverse the increase in plasma creatinine and reduction in renal function (Ruggenenti et al., 2010); similar effects have also been reported in diabetic animals (Abdel-Wahab et al., 2016). This discrepancy with the results of our study may be attributable to differences in the disease phase of diabetic nephropathy, dose and dosing period, and mechanisms of pathogenesis and progression of nephropathy between diabetic patients and our animal model. Further investigation is necessary to clarify these issues. The results of the present study suggest that the SGLT2 inhibitor ipragliflozin may reverse or prevent the pathological deterioration from incipient to overt diabetic nephropathy, including a decline in renal function. Therefore, SGLT2 inhibitors may represent a promising therapeutic option for the management of type 2 diabetes to slow the progression of nephropathy.
pressure (Majewski and Bakris, 2015). In contrast, a SGLT2 inhibitor did not decrease blood pressure in subtotally nephrectomized rats (Li et al., 2018), a non-diabetic chronic kidney disease model, while RAAS blockers including losartan potently decreased blood pressure in this model. These findings suggest that the ipragliflozin-induced decrease in blood pressure observed in the present study may be secondary to the improved renal function and diuresis under diabetic conditions. Since hypertension is highly correlated with the initiation and deterioration of diabetic nephropathy, particularly glomerular hyperfiltration (Mogensen, 1982), the antihypertensive effect of ipragliflozin might partially contribute to improving diabetic nephropathy. Recent reports have also suggested that SGLT2 inhibitor-induced alleviation of diabetic nephropathy might be attributed to not only direct amelioration of hyperglycemia, but also several other mechanisms, including tubuloglomerular feedback system (TGF)-mediated alleviation of glomerular hyperfiltration (Škrtić and Cherney, 2015) and amelioration of renal tissue disorders, such as glomerulosclerosis, that arise due to inflammation associated with insulin resistance/hyperinsulinemia or increased oxidative stress (Whaley-Connell and Sowers, 2017). Therefore, further studies are necessary to clarify the mechanisms underlying SGLT2 inhibitor-induced alleviation of diabetic nephropathy. Our findings that ipragliflozin reverses the incipient to overt stage progression of diabetic nephropathy constitute the first report of the efficacy of SGLT2 inhibitors not only for incipient diabetic nephropathy but also overt diabetic nephropathy. In our mouse model, losartan slightly but non-significantly improved hyperglycemia and various other type 2 diabetes symptoms. Since the RAAS has been linked to insulin resistance in skeletal muscle and adipocytes (Underwood and Adler, 2013), the mild improvements in diabetic symptoms resulting from losartan administration might be due to improvements to insulin resistance; however, the full mechanism remains to be discerned. In contrast, losartan was effective for improving diabetic nephropathy, with a similar efficacy for urinary
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Fig. 5. Effects of 4-week intervention with ipragliflozin or losartan on renal tubular injury markers and glomerular podocyte injury markers in uninephrectomized type 2 diabetic mice. Graphs show levels of renal tubular injury markers, urinary (A) N-acetyl-β-D-glucosaminidase (NAG) activity and (B) kidney injury molecule-1 (KIM-1) excretion, and glomerular podocyte injury markers, urinary (C) nephrin and (D) podocalyxin excretions. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
Fig. 6. Effects of 4-week intervention with ipragliflozin or losartan on renal injury in uninephrectomized type 2 diabetic mice. Graphs show the histopathological scores for (A) glomerulosclerosis, (B) interstitial fibrosis, (C) cell infiltration, and (D) basophilic change. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.D. for six animals per group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
Acknowledgments The authors thank Drs. Yuichi Tomura, Akiyoshi Shimaya, Shoji Takakura, Toshihide Yokoyama, and Wataru Uchida (Astellas Pharma Inc.) and Drs. Hiroshi Tomiyama, Akira Tomiyama, Yoshihiko Haino, and Yoshinori Kondo (Kotobuki Pharmaceutical Co., Ltd.) for their valuable comments and continuing encouragement.
Conflict of interest The authors have no conflict of interest other than being employees of Astellas Pharma Inc.
