DiEtary Sodium Intake Effects on Ertugliflozin-induced Changes in GFR, reNal Oxygenation and Systemic Hemodynamics: the DESIGN Study, a Randomized, Placebo-controlled, Cross-over Study With Ertugliflozin in People With Type 2 Diabetes
Phase: PHASE4
Conditions: Diabetes Mellitus, Diabetic Kidney Disease, Hypertension
Sponsor: Amsterdam UMC, location VUmc

NL80772.029.22 / DC2022ERTU 
 
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Protocol DESIGN, Study NO. 2022.0737 
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PROTOCOL TITLE DiEtary Sodium Intake effects on ertugliflozin-induced changes in GFR, 
reNal oxygenation and systemic hemodynamics: the DESIGN study, a randomized, placebo-
controlled, cross-over study with ertugliflozin in people with type 2 diabetes 
 
Protocol ID 
DC2022ERTU 
Short title 
DESIGN study 
EudraCT number  
2021-005474-25 
Version 
5 .0 
Date 
22-02-2024  
Coordinating investigator/project 
leader 
 
Dr. D.H. van Raalte 
Department of Internal Medicine, Amsterdam UMC 
De Boelelaan 1117 1081 HV Amsterdam Room ZH 
4A-58 
TEL: +31-20-4440534 
E-mail: d.vanraalte@amsterdamumc.nl 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
 
 
 
 
Multicenter research: per site 
 
Dr. D.H. van Raalte 
Department of Internal Medicine, Amsterdam UMC 
De Boelelaan 1117 1081 HV Amsterdam Room ZH 
4A-58 
TEL: +31-20-4440534 
E-mail: d.vanraalte@amsterdamumc.nl 
 
Dr. Petter Bjornstad 
Department of Medicine, Division of Nephrology and 
Hypertension; Department of Pediatrics, Section 
Endocrinology and Diabetes. University of Colorado 
School of Medicine. 13123 16th Ave Box B265 
Aurora, CO 80045, USA 
 
TEL: +1 720 777 4659 
E-mail: Petter.Bjornstad@childrenscolorado.org 
 

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Sponsor (in Dutch: 
verrichter/opdrachtgever) 
Diabetes Center, Department of internal medicine, 
Amsterdam UMC, location VUMC 
 
 
Subsidising party 
MSD, the proposal is an investigator-initiated study 
Independent expert 
Dr. E. Peters 
Department of Internal Medicine, Amsterdam UMC 
De Boelelaan 1117 1081 HV Amsterdam Room PK-
1Z-108 
TEL: +31-20-4440596 
E-mail: e.peters@amsterdamumc.nl 
Laboratory sites 
Department of Clinical Chemistry and Laboratory 
of Endocrinology Amsterdam UMC 
 
 
Pharmacy 
Trial pharmacy Amsterdam UMC 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Prof. J. Prins 
Head of department Internal Medicine 
 
 
 
Dr. D.H. van Raalte 
Principle Investigator 
 
 
 
 
 

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TABLE OF CONTENTS 
 
INTRODUCTION AND RATIONALE ..................................................................................... 9 
3. 
STUDY DESIGN ...........................................................................................................12 
4. 
STUDY POPULATION ..................................................................................................13 
4.1 
Population (base) ...................................................................................................13 
4.2 
Inclusion criteria .....................................................................................................13 
4.3 
Exclusion criteria ....................................................................................................13 
4.4 
Sample size calculation ..........................................................................................14 
5. 
TREATMENT OF SUBJECTS .......................................................................................16 
5.1 
Investigational product/treatment ............................................................................16 
Table 1. Overview treatment allocation ..........................................................................16 
5.2 
Use of co-intervention (if applicable) ......................................................................16 
5.3 
Escape medication .................................................................................................16 
6. 
INVESTIGATIONAL PRODUCT ....................................................................................17 
6.1 
Name and description of investigational  product(s) ...............................................17 
6.2 
Summary of findings from non-clinical studies ........................................................17 
6.3 
Summary of findings from clinical studies ...............................................................17 
6.4 
Summary of known and potential risks and benefits ...............................................17 
6.5 
Description and justification of route of administration and dosage .........................17 
6.6 
Dosages, dosage modifications and method of administration ...............................17 
6.7 
Preparation and labelling of Investigational Medicinal Product ...............................17 
6.8 
Drug accountability .................................................................................................17 
7. 
NON-INVESTIGATIONAL PRODUCT ...........................................................................20 
7.1 
Name and description of non-investigational product(s) .........................................20 
7.2 
Summary of findings from non-clinical studies ........................................................21 
7.3 
Summary of findings from clinical studies ...............................................................21 
7.4 
Summary of known and potential risks and benefits ...............................................21 
7.5 
Description and justification of route of administration and dosage .........................21 
7.6 
Dosages, dosage modifications and method of administration ...............................21 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ........................21 
7.8 
Drug accountability .................................................................................................21 
8. 
METHODS ....................................................................................................................22 
8.1 
Study parameters/endpoints ...................................................................................22 
8.1.1 
Main study parameter/endpoint .......................................................................22 
8.1.2 
Secondary study parameters/endpoints (if applicable) ....................................22 
8.1.3 
Other study parameters (if applicable) .............................................................22 
8.2 
Randomisation, blinding and treatment allocation ..................................................22 
8.3 
Study procedures ...................................................................................................23 
8.4 
Withdrawal of individual subjects ............................................................................28 
8.4.1 
Specific criteria for withdrawal (if applicable) ...................................................28 
8.5 
Replacement of individual subjects after withdrawal ...............................................28 
8.6 
Follow-up of subjects withdrawn from treatment .....................................................28 

