Typical features of type 2 diabetes mellitus (T2DM) are insulin resistance of various organs and reduced glucose-stimulated insulin secretion [
]. In addition, the kidney plays a crucial role in regulating glucose levels by mediating the reabsorption of glucose filtered through glomeruli. Renal glucose reabsorption is mediated by sodium glucose co-transporter 2 (SGLT2) and SGLT1, with SGLT2 playing the major role because of its specific localization in the kidney and higher capacity than SGLT1. SGLT2 expression is increased in T2DM; hence, higher amounts of glucose are reabsorbed by the kidney, thereby contributing to the sustained elevation of serum glucose levels in patients with diabetes [
]. SGLT2 inhibitors reduce blood glucose levels by suppressing this glucose reabsorption in an insulin-independent manner. Furthermore, SGLT2 inhibitors have demonstrated not only a hypoglycemic effect but also a body weight-reducing effect through calorie loss associated with glycosuria and a hypotensive effect [
]. In addition to control of blood glucose, control of body weight and blood pressure play important roles in preventing complications associated with T2DM [
]. Thus, a series of SGLT2 inhibitors have become new therapeutic options for T2DM [
]. Several studies of SGLT2 inhibitors have been conducted to assess the cardiovascular outcomes [
], and it was reported that the SGLT2 inhibitor empagliflozin reduced heart failure hospitalization as well as cardiovascular and all-cause deaths in T2DM patients with high CVD risk (EMPA-REG OUTCOME trial) [
]. One possible mechanism underlying the cardiovascular benefit of empagliflozin is related to its osmotic diuretic effect by glycosuria [
]. However, osmotic diuresis-induced increase in urine volume, loss of body fluid, and risk of dehydration have been of concern with SGLT2 inhibitor treatment [
Canagliflozin is an SGLT2 inhibitor approved in North America, Europe, Latin America, and Asia–Pacific regions for treatment of T2DM [
]. We previously conducted a pharmacokinetic and pharmacodynamic study of canagliflozin in Japanese patients with T2DM, in which administration of canagliflozin at 25, 100, 200, or 400 mg once-daily increased 24-h urine volume by 400–700 mL on Day 1, with a return to baseline from Day 2 onward. There was also an increase in urinary sodium (Na) excretion on Day 1 with a return to baseline thereafter, while water intake increased in the 400-mg group on Day 1, but did not change in the other dosage groups [
] (ESM Table 1). Although some previous studies reported that SGLT2 inhibitor-induced increases in urine volume were short-lived [
], the detailed nature of the transient effect remains unknown. For the proper clinical use of the SGLT2 inhibitor canagliflozin, it is important to confirm this transient increase in urine volume and elucidate the underlying mechanism. Therefore, the purpose of this study was to investigate the mechanism underlying canagliflozin-induced diuresis in Japanese T2DM patients by measuring factors that affect homeostasis of body fluid and mineral balance.
Subjects and Study Design
This single-arm, exploratory study was carried out to investigate the factors involved in the canagliflozin-induced transient change in urine volume in Japanese T2DM patients by examining major endogenous factors that affect urine volume. Eligible subjects were men and women with stable T2DM with the following characteristics: age of ≥25 and <75 years at the time of providing written consent; inadequate glycemic control, comprising HbA
1c ≥7% (53.01 mmol/mol) and ≤10% (85.8 mmol/mol); BMI ≥20 and ≤35 kg/m
2; on unchanged single or dual oral antidiabetic drug therapy for ≥3 months; systolic blood pressure of ≥95 and ≤160 mmHg and diastolic blood pressure of ≥50 and ≤100 mmHg; and an estimated GFR (eGFR) of ≥60 mL/min/1.73 m
2. The major exclusion criteria were as follows: no treatment for T2DM; history of type 1 diabetes mellitus or secondary diabetes; history of repeated severe hypoglycemia, or on therapy with insulin, glucagon-like peptide 1 (GLP-1) receptor agonist, SGLT2 inhibitor, or diuretic; history of serious diabetic complications requiring treatment; history or complication of kidney disease, nephrolithiasis, prostatitis, or complication of urinary tract infection; history of drug or food allergy; myocardial infarction, congestive heart failure, unstable angina pectoris, or cerebrovascular disorder (except for lacuna infarction) within 6 months prior to screening; history of transient ischemic attack or brain infarction with apparent neurological symptoms; complication of arteriosclerosis obliterans (stage III or IV of Fontaine classification); history of atrial fibrillation; or patients whom the investigator judged to be inappropriate for this study.