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References
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Prevention of progression of diabetic nephropathy by the SGLT2 inhibitor ipragliflozin in uninephrectomized type 2 diabetic mice
T
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Atsuo Tahara , Toshiyuki Takasu Candidate Discovery Science Laboratories, Astellas Pharma Inc., Ibaraki, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: SGLT2 inhibitor Ipragliflozin Hyperglycemia Type 2 diabetes Nephropathy
Diabetic nephropathy is the leading cause of end-stage renal disease in the world. Although recent development of sodium-glucose cotransporter (SGLT) 2 inhibitors offers a new antidiabetic therapeutic strategy, it remains unclear whether such treatments are beneficial for limiting the progression of type 2 diabetic overt nephropathy. This study examined the effect of the SGLT2 inhibitor ipragliflozin on the progression of nephropathy in uninephrectomized KK/Ay type 2 diabetic mice, which exhibit not only typical diabetic symptoms such as hyperglycemia, hyperinsuemia, glucose intolerance, insulin resistance, hyperlipidemia, inflammation, and obesity, but also moderate hypertension and overt nephropathy with decline in renal function. Four-week repeated administration of ipragliflozin improved various diabetic symptoms, including hyperglycemia, insulin resistance, and inflammation by increasing urinary glucose excretion. In addition, ipragliflozin ameliorated albuminuria/ proteinuria; decline in renal function, as measured by creatinine clearance; hypertension; and renal injury, including glomerulosclerosis and interstitial fibrosis. These effects were significant at doses of 1 mg/kg or higher and were similar to those observed following administration of losartan (30 mg/kg). These results suggest that the SGLT2 inhibitor ipragliflozin prevents progression to diabetic overt nephropathy in uninephrectomized type 2 diabetic mice. SGLT2 inhibitors may therefore represent a promising therapeutic option for the management of type 2 diabetes to slow the progression of diabetic nephropathy.
1. Introduction Diabetic nephropathy is one of the most common complications of diabetes and the leading cause of end-stage renal disease. It is also associated with high cardiovascular risk and significant morbidity and mortality worldwide (Pálsson and Patel, 2014). In the past, several mechanisms have been implicated in the initiation and deterioration of diabetic nephropathy, including hyperglycemia, hypertension, dyslipidemia, and obesity, as well as ethnic and genetic factors (Tavafi, 2013). Mounting evidence suggests that proper control of blood glucose and blood pressure can reduce the risk of developing this complication. While current antidiabetic therapies to control blood glucose and administration of renin-angiotensin-aldosterone system (RAAS) inhibitors, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor antagonists (ARBs) to control blood pressure can slow the progression of diabetic nephropathy, they do not sufficiently prevent it (Fioretto et al., 2010). Therefore, there is a strong need to explore additional therapies for limiting the progression of diabetic nephropathy. In recent years, sodium-glucose cotransporter (SGLT) 2 inhibitors, which improve hyperglycemia via the stimulation of glucose excretion
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into the urine, have been proposed as novel drugs for treating type 2 diabetes (Chao, 2014). As chronic hyperglycemia is thought to contribute to the progression of diabetic nephropathy, the potent antihyperglycemic effects of SGLT2 inhibition may be promising for treating diabetic nephropathy. Several SGLT2 inhibitors including ipragliflozin have been shown to reduce urinary albumin excretion in type 2 diabetic animals with incipient nephropathy, which, at least in part, was attributed to inflammation, oxidative stress, and insulin resistance (Škrtić and Cherney, 2015; Tahara et al., 2013). However, the potential effects of SGLT2 inhibitors on the progression of overt nephropathy in type 2 diabetic animal models have not been determined in detail. Here, we investigated the effects of the SGLT2 inhibitor ipragliflozin on the progression of nephropathy in uninephrectomized type 2 diabetic mice, which exhibit not only typical diabetic symptoms such as hyperglycemia, insulin resistance, hyperlipidemia, hepatic steatosis, inflammation and obesity, but also moderate hypertension and overt nephropathy with decline in renal function. In addition, we compared the effects of ipragliflozin with those of the ARB losartan.
Correspondence to: Candidate Discovery Science Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. E-mail address: [email protected] (A. Tahara).
https://doi.org/10.1016/j.ejphar.2018.04.024 Received 10 January 2018; Received in revised form 19 April 2018; Accepted 20 April 2018 Available online 25 April 2018 0014-2999/ © 2018 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 830 (2018) 68–75
A. Tahara, T. Takasu
2. Materials and methods 2.1. Materials Ipragliflozin was synthesized at Astellas Pharma Inc. (Ibaraki, Japan) and losartan was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). These drugs were suspended or dissolved in 0.5% methylcellulose solution and administered orally via a stomach tube. Doses of these drugs were expressed as the free base form. 2.2. Animals Male C57BL/6 (normal) and KK/Ay type 2 diabetic mice were purchased from CLEA Japan (Kanagawa, Japan) at age 6 weeks and used at age 14 weeks. The left kidney of diabetic mice was removed under isoflurane anesthesia. Two weeks after uninephrectomy, diabetic mice were grouped such as to attain uniform mean blood glucose levels among the groups. All animals were housed under standard conditions of controlled temperature, humidity, and light (12-h light-dark cycle), and were given free access to standard commercial chow and water. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. Astellas Pharma Inc., Tsukuba Research Center has been awarded Accreditation Status by the AAALAC International.