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8.7 
Premature termination of the study .........................................................................28 
9. 
SAFETY REPORTING ..................................................................................................29 
9.1 
Temporary halt for reasons of subject safety ..........................................................29 
9.2 
AEs, SAEs and SUSARs ........................................................................................29 
9.2.1 
Adverse events (AEs) ......................................................................................29 
9.2.2 
Serious adverse events (SAEs) .......................................................................29 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .........................30 
9.3 
Annual safety report ...............................................................................................31 
9.4 
Follow-up of adverse events ...................................................................................31 
10. 
STATISTICAL ANALYSIS ..........................................................................................33 
10.1 
Primary study parameter(s) ....................................................................................33 
10.2 
Secondary study parameter(s) ...............................................................................34 
10.3 
Other study parameters ..........................................................................................35 
10.4 
Interim analysis (if applicable) ................................................................................35 
11. 
ETHICAL CONSIDERATIONS ...................................................................................36 
11.1 
Regulation statement .............................................................................................36 
11.2 
Recruitment and consent........................................................................................36 
11.3 
Benefits and risks assessment, group relatedness .................................................37 
11.4 
Compensation for injury .........................................................................................37 
11.5 
Incentives ...............................................................................................................37 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .........................38 
12.1 
Handling and storage of data and documents ........................................................38 
12.2 
Amendments ..........................................................................................................38 
12.3 
Annual progress report ...........................................................................................39 
12.4 
Temporary halt and (prematurely) end of study report ............................................39 
12.5 
Public disclosure and publication policy ..................................................................39 
13. 
STRUCTURED RISK ANALYSIS...............................................................................40 
13.1 
Potential issues of concern .....................................................................................40 
13.2 
Synthesis ...............................................................................................................42 
14. 
REFERENCES ..........................................................................................................42 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; in 
Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-formulier) 
AE 
Adverse Event 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics; in Dutch: officiële productinformatie 
IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or performance 
of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A party 
that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; in 
Dutch: Uitvoeringswet AVG 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
 
Rationale: SGLT2 inhibitors such as ertugliflozin improve blood pressure and kidney 
outcomes in people living with diabetes through incompletely understood mechanisms, 
however, not all patients treated with SGLT2 inhibition have improved outcomes. Changes in 
kidney sodium handling is among the mechanisms by which SGLT2 inhibition may reduce 
blood pressure and drive beneficial kidney outcomes. This process is heavily dependent on 
daily sodium intake by patients receiving SGLT2 inhibitor treatment. In this study, the effect 
of daily sodium intake on SGLT2-inhibitor induced physiological effect is studied, including 
blood pressure regulation and kidney physiology. 
Objective: Primary outcome: to investigate the modifying effects of WHO-recommended 
sodium intake (90 mmol per day) vs. high sodium intake (targeted at 250 mmol per day) on 
the effect of ertugliflozin 15 mg daily, versus placebo, on 24-hour blood pressure in 
overweight/obese adults with type 2 diabetes. 
Study design: The study is a bi-center randomized, placebo-controlled, crossover 
intervention study. Total number of included participants is 34, which undergo 4 conditions in 
randomized order (randomization list generated by computer and supervised by pharmacy; 
blinded to investigator). While the treatment will be blinded for all participants, the sodium 
interventions are open-label.  
 
Study population: 34 Adults living with type 2 diabetes.  18 - 85 years of age. Obese or 
overweight with BMI > 25 kg/m2. 
Intervention (if applicable): Cross-over design during 4 conditions. The 4 conditions are: 
1) low-sodium diet; placebo; 2) low-sodium diet; ertugliflozin 15 once daily; 3) high-sodium 
diet; placebo; 4) high-sodium diet; ertugliflozin 15 mg once daily 
Main study parameters/endpoints:  
Primary endpoint is indicated above. 
Secondary endpoint: To investigate the efficacy of ertugliflozin 15 mg daily, versus placebo, 
in overweight/obese adults with type 2 diabetes to reduce the hypertensive effects of a high-
sodium diet (250 mmol per day) versus 24-h blood pressure measurement during 
participant’s normal diet (170 mmol/per day) obtained at screening visit. 
Exploratory endpoints: To investigate the modifying effects of WHO-recommended sodium 
intake (90 mmol per day) vs. high sodium intake (targeted at 250 mmol per day) on the effect 
of ertugliflozin 15 mg daily, versus placebo, on the following parameters in overweight/obese 
adults with type 2 diabetes on GFR by iohexol clearance, Hematocrit, Office blood pressure, 
Kidney oxygenation, Parameters of intra kidney hemodynamic function including ERPF by p-
aminohippurate (PAH) clearance and renal vascular resistance, Body anthropometrics, 
Fasting plasma glucose, Albumin excretion rate (24 hour), 24-hr urinary glucose excretion, 
Urinary and plasma biomarkers. 