Eligible patients were hospitalized for 8 days for the study. From the third day (Day 1) to the eighth day (Day 6), each patient received an oral daily dose of canagliflozin 100 mg at 8:00 am before breakfast and was asked to finish their breakfast within 20 min. Blood and urine samples were collected at predetermined time points for laboratory tests and analyses of factors affecting urine volume. Efficacy was evaluated using data from Day 0 to Day 5, and safety was evaluated using data from day of admission to Day 6.
Endpoints and Assessments
The primary endpoints were correlations between changes from baseline in urine volume and factors known to affect urine volume, and between actual urine volume and these factors. The factors examined included water intake, urinary glucose excretion, urinary electrolyte excretion (Na and K), urine osmolality, and plasma concentrations of atrial natriuretic peptide (ANP), N-terminal pro-b-type natriuretic peptide (NT-proBNP), vasopressin, aldosterone, and renin activity. The blood samples for measuring these plasma hormones and enzyme (ANP, NT-proBNP, vasopressin, aldosterone, and renin) were obtained from the recumbent position. The secondary endpoints were blood glucose over time, changes in total GLP-1 concentration after breakfast, and safety such as adverse events (AEs), laboratory tests, and 12-lead ECGs. The daily urine osmolality, AUCs of blood glucose, and AUCs of total GLP-1 were determined by post hoc analysis. Laboratory tests including biochemical and hematological examinations, measurements of hormone levels, immunological tests, and urinalysis were performed by the Sapporo Clinical Laboratory Inc. (Sapporo, Japan). The 12-lead ECGs were monitored at the Clinical Pharmacology Center, Medical Corporation Hokubukai Utsukushigaoka Hospital (Sapporo, Japan).
Ethics Approval and Consent to Participate
The study was conducted in accordance with ethical principles that complied with the Declaration of Helsinki (revised October 2013), ethical guidelines for medical and health research involving human subjects, and conflict of interest management rules for clinical studies at the Medical Corporation Hokubukai Utsukushigaoka Hospital (Sapporo, Japan). The study protocol was approved by the Institutional Review Board at the Utsukushigaoka Hospital. All participants provided written informed consent before taking part in the study. Clinical trial registration: UMIN000019462.
To analyze the primary endpoint, Spearman’s correlation coefficients between changes from baseline in urine volume and factors known to affect urine volume, as well as between actual urine volume and each factor, were calculated, and a statistical test for absence of correlation was performed. Regarding continuous variables including urine volume, summary statistics (mean, SD, median, minimum, and maximum) for actual values and changes from baseline at each time point were calculated. For changes from baseline, 95% confidence intervals (CIs) were also calculated. For each of the secondary endpoints, actual values and changes from baseline were obtained at each time point and the data were analyzed in the same manner. To identify factors affecting a transient increase in urine volume, a post hoc multiple regression analysis (stepwise variable selection method; both entry and retention criteria were set at
p = 0.05.) was performed with change in urine volume as the dependent variable and related factors as independent variables.
Statistical analyses of the present study were performed with SAS 9.3 (SAS Institute Inc., Cary, NC, USA) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA). Values of
p < 0.05 were considered statistically significant. Data management and statistical analyses were carried out by IBEC Co. Ltd. (Osaka, Japan).
To evaluate the reproducibility of the present study, correlation and multiple regression analyses of a previous study [
] were performed by Takumi Information Technology (Tokyo, Japan) using SAS 9.4 (SAS Institute Inc.).