Fig. 1. Effects of 4-week intervention with ipragliflozin or losartan on (A) blood pressure and (B) heart rate in uninephrectomized type 2 diabetic mice. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. nonNx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $ P < 0.05 vs. Nx diabetic vehicle group.
2.3. Repeated administration study Groups comprised the following: (1) normal mice: normal (vehicle); (2) non-nephrectomized (non-Nx) diabetic mice: control (vehicle); (3) nephrectomized (Nx) diabetic mice: (i) vehicle, (ii) ipragliflozin (0.1 mg/kg), (iii) ipragliflozin (0.3 mg/kg), (iv) ipragliflozin (1 mg/kg), (v) ipragliflozin (3 mg/kg), (vi) losartan (30 mg/kg). Six mice were used in each group/subgroup. To measure pretreatment creatinine clearance values, mice were transferred to metabolic cages where spontaneously voided urine was collected for 24 h, and blood samples were obtained from the tail vein. Subsequently, each drug was orally administered to Nx diabetic mice once daily (at night) for 4 weeks. Body weight and food intake were measured weekly. An oral glucose tolerance test (OGTT) was performed at Week 3. Mice were fasted over the inactive period (from 7:00–19:00) prior to drug administration. Two hours later, blood was sampled from a tail vein for the evaluation of fasting blood glucose and plasma insulin levels. A glucose solution (2 g/kg) was then orally administered, and blood sampling was conducted for 2 h. After drug administration on day 25, spontaneously voided urine was collected for 24 h. The morning after the final drug administration on day 28, blood samples were collected from the abdominal vena cava and tissues (kidneys, pancreas, liver, and epididymal adipose tissue) were isolated under isoflurane anesthesia. To measure blood pressure, a second set of animals was subjected to the same group composition and drug administration schedule. The morning after the final drug administration on day 29, systolic blood pressure and heart rate were measured using a photoelectric tail cuff pulse detection system (BP-98A-L; Softron, Tokyo, Japan).
using the Triglyceride E-test Wako, NEFA C-test Wako, and Cholesterol E-test Wako kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Hepatic lipid (triglyceride and cholesterol) content levels were measured according to a previously reported method (Tahara et al., 2011). Levels of aminotransferases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the Transaminase CII test reagent (Wako Pure Chemical Industries, Ltd.). Plasma levels of leptin, fibroblast growth factor 21 (FGF-21), adiponectin, interleukin (IL)− 1β, IL-6, tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and C-reactive protein (CRP) were measured using commercial ELISA kits (R&D Systems Inc., Minneapolis, MN, USA). Urinary albumin and protein excretion were measured using mouse albumin ELISA and a protein assay reagent (BioRad, Hercules, CA, USA), respectively. Plasma and urinary creatinine levels were measured using Determiner L CRE (Kyowa Medex, Tokyo, Japan), and creatinine clearance (µl/g body weight/min) was calculated based on urinary and plasma creatinine levels, urine volume, and body weight. Urinary concentrations of nephrin and podocalyxin were measured using ELISA kits (Exocell Inc., PA, USA). Urinary concentrations of kidney injury molecule-1 (KIM-1) were measured using an ELISA kit (R&D Systems Inc.). Urinary N-acetyl-β-D-glucosaminidase (NAG) activity was measured using NAG Test Shionogi (Shionogi Co. Ltd, Osaka, Japan).