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Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness:  
There is no clear health benefit for the study participants, except that participation will 
provide insight into their sodium intake and knowledge how to reduce sodium intake to WHO-
recommended amounts, which may benefit their long-term health. The short-term treatment 
with SGLT2 inhibitor ertugliflozin will not provide lasting benefits. On the other hand, the drug 
has little side effects, especially during short-term treatment, which will be closely monitored. 
The most commonly observed side effect (usually after more prolonged treatment) involves 
genital mycotic infections, which can easily be treated with antifungal topicals. Glucose levels 
and blood pressure are lowered during treatment. There are no health risks to short-term 
changes in sodium diet. Potential changes in blood pressure during the high-sodium diet are 
closely monitored. The study procedures are safe. There are no risks with undergoing MRI 
procedures and the use of iohexol and PAH to measure kidney hemodynamic function bears 
no risk, with >1500 tests carried out in our department. Burden of participants includes 5 
visits (including screening); 4 visits where an MRI scan (duration 30 min) is performed and 
where 2 intravenous catheters are placed for tracer infusion during 2,5 hours; 13 24-hr urine 
collections and 5 24-hr blood pressure collections. A total of maximally 500 mL blood will be 
withdrawn over the course of a number of months. The study team will mitigate travel for the 
participants by collecting 24-hr blood pressure and 24-urine samples at home. 

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INTRODUCTION AND RATIONALE 
 
Diabetic kidney disease (DKD) remains the leading cause of chronic kidney disease (CKD) 
and dialysis in the developed world, and a major cause of premature mortality. Sodium-
glucose cotransporter 2 (SGLT2) inhibitors confer cardiovascular and renal protection in 
many, but not all people with type 2 diabetes (T2D) (1). Yet, the mechanisms of these 
salutary cardiorenal benefits remain unclear, and are incompletely explained by the modest 
improvements in blood pressure, glycemic control, body weight, and serum uric acid (2). A 
better understanding of the mechanism of cardio-renal protection in response to SGLT2 
inhibition is therefore needed to determine which patients would see the greatest benefit from 
this drug class and also how to augment the beneficial effects with complimentary drugs 
and/or lifestyle changes such as changes in dietary sodium intake.  
 
As stated, the mechanisms by which SGLT2 inhibitors such as ertugliflozin induce 
cardiorenal benefits are uncertain. With respect to cardiovascular outcomes, hematocrit was 
statistically the most important mediator of heart failure reduction in the EMPA REG 
OUTCOME study (3). This increment in hematocrit likely reflects a reduction in plasma 
volume, which has originally been proposed to be caused by natriuresis. However, several 
trials (4,5), including the recent DAPASALT study (6) have indicated that SGLT-2 induced 
natriuresis is mild in terms of absolute amount of sodium excretion, is very transient (e.g. 
likely only present within 12 hour of the first dose) and therefore has been doubted to a) be 
the cause for the increment in hematocrit and b) to mediate the cardiovascular benefits.  
 
This may be different for kidney outcomes, as SGLT2-induced changes in the location of 
tubular sodium reabsorption (thus even with neutral effects on total body sodium excretion), 
can largely impact kidney physiology. As such, more distal tubular sodium reabsorption 
(distal of the macula densa) triggers activation of tubuloglomerular feedback reducing 
glomerular pressure (7,8). In addition to changes in glomerular pressure, SGLT-2 induced 
changes in kidney physiology may drive changes in kidney oxygen metabolism. 
 
From a metabolic point of view, the kidneys are highly active and are second only to the 
heart with respect to oxygen (O2) consumption per tissue mass. To sustain this activity, the 
kidneys rely on various substrates to generate adenosine triphosphate (ATP) including 
citrate, glutamine, glucose and free fatty acids (FFA) (9). The vast majority of ATP 
consumption in the kidney relates to sodium reabsorption, and glucosuria and single-nephron 
hyperfiltration results in increased filtered sodium and activity of Na+/K+ ATPase pump due to 
high tubular glucose and sodium reabsorption (10-12). In fact, animal models suggest that 
renal O2 consumption is increased by 40% in all cortical segments and by 160% each in the 
S3 segment and medullary collecting duct (13-17). In animal models, SGLT2 inhibition 
attenuates single-nephron hyperfiltration (18) and have also been shown to improve renal 
oxygenation and ameliorate renal hypoxia (19). Thus the SGLT2-induced renal benefit may 
be driven by improvements in renal oxygen availability. In addition, changes in tubular 
sodium reabsorption increase renal EPO production, which, through enhanced 
erythropoiesis, is the most likely explanation for the increase in hematocrit. 
 
Thus, whether SGLT2 inhibitors modulate natriuresis or not, changes in tubular sodium 
handling lead to reduction of hyperfiltration, renal oxygen consumption and enhanced EPO 
secretion that collectively are thought to drive beneficial cardiorenal outcomes, amongst 
several hypotheses; effects that may be dependent of the amount of dietary sodium intake as 
argued below. 
 
SGLT2 inhibitors may alter other aspect of sodium homeostasis as well. People with T2D are 
known to be sensitive to excess sodium intake, which may results in extracellular volume 
(ECV) expansion and hypertension. This is different for healthy, lean people in whom 

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increased salt intake usually does not increase blood pressure. These individuals are termed 
sodium-resistant (20,21). High-sodium intake suppresses SGLT2 expression leading to 
natriuresis. An effect not observed in rodents with hyperglycemia (22). Thus, SGLT2 
inhibition during high-sodium diet  could restore this response.  
While it was previously thought that sodium sensitivity is exclusively driven by differences in 
renal sodium excretion, in salt-resistant people, sodium may bind non-osmotically in tissues 
such as endothelial surface layers and the glycocalyx, preventing water retention and 
hypertension (23). Integrity of the endothelial surface layer is important for vascular 
dynamics, and glycocalyx injuries have been implicated in the progression of DKD and 
cardiovascular disease. Interestingly, the SGLT2 inhibitor empagliflozin was shown to 
decrease tissue sodium content as measured by MRI, which could theoretically reduce non-
osmotic sodium storage (24). On the other hand, SGTL2-inhibitor induced improvements in 
endothelial function could drive opposite effects with improved non-osmotic sodium storage. 
Currently, the effects of SGLT2 inhibition on sodium sensitivity are unknown, while its effect 
on sodium sensitivity may become very relevant in people that consume excess sodium. 
Given the blood pressure lowering effects of SGLT2-inhibitors, that may be driven by 
improvements in endothelial function, one could hypothesize that SGLT2 reduce sodium 
sensitivity. 
 