The present study demonstrated that canagliflozin produced a mild increase in urine volume in patients with T2DM on Day 1. Other changes included increased urinary glucose excretion and urine osmolality on Day 1, which were sustained during the study. A transient increase in urinary Na excretion on Day 1, decrease in NT-proBNP AUC
from Day 1 onward, decrease in ANP AUC
, and increase in renin activity AUC
on Day 5, but no appreciable changes in plasma vasopressin and aldosterone, were observed. We performed correlation and multiple regression analyses using the data of present and previous studies [
]. Regarding changes from baseline, Na, and K were correlated with urine volume. Meanwhile, the actual value of urine volume was correlated with water intake on Days 0, 1, and 5. Multiple regression analysis showed changes in Na excretion and water intake as factors that affected urine volume change on Day 1. Interestingly, two same factors were selected and the regression coefficients were closed in both studies, which suggested the reproducibility of the present study. Water intake was highly correlated with actual urine volume before and during treatment, and was not affected by the treatment; the correlation is thought to reflect the general body response. Meanwhile, canagliflozin induced transient Na excretion on Day 1, and the change in Na excretion was closely correlated with change in urine volume. Thus, the transient increase in Na excretion is thought to have played a major role in the canagliflozin-induced increase in urine volume.
In the present study, change in urinary glucose excretion was correlated with change in urine volume on Day 1. It should be noted that urinary glucose excretion was fairly constant from Day 1 to Day 5, despite urine volume, as well as urinary Na excretion, returning to baseline from Day 2 to Day 5. In addition, there is no correlation between urine volume and urinary glucose excretion in the previous study. Furthermore, urinary glucose excretion was not selected by multiple regression analysis. Collectively, canagliflozin-induced diuresis is likely to reflect natriuresis, not glucose-driven osmotic diuresis. This is consistent with the results of a recent simulation study using a mathematical model of the rat nephron, which demonstrated that SGLT2 inhibition-induced increase in urine volume is expected to reflect natriuresis rather than osmotic diuresis [
]. Increases in urine output and urinary Na excretion were also reported in Japanese patients with T2DM and heart failure after treatment with ipragliflozin for 4 days [
], although the patient profile was different from that in the present study.
In the present and previous studies, the actual value of urine osmolality was negatively correlated with the actual value of urine volume before and during treatment. In general, increasing urine volume dilutes the solutes, resulting in decreased urine osmolality. The negative correlation between increased urine volume and urine osmolality is also likely to be attributed to the physiological response.
The present study also demonstrated that canagliflozin decreased plasma ANP and NT-proBNP levels and increased plasma renin activity. Change in plasma aldosterone was negatively correlated with change in urine volume on Day 5. As these factors are involved in regulating Na excretion, working in concert or counteracting each other in the distal portion of the nephron, these changes may contribute to increased renal tubular reabsorption of Na in a compensatory manner. A similar compensatory mechanism was reported for the action of diuretics such as furosemide and hydrochlorothiazide, with the natriuretic action abolished by continued treatment [
], which is considered to be mediated through a decrease in plasma ANP and activation of the renin-angiotensin system.
The 4-day ipragliflozin treatment significantly decreased plasma ANP and BNP and increased plasma renin activity, while plasma aldosterone tended to increase, but without statistical significance [
]. A 12-week treatment with dapagliflozin was reported to be associated with increases in plasma renin activity and aldosterone [
]. The study also demonstrated a slight increase in NT-proBNP by dapagliflozin. The authors offered a possible explanation for these discordant results through a transient rise in erythropoietin, which is known to enhance BNP secretion [
]. Interestingly, in the present study, NT-proBNP was decreased, which generally indicates decreased body fluids, and Ht, BUN/CRE, serum osmolality, or vasopressin did not change appreciably during the study. Furthermore, plasma aldosterone did not increase. Accordingly, the effects of SGLT2 inhibitors on plasma aldosterone have been inconsistent. Furthermore, change in plasma aldosterone was negatively correlated with change in urine output on Day 5, and thus the physiological role of this relationship remains unclear.
Treatment with canagliflozin induced glycosuria, decreased blood glucose throughout the day, and suppressed postprandial hyperglycemia, with the largest effect after breakfast. In addition, oral administration of 100 mg of canagliflozin enhanced the increase in plasma total GLP-1 after breakfast. Although similar results with higher doses were reported in previous studies on non-Japanese healthy subjects (300 mg) and Japanese healthy subjects (200 mg) [
], this is the first report to show that a lower dose of canagliflozin(100 mg) increased the total GLP-1 in T2DM patients. As canagliflozin has modest potency for SGLT1 inhibition relative to that of other highly-selective SGLT2 inhibitors [
], and intestinal SGLT1 inhibition enhanced GLP-1 secretion in a rodent model [
], the increase in total GLP-1 may have arisen through inhibition of SGLT1 in the intestine.