2.4. Biochemical measurements Blood and urinary glucose concentrations were measured using Glucose CII test reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Hemoglobin A1c (HbA1c) levels were measured using a DCA2000 System (Bayer Medical, Tokyo, Japan). Plasma insulin levels were measured using an ultra-high-sensitivity mouse insulin enzymelinked immunosorbent assay (ELISA) kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). Pancreatic insulin content was measured using an insulin ELISA kit according to a previously reported method (Tahara et al., 2013). Plasma lipid [triglycerides, nonesterified fatty acids (NEFAs), and cholesterol] levels were measured
2.5. Histopathology Specimen preparation and histopathological examination were performed at CMIC Bioresearch Center Co., Ltd. (Yamanashi, Japan). Sagittal slices of renal tissue were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 2-μm thick sections for morphological study. These sections were stained with hematoxylin and eosin, and periodic acid Schiff. All tissue samples were evaluated by an independent investigator blinded to group information. All glomeruli and the entire microscopic area in each specimen were examined. 69
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Fig. 2. Effects of 4-week intervention with ipragliflozin or losartan on glucose tolerance and insulin resistance in uninephrectomized type 2 diabetic mice. Time course of changes in (A) blood glucose and (C) plasma insulin levels, and area under the (B) blood glucose and (D) plasma insulin concentration-time curves (AUCs) during the oral glucose tolerance test (OGTT). The (E) Matsuda and (F) disposition indexes were used as parameters of insulin resistance and secretion, respectively. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. OGTT was performed at Week 3. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group. Table 1 Effects of 4-week intervention with ipragliflozin or losartan on various diabetic parameters in uninephrectomized type 2 diabetic mice. Parameter
Body weight (g) Food intake (g/day) HbA1c (%) Blood glucose (mg/dL) Plasma insulin (ng/mL) Pancreatic insulin content (ng/mg tissue) Urine volume (mL/day) Urinary glucose excretion (mg/day) Epididymal adipose tissue weight (g) Plasma leptin (ng/mL) Plasma FGF-21 (ng/mL) Plasma adiponectin (µg/mL)
Normal
Diabetic (Control)
Nx diabetic
Vehicle
Vehicle
Vehicle
41.7 ± 0.6 3.52 ± 0.14 3.15 ± 0.11 125 ± 5 1.6 ± 0.2 99 ± 11 1.09 ± 0.15 0.9 ± 0.3 0.41 ± 0.02 6.8 ± 1.7 1.19 ± 0.13 9.53 ± 0.93
48.5 ± 0.6a 6.09 ± 0.11a 8.07 ± 0.40a 497 ± 24a 68.5 ± 8.5a 63 ± 9a 4.57 ± 0.44a 425 ± 52a 1.61 ± 0.09a 41.0 ± 5.6a 2.61 ± 0.12a 4.48 ± 0.50a
Ipragliflozin
47.5 ± 0.3a 6.21 ± 0.15a 7.98 ± 0.36a 472 ± 28a 74.2 ± 6.9a 65 ± 6a 4.32 ± 0.44a 478 ± 41a 1.77 ± 0.10a 38.8 ± 3.9a 2.80 ± 0.29a 4.31 ± 0.23a
Losartan
0.1 mg/kg
0.3 mg/kg
1 mg/kg
3 mg/kg
30 mg/kg
47.0 ± 0.9 6.17 ± 0.14 7.50 ± 0.35 446 ± 26 68.3 ± 7.7 72 ± 4 4.20 ± 0.50 531 ± 49 1.75 ± 0.09 33.3 ± 5.5 2.66 ± 0.28 4.84 ± 0.27
45.9 ± 0.7 6.29 ± 0.14 6.62 ± 0.37b 357 ± 15b 52.2 ± 5.1 81 ± 11 5.66 ± 0.38 745 ± 51b 1.64 ± 0.10 26.3 ± 4.1 2.44 ± 0.28 6.12 ± 0.61
44.7 ± 0.7b 6.32 ± 0.14 5.78 ± 0.38b 271 ± 15b 34.2 ± 5.2b 109 ± 3b 5.89 ± 0.56 946 ± 83b 1.35 ± 0.09b 20.6 ± 3.0b 1.85 ± 0.20b 7.28 ± 0.56b
43.4 ± 0.6b 6.25 ± 0.11 5.47 ± 0.33b 221 ± 14b 29.8 ± 4.5b 132 ± 13b 6.84 ± 0.50b 1109 ± 92b 1.31 ± 0.11b 18.8 ± 4.1b 1.59 ± 0.19b 8.13 ± 0.66b
47.8 ± 0.7 6.09 ± 0.15 7.57 ± 0.34 452 ± 23 64.9 ± 7.0 76 ± 6 3.96 ± 0.40 410 ± 42 1.86 ± 0.12 30.3 ± 5.0 2.40 ± 0.22 5.59 ± 0.60
HbA1c: hemoglobin A1c, FGF-21: fibroblast growth factor 21. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. a P < 0.05 vs. normal group (Tukey's test). b P < 0.05 vs. Nx diabetic vehicle group (Dunnett's test).
Histopathological changes (glomerulosclerosis, interstitial fibrosis, cell infiltration, and basophilic change) were evaluated using a semiquantitative scoring system according to the percentage of affected renal tissue: 0, no lesion; 1, very slight (< 10%); 2, slight (10% ≤ X < 25%); 3, moderate (25% ≤ X < 50%); 4, marked
(≥ 50%).