Dietary sodium intake may modulate the effects of SGLT2 on the kidney and beyond through 
several mechanisms, as excessive sodium intake has kidney hemodynamic effects. As such, 
high sodium intake has been shown to increase GFR and effective renal plasma flow (ERPF) 
in normoglycemic individuals, while blood pressure (MAP) increased to a smaller extent. 
Given that renal vascular resistance = MAP/ERPF, this parameter declined. Filtration fraction 
(FF=GFR/ERPF) increased in the overweight individuals, but not in lean persons (25). As 
people with T2D are almost all overweight or obese and are usually sensitive to the effects of 
high-sodium intake (26), the renal hemodynamic effects of SGLT2 inhibitors could be off-set 
by high sodium intake. This could be secondary to impairment of tubuloglomerular feedback 
as shown recently for ertugliflozin in rodent studies (27). In addition, given the potential 
beneficial effect of SGLT2 inhibitors on kidney oxygenation, large amounts of filtered sodium 
could off-set this mechanisms of action. 
 
Observations have been done with this reasoning for RAAS blockers. They are 
renoprotective in people with diabetes, likely secondary to reducing glomerular pressure 
through efferent vasodilation and lowering of filtration fraction (26). In people with diabetic 
kidney disease, this renoprotective effect was markedly affected by sodium consumption, 
with better protection afforded with low-sodium intake (27,28). Also for vitamin D (29) and 
neprilysin (30), interactions with sodium diet have been observed, with less efficacy at higher 
sodium intake. Based on these observations, it is plausible that baseline sodium intake is an 
important mediator for the kidney hemodynamic response and kidney protective effect of 
ertugliflozin. 
 
In conclusion, although SGLT2 inhibitors mitigate cardiorenal risk in people with T2D, the 
protective effects are incomplete in the majority of people, which may be drive due to co-
medication and dietary factors such as sodium intake. Therefore, carefully designed 
mechanistic trials are needed to better understand the interplay between ertugliflozin and salt 
intake. Studies focusing on these interactions will allow researchers to assess the effects of 
SGLT2 inhibitors in combination with other drugs that affect sodium homeostasis and 
potentially modify salt intake to maximize treatment response. In addition, such studies may 
also help to explain differences observed between (outcome) trials, which have included 
different ethnicities that may also contribute to differences observed in renal outcomes due to 
differences in dietary habits.  
 

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2. OBJECTIVES 
 
2.1 Primary objective:  
 
1: To investigate the modifying effects of WHO-recommended sodium intake (90 mmol per 
day) vs. high sodium intake (targeted at 250 mmol per day) on the effect of ertugliflozin 15 
mg daily, versus placebo, on 24-hour blood pressure in overweight/obese adults with type 2 
diabetes. 
 
2.2 Secondary objective: 
 
To investigate the efficacy of ertugliflozin 15 mg daily, versus placebo, in overweight/obese 
adults with type 2 diabetes to reduce the hypertensive effects of a high-sodium diet (250 
mmol per day) versus 24-hour blood pressure measurement during participant’s normal diet 
(170 mmol/per day) obtained at screening visit. 
 

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3. STUDY DESIGN 
 
The study is a bi-center randomized, placebo-controlled, crossover intervention study. Total 
number of included participants is 34, which undergo 4 conditions in randomized order 
(randomization list generated by computer and supervised by pharmacy; blinded to 
investigator). While the treatment will be blinded for all participants, the sodium interventions 
are open-label. Study design is shown in the figure below. 
 
The 4 conditions are: 
1) low-sodium diet; placebo 
2) low-sodium diet; ertugliflozin 15 once daily 
3) high-sodium diet; placebo 
4) high-sodium diet; ertugliflozin 15 mg once daily 
 
 
 
 
Plac/Ertu 15 mg
10-days Intervention
Plac/Ertu 15 mg
10-days
Diet run in
Sodium diet 90 mmol per day
Sodium diet 250 mmol per day
10-days Intervention
10-days
Diet run in
24-hr urine  
(compliance)
endpoints
endpoints
Plac/Ertu 15 mg
10-days Intervention
Plac/Ertu 15 mg
10-days
Diet run in
10-days Intervention
10-days
Diet run in
endpoints
endpoints
24-hr urine  
(compliance)
24-hr urine  
(compliance)
24-hr urine  
(compliance)
4 weeks 
washout
4 weeks 
washout
4 weeks 
washout

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4. STUDY POPULATION 
 
4.1 Population (base)  
 
 
 
Participants will be recruited from the database at Amsterdam Diabetes Center at 
 
Amsterdam UMC and by advertisement in local papers. This database has currently over  
 
500 individuals with diabetes that have agreed to be approached for new studies and that  
 
have been phenotyped in dept. University of Colorado has a similar database ensuring 
 
rapid inclusion of required participants. 
 