The magnitude of the increase in serum ketone bodies in the present study was larger than that in a previous study [
]. This may be attributed to the fact that ketone body levels were elevated immediately after hospitalization (the average of ketone body levels increased from 123.37 μmol/L at screening to 222.29 μmol/L before dosing), possibly through the dietary restriction.
The limitations of the study were the small sample size, short-term nature, and lack of control group. Urine volume is variable among patients, days, and ambient conditions. However, the samples in the present study were obtained from hospitalized patients to ensure the controlled and reliable baseline data (before administration). In addition, we performed similar analyses using our previous study data to evaluate the reproducibility of the present study. Therefore, we think this paper has clinical value. The long-term effect and mechanism of canagliflozin for urine volume remains to be addressed.
Sponsorship, article processing charges, and the open access charge for this study were funded by Mitsubishi Tanabe Pharma Corporation. The authors would like to acknowledge the assistance of Ms. M. Goda (Science Communication Department, Mitsubishi Tanabe Pharma Corporation), and Dr. M. Katoh (Ikuyaku Medical Research Department, Mitsubishi Tanabe Pharma Corporation) during interpretation of the study data. The authors thank the ASCA Corporation and Dr. A. Saito (International Medical Translation Service, Inc.) for providing editorial support, which was funded by Mitsubishi Tanabe Pharma Corporation. H.T. contributed to the organization of the study and interpretation of data. K.T. contributed to the acquisition of data. H.I., H.K., N.M., T.H., and K.A. contributed to the study design and interpretation of data. M.T. contributed to the conception of the study. N.I. contributed to interpretation of data. K.K. supervised the study, and contributed to the study design and interpretation of data. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published. All authors had full access to all of the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis.
Nobuya Inagaki has received research support from Eli Lilly Japan. K.K., Mitsubishi Tanabe Pharma Corp., and MSD K.K.; received grant from Astellas Pharma Inc., AstraZeneca K.K., Bristol-Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Ltd., Japan Diabetes Foundation, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corp., MSD K.K., Nippon Boehringer Ingelheim Co., Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho Co., Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co. Ltd., and Takeda Pharmaceutical Co., Ltd.,. has received speakers bureaus or consulting fee from Astellas Pharma Inc., MSD K.K.,Nippon Boehringer Ingelheim Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., and Takeda Pharmaceutical Co., Ltd.
Kohei Kaku has received grant from Nippon Boehringer Ingelheim Co. Ltd., Daiichi Sankyo Co. Ltd., and Mitsubishi Tanabe Pharma Corp.; has received speakers’ bureau from Astellas Pharma Inc., AstraZeneca K.K., Fuji Film Pharma Co. Ltd., Kissei Pharmaceutical Co. Ltd., Kowa Company Ltd., Mitsubishi Tanabe Pharma Corp., MSD K.K., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co. Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co. Ltd., Takeda Pharmaceutical Co. Ltd., and Taisho Toyama Pharmaceutical Co. Ltd. Kazuhiko Takano has nothing to disclose.Hiroyuki Tanaka is an employee of Mitsubishi Tanabe Pharma Corp.Hiroaki Iijima is an employee of Mitsubishi Tanabe Pharma Corp.Hajime Kubo is an employee of Mitsubishi Tanabe Pharma Corp.Nobuko Maruyama is an employee of Mitsubishi Tanabe Pharma Corp.Toshio Hashimoto is an employee of Mitsubishi Tanabe Pharma Corp.Kenji Arakawa is an employee of Mitsubishi Tanabe Pharma Corp.Masanori Togo is an employee of Mitsubishi Tanabe Pharma Corp.
Compliance with Ethical Standards
The study was conducted in accordance with ethical principles that complied with the Declaration of Helsinki (revised October 2013), ethical guidelines for medical and health research involving human subjects, and conflict of interest management rules for clinical studies at the Medical Corporation Hokubukai Utsukushigaoka Hospital (Sapporo, Japan). The study protocol was approved by the Institutional Review Board at the Utsukushigaoka Hospital. All participants provided written informed consent before taking part in the study
The datasets generated during and/or analyzed during the current study are not publicly available because of lack of agreement for disclosing individual raw data in public but are available from the corresponding author on reasonable request.
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