2.6. Statistical analysis The experimental results are expressed as the mean ± standard 70
European Journal of Pharmacology 830 (2018) 68–75
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Table 2 Effects of 4-week intervention with ipragliflozin or losartan on various lipid and inflammatory parameters in uninephrectomized type 2 diabetic mice. Parameter
Plasma triglycerides (mg/dL) Plasma NEFAs (mEq/L) Plasma cholesterol (mg/dL) Liver weight (g) Hepatic triglyceride content (mg/g liver) Hepatic cholesterol content (mg/g liver) ALT (IU/L) AST (IU/L) Plasma IL-1β (pg/mL) Plasma IL-6 (pg/mL) Plasma TNF-α (pg/mL) Plasma MCP-1 (pg/mL) Plasma CRP (ng/mL)
Normal
Diabetic (Control)
Nx diabetic
Vehicle
Vehicle
Vehicle
106 ± 7 0.67 ± 0.05 77 ± 6 1.55 ± 0.07 10.7 ± 1.7 4.7 ± 0.8 14.9 ± 2.4 27.5 ± 4.8 71 ± 14 186 ± 21 55 ± 6 103 ± 27 150 ± 53
411 ± 39a 1.24 ± 0.07a 136 ± 7a 2.97 ± 0.09a 29.7 ± 3.8a 16.7 ± 2.4a 55.3 ± 7.2a 74.7 ± 6.7a 335 ± 52a 939 ± 128a 212 ± 25a 469 ± 68a 362 ± 29a
Ipragliflozin
395 ± 34a 1.21 ± 0.06a 139 ± 10a 3.09 ± 0.12a 30.9 ± 3.3a 16.9 ± 2.0a 64.4 ± 7.0a 79.1 ± 8.4a 397 ± 75a 959 ± 100a 220 ± 20a 534 ± 76a 426 ± 41a
Losartan
0.1 mg/kg
0.3 mg/kg
1 mg/kg
3 mg/kg
30 mg/kg
383 ± 30 1.21 ± 0.09 140 ± 7 2.94 ± 0.15 30.2 ± 3.2 16.9 ± 1.4 53.5 ± 10.5 69.8 ± 11.8 352 ± 49 837 ± 110 225 ± 31 455 ± 69 400 ± 33
302 ± 35 1.05 ± 0.07 141 ± 11 2.64 ± 0.13 24.2 ± 2.4 12.4 ± 1.5 40.5 ± 6.6 65.0 ± 5.8 282 ± 64 669 ± 100 175 ± 19 386 ± 76 325 ± 36
208 ± 29b 0.98 ± 0.07 115 ± 9 2.23 ± 0.13b 19.0 ± 2.8b 8.9 ± 1.5b 24.3 ± 6.3b 52.4 ± 7.3 187 ± 41b 564 ± 68b 134 ± 15b 267 ± 44b 212 ± 30b
220 ± 24b 0.87 ± 0.04b 96 ± 8b 2.22 ± 0.15b 15.7 ± 1.5b 6.6 ± 0.7b 21.5 ± 4.2b 40.0 ± 7.0b 150 ± 28b 510 ± 56b 117 ± 10b 245 ± 36b 197 ± 39b
388 ± 28 1.11 ± 0.08 147 ± 14 3.01 ± 0.10 27.5 ± 3.3 14.6 ± 1.8 50.9 ± 7.2 60.0 ± 7.8 313 ± 41 990 ± 93 200 ± 31 478 ± 44 359 ± 51
NEFAs: non-esterified fatty acids, ALT: alanine aminotransferase, AST: aspartate aminotransferase, IL: interleukin, TNF-αː tumor necrosis factor α, MCP-1: monocyte chemotactic protein-1, CRP: C-reactive protein. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. a P < 0.05 vs. normal group (Tukey's test). b P < 0.05 vs. Nx diabetic vehicle group (Dunnett's test).
improved obesity (body and fat weights), dyslipidemia, steatosis, and inflammatory symptoms without affecting food intake. Plasma levels of leptin, FGF-21, and adiponectin, which are highly correlated with insulin resistance, returned to normal levels, suggesting that ipragliflozin significantly and dose-dependently improved insulin resistance. Pancreatic insulin content was also significantly and dose-dependently increased to normal levels. In contrast, while losartan slightly improved these type 2 diabetes symptoms, the changes were not significant. Compared to normal mice, non-Nx diabetic mice exhibited increased urinary albumin excretion, creatinine clearance (hyperfiltration), tubular injury markers (urinary NAG activity and KIM-1 excretion) and glomerular podocyte injury markers (urinary excretion of nephrin and podocalyxin), as well as histopathological features of glomerulosclerosis, with little interstitial fibrosis (Figs. 3–6). Renal function was not reduced, as evidenced by the lack of a significant decrease in creatinine clearance (Fig. 4A) and changes in urinary protein excretion (Fig. 3B) and plasma creatinine levels (Fig. 4B). These results demonstrate that non-Nx diabetic mice exhibit symptoms associated with incipient diabetic nephropathy. In contrast, Nx diabetic mice exhibited reduced renal function at the end of the 4-week study, as evidenced by significantly increased urinary albumin and protein excretions, decreased creatinine clearance, and increased plasma creatinine levels. In addition, further increases were observed in tubular and glomerular podocyte injury markers and interstitial fibrosis, which was an additional pathological change to glomerulosclerosis. Since there was no evidence of reduced renal function in these mice at the start of the study, these findings indicate that Nx diabetic mice show rapid progression from incipient to overt nephropathy. Ipragliflozin significantly and dose-dependently decreased albumin and protein urinary excretions, alleviated renal hypertrophy (Fig. 3), reversed the decline in renal function (decreased creatinine clearance and increased plasma creatinine) (Fig. 4), decreased markers of renal tubular and glomerular podocyte injury (Fig. 5), and improved histopathological changes, including glomerulosclerosis and interstitial fibrosis (Fig. 6). Losartan also significantly improved nephropathy, almost as effectively as ipragliflozin. However, unlike with ipragliflozin, losartan significantly decreased creatinine clearance and slightly increased plasma creatinine.