 
4.2 Inclusion criteria 
 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
 
• 
Adults with previously diagnosed T2DM according to American Diabetes 
Association (ADA) criteria  
• 
HbA1c 6.5-10% 
• 
Age 18 – 85 years of age 
• 
Overweight or obese with BMI: >25 kg/m2 
• 
We will make every effort to enrol participants of all races/ethnicities.” 
• 
Both sexes (females must be post-menopausal; no menses >1 year; in 
case of doubt, Follicle-Stimulating Hormone (FSH) will be determined with cut-off defined 
as >31 U/L) 
• 
Ability to provide signed and dated, written informed consent prior to any 
study procedures 
• 
Estimated GFR 60-90 ml/min/1.73m2 by CKD-EPI matching the eGFR 
range of most participants in VERTIS-CV 
• 
Sodium intake at baseline < 200 mmol/day 
• 
UACR < 30 mg/mmol 
•  
All participants need to be on a stable dose of diabetes medication, 
including Metformin, SU, DPP4-inhibitors, or insulin. 
• 
Participants suffering from hypertension need to be on a stable dose of 
RAS inhibitors. In case RAS inhibition is not tolerated, the participant should to be on a 
stable dose of other antihypertensive treatment.  
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
 
• 
History of unstable or rapidly progressing renal disease 

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• 
Estimated GFR <60 mL/min/1.73m2  or eGFR > 90 mL/min/1.73m2 
determined by CKD-EPI 
• 
UACR > 30 mg/mmol 
• 
Current/chronic use of the following medication: SGLT2 inhibitors, TZD, 
GLP-1RA, glucocorticoids, immune suppressants, antimicrobial agents, 
chemotherapeutics Participants should be on a stable dose of antipsychotics, tricyclic 
antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs). Subjects on diuretics 
will only be excluded when these drugs cannot be stopped for the duration of the study. 
• 
Chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) will not be 
allowed, unless used as incidental medication (1-2 tablets) for non-chronic indications (i.e. 
sports injury, headache or back ache). However, no such drug can be taken within a 
timeframe of 2 weeks prior to renal testing 
• 
History of diabetic ketoacidosis (DKA) requiring medical intervention (e.g. 
emergency room visit and/or hospitalization) within 1 month prior to the Screening visit. 
• 
Current urinary tract infection and active nephritis 
• 
Recent (<6 months) history of cardiovascular disease, including: 
o 
Acute coronary syndrome 
o 
Chronic heart failure (New York Heart Association grade II-IV) 
o 
Stroke or transient ischemic neurologic disorder 
• 
Severe hepatic insufficiency and/or significant abnormal liver function 
defined as aspartate aminotransferase (AST) >3x upper limit of normal (ULN) and/or 
alanine aminotransferase (ALT) >3x ULN 
• 
 Active malignancy. History of malignancy is allowed unless the participant 
still has active treatment other than hormonal therapy.• History of or actual severe mental 
disease  
• 
Substance abuse (alcohol: defined as >4 units/day) 
• 
Allergy to any of the agents used in the study  
• 
Individuals who are investigator site personnel, directly affiliated with the 
study, or are immediate (spouse, parent, child, or sibling, whether biological or legally 
adopted) family of investigator site personnel directly affiliated with the study 
• 
Inability to understand the study protocol or give informed consent 
 
4.4 Sample size calculation 
Based on previous studies in patients treated with SGLT2 inhibitors, we expect a change in 
systolic blood pressure of 6-8 mmHg during normal sodium intake (150 mmol/day) (6,7). 
Based on our hypothesis, this effect is increased on a low-sodium intake and reduced during 
high-sodium intake. 
 
We expect a difference of 4 mmHg between the placebo-controlled effects of ertugliflozin on 
a low- versus high-sodium diet, with a conservative SD on this difference of 8 mmHg. 
 
A total of 34 patients completing the study provide 80% power at a two-sided alpha of 0.05, 
to detect a 4 mmHg difference with the indicated SD of 8 mmHg. Drop-outs will be replaced. 

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Based on the RED study (7) and DAPASALT(6) study, the power needed to detect changes 
in GFR and hematocrit are of sufficient magnitude and therefore we expect sufficient power 
for these parameters as well. We will include 34 individuals. 
 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
 
In addition to their ongoing oral glucose-lowering medication (metformin or a SU-
derivative) and drugs for cardiovascular risk management (e.g. ARB and/or statin), 
subjects will randomly be assigned to receive ertugliflozin 15mg or a matched 
placebo treatment during the first intervention phase of the study. Following a 
wash-out phase the participants will than start with the second intervention phase 
and receive the other study medication (see table 1). 
 
Table 1. Overview treatment allocation 
Product and dosage  
Dosage form and strength 
Manufacturer 
Ertugliflozin 15mg QD 
15 mg tablets (orally) 
Merck 
Placebo ertugliflozin-
matching tablet QD 
Matched tablets (orally) 
Merck 
 
The study medication (ertugliflozin and matching placebo) will be provided by 
Merck. The pharmacy department of Amsterdam UMC, location VUMC, will be 
responsible for randomization and distribution of study medication.  
 
Study medication details: ertugliflozin (Steglatro® by Merck Sharp& Dohme BV., 
USA). Ertugliflozin L-pyroglutamic acid: (1S,2S,3S,4R,5S)-5-(4-chloro- 3-(4-
ethoxybenzyl)phenyl)-1- (hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octane-2,3,4-triol, 
compound with (2S)-5- oxopyrrolidine-2- carboxylic acid. 
 
5.2 Use of co-intervention (if applicable) 
During the treatment phases, participants will be subjected to a sodium diet. While a 
regular sodium intake in the Netherlands, based on previous studies and literature, is 
around 170 mmol/day; we will coach participants by providing food lists and counselling, 
to lower their intake to WHO-recommended amounts of 90 mmol/day. This will be done 
during 10 days prior to start study medication (compliance checked by 24-hour sodium 
analysis) and during the 10-day intervention. Thus for a total of 20 days per treatment 
block. Low-sodium intake will be effected during both ertugliflozin and placebo. Similarly, 
using dietary counselling, participants will be instructed to follow a high-sodium diet aimed 
at 250 mmol/day. 
5.3 Escape medication 
Not applicable 

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6. INVESTIGATIONAL PRODUCT  
 
6.1 Name and description of investigational  product(s) 
Study medication details: ertugliflozin (Steglatro® by Merck Sharp& Dohme 
BV., USA). Ertugliflozin L-pyroglutamic acid: (1S,2S,3S,4R,5S)-5-(4-chloro- 3-
(4-ethoxybenzyl)phenyl)-1- (hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octane-
2,3,4-triol, compound with (2S)-5- oxopyrrolidine-2- carboxylic acid. 
 
6.2 Summary of findings from non-clinical studies 
Please refer to the IB 
 
6.3 Summary of findings from clinical studies 
Please refer to the IB 
6.4 Summary of known and potential risks and benefits 
Please refer to the IB. 
In addition, a structured risk analysis is provided in chapter 13. 
6.5 Description and justification of route of administration and dosage 
The standard clinical dosage is 15 mg once daily. It is oral administration. Please refer to 
IB. 
6.6 Dosages, dosage modifications and method of administration 
The dosage in this study is 15 mg once daily, oral. This is conform the manufacturer’s 
instructions and what has been extensively studies in clinical trials. 
6.7 Preparation and labelling of Investigational Medicinal Product 
The investigational product (ertugliflozin and placebo) are provided by MSD according to 
GMP production. Labelling will be done in collaboration with the trial pharmacy at 
Amsterdam UMC. 
 
 
6.8 Drug accountability 
Medication is shipped to both study sites by MSD and will be received by the trial 
pharmacy at Amsterdam UMC according to guidelines.  
 
Randomization 
The treatment assignment will be performed by the trial pharmacists of Amsterdam UMC- 
location VUmc (who have ample experience with this process), in order to maintain 
blinding for the investigator, site personnel, and patients. A randomization sequence will 
be generated and sent through an automated system (Castor EDC) by the investigator to 
the trial pharmacy, ensuring that the investigator remains blinded.  

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Labeling, Drug dispensation, and Accountability 
Merck will provide the trial drug and the placebo. The trial products will be labelled with 
participant identifying information to our institutional trial pharmacist, who subsequently 
will store the trial products according to prevailing guidance until they are needed. The 
trial pharmacist will deliver the study medication to the research personnel (who remain 
blinded). These will then dispense the study medication to the participants at the 
designated visits, combined with extensive instructions on use and administration of the 
study drug. The number of daily dosages dispensed will be sufficient to cover the 
complete expenditure of the study. No trial products will be dispensed to any person not 
enrolled in the trial.  
 
On the designated days, the participants will bring their trial medication. In order to 
perform proper drug accountability, the investigator will keep track of all received, used 
and unused trial products and -if possible- all empty packaging. Non-used medication will 
be returned to the pharmacy department where it will be destroyed. 
 
Drug storage 
Ertugliflozin does not require any special storage conditions (Agency, 2018). 
Nevertheless, when not distributed to participants, drugs will be stored at the institutional 
trial pharmacy. Proper storage conditions will be ensured and records and evaluations will 
be kept as required by International Conference on Harmonisation – Good Clinical 
Practice (ICH-GCP).  
 
Blinding and Unblinding 
Throughout the study duration, all study personnel will remain blinded with regard to the 
medication used. The randomization –in order to achieve blinding for study personnel- will 
be performed by the trial pharmacist of the Amsterdam UMC-VUmc. The investigator will 
receive the subjects blinding information concerning the study medication in the form of a 
sealed envelope; this envelope will be stored on a secure location in the Diabetes Center 
Amsterdam UMC-VUmc. The blind shall not be broken by the investigator unless 
information concerning the study medication is necessary for the medical treatment of the 
subjects. Procedures regarding emergency unblinding are described in Chapter 9. If the 
investigator is unblinded, study medication will be stopped immediately in addition to the 
withdrawal of the study subject from the study. At the end of the study, following the last 
patients last visit and database lock (which will only occur after medical / scientific review), 
the blind will be broken by the trial pharmacist. 