error of the mean (S.E.M.) or standard deviation (S.D.). Differences between normal, diabetic (non-Nx) control, and Nx diabetic vehicle groups were assessed using Tukey's multiple comparisons test. Differences between the Nx diabetic vehicle and ipragliflozin-treated groups were assessed using a Dunnett's multiple comparisons test, while those between the Nx diabetic vehicle and losartan-treated groups were assessed using a Student's t-test. Histopathological scores were compared using a Mann-Whitney test to analyze differences between two groups and Dunn's multiple comparisons test among multiple groups. A value of P < 0.05 was considered to be significant. Statistical and data analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
3. Results Compared to normal mice, non-Nx diabetic mice exhibited a slight but non-significant increase in blood pressure (Fig. 1). In contrast, Nxdiabetic mice exhibited a significant increase in blood pressure in the absence of changes in heart rate. Ipragliflozin slightly but significantly and dose-dependently decreased blood pressure, and losartan markedly decreased blood pressure. Nx diabetic mice exhibited glucose intolerance and insulin resistance during the OGTT (Fig. 2). In addition, these mice exhibited hyperglycemia, hyperinsulinemia, glycosuria and polyuria, obesity with overeating, dyslipidemia (increased plasma levels of triglycerides, NEFAs, and cholesterol), steatosis (hepatic hypertrophy, increased hepatic contents of triglycerides and cholesterol, and increased plasma ALT/AST levels), and inflammatory symptoms (increased plasma levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1, and the inflammatory marker CRP) (Tables 1 and 2). Nx diabetic mice also showed increased plasma leptin and FGF-21 and decreased plasma adiponectin levels, suggesting the development of insulin resistance. In addition, the mice showed decreased pancreatic insulin content (pancreatic exhaustion), probably due to insulin resistance and hyperinsulinemia. This pathology was not markedly different from that in non-Nx diabetic (control) mice. Ipragliflozin improved hyperglycemia and hyperinsulinemia by significantly and dose-dependently stimulating urinary glucose excretion. In addition, ipragliflozin attenuated glucose intolerance and insulin resistance. Ipragliflozin also significantly and dose-dependently 71
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albuminuria, glomerular hyperfiltration, and glomerulosclerotic lesions in addition to symptoms of type 2 diabetes such as obesity, hyperglycemia, and insulin resistance. However, nephropathy in these mice does not progress over time, unlike that in diabetic patients. A model of more severe nephropathy with reduced renal function was successfully created in uninephrectomized type 2 diabetic mice to increase the burden and contribution of various factors/mechanisms to exacerbate nephropathy in the kidney (O'Brien et al., 2013; Zheng et al., 2011). Based on these reports, we attempted to create a model of overt nephropathy in uninephrectomized KK/Ay type 2 diabetic mice. Compared with nonNx diabetic mice, Nx diabetic mice not only exhibited increased urinary protein excretion, decreased creatinine clearance, increased plasma creatinine levels and glomerulosclerosis, but also an additional pathological change in the form of interstitial fibrosis as well as increased blood pressure. These mice showed notable renal function decline and exacerbated renal pathological changes that appeared to correspond to those observed in overt diabetic nephropathy. Since there was no clear evidence of reduced renal function in these mice at the start of the study, these findings suggest that this model may represent a rapid form of diabetic nephropathy, progressing from incipient to overt nephropathy in a relatively short period of 4 weeks. In this animal model, ipragliflozin improved various symptoms of type 2 diabetes, including hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, obesity, dyslipidemia, steatosis, and inflammation. In addition, ipragliflozin significantly increased pancreatic insulin content. We hypothesize that this was mainly induced by preventing the exhaustion of pancreatic insulin stores through alleviation of hyperglycemia-induced excess insulin secretion. In addition, this effect might also be correlated with the ipragliflozin-induced improvement in glucose tolerance and insulin resistance caused by glucose toxicity. These improvements by ipragliflozin are similar to those shown previously in other type 2 diabetes models (Tahara et al., 2013), demonstrating that ipragliflozin is also effective for treating the symptoms of type 2 diabetes in this model. In addition, ipragliflozin significantly decreased the urinary excretion of albumin and protein and reversed the decline in renal function, including increasing plasma creatinine levels and decreasing creatinine clearance. Ipragliflozin also improved the characteristic pathological changes of diabetic nephropathy, including glomerulosclerosis, interstitial fibrosis, inflammatory cell infiltration, and basophilic change (Fioretto and Mauer, 2007). Therefore, ipragliflozin improves the histopathological changes associated with diabetic nephropathy throughout the whole kidney, including the glomeruli, tubules, and interstitium. It also showed efficacy for improving tubular disorder, as indicated by significant decreases in the tubular injury markers, urinary NAG activity and KIM-1 excretion, following ipragliflozin