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7. NON-INVESTIGATIONAL PRODUCT 
7.1 Name and description of non-investigational product(s) 
Iohexol (Omnipaque® by GE Healthcare B.V.) (1) 
Supplied as: 200 mL ampoules with a clear, colorless solution containing 300 mg iohexol 
per mL. 
Approved agent: Approved by the Dutch drug authority (College ter Beoordeling van 
Geneesmiddelen; RVG 09821) 
Action: Iohexol is a non-ionic, monomeric, triiodinated, water-soluble substance mostly 
used as X-ray contrast medium. In the concentration of 140 mg I/ml it is isotonic with 
blood and tissue fluid. Close to 100 per cent of the intravenously injected iohexol is 
excreted unchanged through the kidneys within 24 hours in patients with normal renal 
function. The maximum urinary concentration of iohexol appears within approximately 1 
hour after injection. No metabolites have been detected. The protein binding of iohexol is 
very low (less than 2%). 
Side effects: The risk of serious reactions in connection with use of Omnipaque is 
regarded as minor. However, iodinated contrast media may provoke serious, life-
threatening, fatal anaphylactic/anaphylactoid reactions or other manifestations of 
hypersensitivity. 
Dosing: For the steady-state iohexol-clearance test, an initial bolus dose is given, 
followed by continuous infusion with a dose calculated to maintain the plasma 
concentration between 100 mg/L and 150 mg/L. 
Storage: Iohexol can be stored up to 3 years below 30°C. 
 
Sodium-4 amino hippurate (PAH, Basic Pharma Manufacturing BV) (2) 
Supplied as: PAH is supplied as single use vials of 10mL vials containing sterile 20% 
(2g/10mL) of 4-Aminohippuric acid dissolved with one equivalent of sodium hydroxide 
using water for injection.  
Approved agent: An authorized PAH, USP Grade, manufactured by Merck, was available 
for research for the estaimation of ERPF. However, the authorized product was removed 
from the market in the late ‘90s/ early 20’s and is no longer available for use in clinical 
trials. PAH manufactured by Basic Pharma does not yet have marketing authorization in 
an ICH country. 
Action: PAH is a derivative of hippuric acid, which is derived from the amino acid glycine. 
It is inert in normal doses. 
Side effects: Extremely large doses may cause osmotic diuresis. Some patients may 
have a sensation of warmth, or the desire to urinate or defaecate during or shortly 
following the infusion. Allergic and anaphylactic reactions have been reported with the 
PAH produced via Merck but were very uncommon. 
Dosing: During one PAH-clearance test, an initial bolus will be given followed by a 
continuous infusion with a dose calculated to maintain the plasma concentration between 
15mg/L and 20mg/L. 
Storage: PAH will be stored at room temperature protected from light. 
 

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7.2 Summary of findings from non-clinical studies 
 
Please refer to IB 
 
7.3 Summary of findings from clinical studies 
 
Please refer to IB 
 
7.4 Summary of known and potential risks and benefits 
See above and in the structured risk analysis. At our research centre at Amsterdam UMC 
we have done > 500 iohexol and PAH clearances in the last 5 years and not encountered 
a single problem. 
 
7.5 Description and justification of route of administration and dosage 
Intravenous usage. Dosage are indicated above. 
 
7.6 Dosages, dosage modifications and method of administration 
Please see above. 
 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
Not applicable 
 
7.8 Drug accountability 
Not applicable 
 

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8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
To investigate the modifying effects of WHO-recommended sodium intake (90 mmol per day) 
vs. high sodium intake (targeted at 250 mmol per day) on the effect of ertugliflozin 15 mg 
daily, versus placebo, on 24-hour blood pressure in overweight/obese adults with type 2 
diabetes. 
8.1.2 Secondary study parameters/endpoints (if applicable) 
To investigate the efficacy of ertugliflozin 15 mg daily, versus placebo, in overweight/obese 
adults with type 2 diabetes to reduce the hypertensive effects of a high-sodium diet (250 
mmol per day) versus 24-hour blood pressure measurement during participant’s normal diet 
(170 mmol/per day) obtained at screening. 
8.1.3 Other study parameters (if applicable) 
To investigate the modifying effects of WHO-recommended sodium intake (90 mmol per day) 
vs. high sodium intake (targeted at 250 mmol per day) on the effect of ertugliflozin 15 mg 
daily, versus placebo, on the following parameters in overweight/obese adults with type 2 
diabetes: 
 
 
GFR by iohexol clearance 
 
Hematocrit 
 
Office blood pressure 
 
Kidney oxygenation 
 
Parameters of intra kidney hemodynamic function including ERPF by p-
aminohippurate (PAH) clearance and renal vascular resistance 
 
Body anthropometrics 
 
Fasting plasma glucose 
 
Albumin excretion rate (24 hour) 
 
24-hr urinary glucose excretion 
 
Urinary and plasma biomarkers 
 
 
8.2 Randomisation, blinding and treatment allocation 
The study is blinded with ertugliflozin and matching placebo’s being provided by 
MSD. The investigator will send a computer-generated randomization list (block 
randomization of 4) via an automated system (Castor EDC) to the trial pharmacy of 
Amsterdam UMC. In this way, all study participants and trial personnel remain blinded 
at all times. Unblinding will only occur in a medical emergency. The sodium diets are 

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of course open label. Each participant receives sufficient tablets (ertugliflozin or 
matching placebo) for each treatment period of n=10 days. 
 