administration. In addition, ipragliflozin significantly decreased urinary nephrin and podocalyxin excretions, glomerular podocyte injury markers that are highly correlated with the exacerbation of nephropathy (Lioudaki et al., 2015). This suggests that the actions of ipragliflozin in the glomeruli are not only to improve glomerulosclerosis, which arises from extracellular matrix accumulation in the mesangial region, but also to restore podocyte function, which plays an important role in the barrier mechanism of the basement membrane. Ipragliflozin may improve diabetic nephropathy by primarily ameliorating hyperglycemia, and thereby glomerular hyperfiltration, to improve glycemic control and reduce complications, such as nephropathy, as observed in a large clinical study (ADVANCE Collaborative Group, 2008; Neal et al., 2017; Wanner et al., 2016). Compared with normal mice, non-Nx and Nx diabetic mice showed little change in and significantly increased blood pressure, respectively. Since nephrectomy-induced reduction in renal function is widely known to result in hypertension (Gava et al., 2012), our model may exhibit hypertension-induced chronic renal failure. Ipragliflozin significantly and dose-dependently improved hypertension. In clinical settings, SGLT2 inhibitors have been shown to mildly decrease blood
Fig. 3. Effects of 4-week intervention with ipragliflozin or losartan on urinary (A) albumin and (B) protein excretions and (C) kidney weight in uninephrectomized type 2 diabetic mice. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, # P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
4. Discussion Many studies have reported that various SGLT2 inhibitors, including ipragliflozin, are effective for improving nephropathy in type 2 diabetic models (Tahara et al., 2013; Gembardt et al., 2014; Terami et al., 2014; Kojima et al., 2013; Nagata et al., 2013; Abdel-Wahab et al., 2016). Most of these studies have used animals with genetic type 2 diabetes, such as ob/ob mice, which exhibit characteristics of incipient nephropathy, characterized by increased urinary albumin excretion and creatinine clearance (glomerular hyperfiltration). Therefore, SGLT2 inhibitors are effective for improving the characteristics of incipient nephropathy, such as decreasing urinary albumin excretion and glomerulosclerotic lesions, by improving hyperglycemia and glomerular hyperfiltration. While this efficacy of SGLT2 inhibitors for incipient diabetic nephropathy is very important, it is also necessary to evaluate their efficacy for overt nephropathy, which is characterized by reduced renal function, and may therefore be more clinically significant. Nevertheless, few studies have evaluated the efficacy of SGLT2 inhibitors for overt diabetic nephropathy due to the scarcity of suitable pathological models (Kojima et al., 2013). Mice with genetic type 2 diabetes, including ob/ob, db/db, and KK/ Ay mice, exhibit typical symptoms of incipient nephropathy such as 72
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Fig. 4. Effects of 4-week intervention with ipragliflozin or losartan on (A) creatinine clearance and (B) plasma creatinine levels in uninephrectomized type 2 diabetic mice. Effects before (pre, left) and after (4 w, right) drug administration. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
albumin and protein excretion and renal tissue lesions, and a more potent antihypertensive effect compared with a high dose of ipragliflozin. These effects of losartan are consistent with the results of previous studies (Weil et al., 2013; Bakris et al., 2008), and may primarily be attributed to the alleviation of glomerular hyperfiltration resulting from stimulated RAAS-mediated efferent arteriolar contraction and partially to its antihypertensive actions. In addition, the RAAS is reportedly correlated with the initiation and progression of diabetic nephropathy not only through hemodynamic mechanisms such as systemic and glomerular hypertension, but also various non-hemodynamic mechanisms, including fibrosis factors such as transforming growth factor-β1 (TGF-β1), proinflammatory cytokines such as TNF-α, and oxidative stress (Wiecek et al., 2003). Unlike ipragliflozin, losartan decreased creatinine clearance and slightly elevated plasma creatinine levels. This may be the result of a sustained and potent reduction in renal blood flow and glomerular filtration resulting from RAAS inhibition, suggesting that ipragliflozin and losartan alleviate glomerular hyperfiltration via different mechanisms of action. Long-term clinical studies have shown that ARB and ACE inhibitors, which inhibit the RAAS, reverse the increase in plasma creatinine and reduction in renal function (Ruggenenti et al., 2010); similar effects have also been reported in diabetic animals (Abdel-Wahab et al., 2016). This discrepancy with the results of our study may be attributable to differences in the disease phase of diabetic nephropathy, dose and dosing period, and mechanisms of pathogenesis and progression of nephropathy between diabetic patients and our animal model. Further investigation is necessary to clarify these issues. The results of the present study suggest that the SGLT2 inhibitor ipragliflozin may reverse or prevent the pathological deterioration from incipient to overt diabetic nephropathy, including a decline in renal function. Therefore, SGLT2 inhibitors may represent a promising therapeutic option for the management of type 2 diabetes to slow the progression of nephropathy.
pressure (Majewski and Bakris, 2015). In contrast, a SGLT2 inhibitor did not decrease blood pressure in subtotally nephrectomized rats (Li et al., 2018), a non-diabetic chronic kidney disease model, while RAAS blockers including losartan potently decreased blood pressure in this model. These findings suggest that the ipragliflozin-induced decrease in blood pressure observed in the present study may be secondary to the improved renal function and diuresis under diabetic conditions. Since hypertension is highly correlated with the initiation and deterioration of diabetic nephropathy, particularly glomerular hyperfiltration (Mogensen, 1982), the antihypertensive effect of ipragliflozin might partially contribute to improving diabetic nephropathy. Recent reports have also suggested that SGLT2 inhibitor-induced alleviation of diabetic nephropathy might be attributed to not only direct amelioration of hyperglycemia, but also several other mechanisms, including tubuloglomerular feedback system (TGF)-mediated alleviation of glomerular hyperfiltration (Škrtić and Cherney, 2015) and amelioration of renal tissue disorders, such as glomerulosclerosis, that arise due to inflammation associated with insulin resistance/hyperinsulinemia or increased oxidative stress (Whaley-Connell and Sowers, 2017). Therefore, further studies are necessary to clarify the mechanisms underlying SGLT2 inhibitor-induced alleviation of diabetic nephropathy. Our findings that ipragliflozin reverses the incipient to overt stage progression of diabetic nephropathy constitute the first report of the efficacy of SGLT2 inhibitors not only for incipient diabetic nephropathy but also overt diabetic nephropathy. In our mouse model, losartan slightly but non-significantly improved hyperglycemia and various other type 2 diabetes symptoms. Since the RAAS has been linked to insulin resistance in skeletal muscle and adipocytes (Underwood and Adler, 2013), the mild improvements in diabetic symptoms resulting from losartan administration might be due to improvements to insulin resistance; however, the full mechanism remains to be discerned. In contrast, losartan was effective for improving diabetic nephropathy, with a similar efficacy for urinary
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Fig. 5. Effects of 4-week intervention with ipragliflozin or losartan on renal tubular injury markers and glomerular podocyte injury markers in uninephrectomized type 2 diabetic mice. Graphs show levels of renal tubular injury markers, urinary (A) N-acetyl-β-D-glucosaminidase (NAG) activity and (B) kidney injury molecule-1 (KIM-1) excretion, and glomerular podocyte injury markers, urinary (C) nephrin and (D) podocalyxin excretions. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.E.M. for six animals in each group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
Fig. 6. Effects of 4-week intervention with ipragliflozin or losartan on renal injury in uninephrectomized type 2 diabetic mice. Graphs show the histopathological scores for (A) glomerulosclerosis, (B) interstitial fibrosis, (C) cell infiltration, and (D) basophilic change. Ipragliflozin and losartan were orally administered to uninephrectomized (Nx) diabetic mice once daily for 4 weeks. Data are expressed as the mean ± S.D. for six animals per group. *P < 0.05 vs. normal group, %P < 0.05 vs. non-Nx diabetic (control) group, #P < 0.05 vs. Nx diabetic vehicle group, $P < 0.05 vs. Nx diabetic vehicle group.
Acknowledgments The authors thank Drs. Yuichi Tomura, Akiyoshi Shimaya, Shoji Takakura, Toshihide Yokoyama, and Wataru Uchida (Astellas Pharma Inc.) and Drs. Hiroshi Tomiyama, Akira Tomiyama, Yoshihiko Haino, and Yoshinori Kondo (Kotobuki Pharmaceutical Co., Ltd.) for their valuable comments and continuing encouragement.
Conflict of interest The authors have no conflict of interest other than being employees of Astellas Pharma Inc.
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