8.3 Study procedures 
 
Recruitment 
Potential participants will be recruited by researcher physicians involved in this trial 
using methods that are established practice for all human studies in the Diabetes 
Center Amsterdam UMC-VUMC:  
1) 
Via advertisements in (local) newspapers  
2) 
Use of a Diabetes Center database to approach volunteers from previous 
studies, who have given written informed consent to be available for future studies  
3) 
Through the Diabetes Center website (www.diabetescentrum.nl)  
4) 
Via general practitioner offices with whom we have collaborations., 
Potential participants will be approached by the general practitioner 
5) 
Where possible, subjects will be recruited from the out-patient clinic of the 
Diabetes Center / Department of Internal Medicine Amsterdam UMC-VUmc or 
affiliated hospitals / pharmacies 
6) 
Online advertising directed at patient support groups 
In case of a positive response, the information letter and informed consent forms will 
be sent to these individuals. They will then be contacted by telephone by the research 
physician to answer any remaining questions and make an appointment for a 
screening visit if the individual wants to participate. 
 
Screening and eligibility (V1 and T1) 
After giving extensive oral and written information, and sufficient time to consider 
study participation (minimally one week), a written informed consent form will be 
obtained from the subjects before screening. Once the participant signs the informed 
consent form he/she will be assigned a unique study number, which will be used for 
identification.  
 
The screening procedure will consist of obtaining an extensive medical history 
(including disease history, current medical conditions and use of medication, 
substance use, employment, education, family history), complete physical 
examination, drawing blood for hematologic and biochemistry testing (full blood count, 
glucose, HbA1c, creatinine, ALT, AST, gamma-glutamyl transferase (GGT), 
creatinine, albumin, and thyroid-stimulating hormone (TSH)), urine screening, urine 
creatinine and albumin, and a 12-lead electrocardiogram (ECG). If subjects are 
eligible, the investigator will inform them within two weeks after screening. Then, 24-
hour urine and 24-hour blood pressure are obtained in order to be able to study 
hypothesis 2 (effect of ertugliflozin on sodium sensitivity). 
 
 

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After confirmation of eligibility during screening, a 24-hr blood pressure measurement 
as well 24-hr sodium excretion is measured prior to the dietary blocks as detailed 
below. This 24-hr sodium and blood pressure measurement will be crucial for 
hypothesis 2. Urine containers and 24-hr blood pressure meters are provided to the 
patient and collected after recording. 
 
Screening 
Day 0 
Day 1 (offered at home of participants to reduce travel 
Medical history 
X 
 
Physical examination 
X 
 
Review of 
medication 
X 
 
Office blood 
pressure 
X 
 
Routine hematology 
and chemistry 
X 
 
HbA1c 
X 
 
Urinary dipstick 
X 
 
Start 24-hr urine 
sodium collection 
X 
 
Start 24-hr BP 
measurement 
X 
 
Return 24-hr urine 
sodium collection 
 
X 
Return 24-hr BP 
measurement 
 
X 
  
Subsequently, 4 of the blocks of 20 days below are conducted in random order with 
differences in study medication (placebo versus ertugliflozin) and sodium diet (90 
mmol/day versus 250 mmol/day). The washout is 4 weeks between the blocks. 
Please refer to the figure on page 11. Urine containers are provided to the patients. 
 
A sodium diet is commenced (either aimed at 90 mmol/day or 250 mmol/day) and it is 
expected that after 10 days this is reached (+/- 20%, due to normal variation in 
sodium excretion). This will be assessed with two 24 hour urine collections. 
Subsequently, the diet is continued and treatment with the study medication 
commences. In the morning of day 9, 24-hour blood pressure recording starts. At the 
study visit at day 10 of treatment, endpoints are collected following an overnight fast 
of 10-hours at our clinical trial unit as indicated below. A flow chart of study activities 
is attached below. 
 
Subject will arrive at Amsterdam UMC location AMC for multimetric MRI following an 
overnight fast. Subjects are then brought to Clinical Research Unit (CRU) location 
VUMC where the 24-hour urine collection is processed (in order to assess sodium 

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excretion and other variables of interest obtained from 24-hr urine collection). The 24-
hour blood pressure recording is stopped.  Two intravenous catheters will be placed 
in both forearms for blood sampling and for the administration of test-agents (e.g. 
iohexol and PAH). A total of 125 mL blood will be drawn during the day for this 
purpose. The renal protocol commences with measurement of GFR and effective 
renal plasma flow (ERPF) based on continuous infusion for 2.5.   
During the infusion, several measurements will be performed including 
anthropometrics, bio-impedance analysis, blood pressure measurement and 
collection of samples for biomarker analysis.  
 
 
Day -10 
Day -2  
Day -1 
Day 0 
Day 3 
Day 10 
Start sodium-diet 
(continue until day 
10) 
X 
 
 
 
 
 
24-hr urine sodium 
(compliance) 
 
X 
X 
 
 
 
Start study 
medication 
(continue until day 
10) 
 
 
 
X 
 
 
Telephone call 
compliance 
 
 
 
 
X 
 
Recent history 
 
 
 
 
 
X 
24-hr blood pressure 
 
 
 
 
 
X 
Measured GFR 
 
 
 
 
 
X 
ERPF 
 
 
 
 
 
X 
Intrarenal 
hemodynamic 
function 
 
 
 
 
 
X 
Office blood 
pressure 
 
 
 
 
 
X 
Hematocrit 
 
 
 
 
 
X 
Renal oxygenation 
(BOLD MRI)  
 
 
 
 
 
X 
RAAS components 
 
 
 
 
 
X 
Collection of 
biomarkers 
 
 
 
 
 
X 
Body 
anthropometrics 
 
 
 
 
 
X 
24-hr urine analysis 
(sodium, UACR) 
 
 
 
 
 
X 
 

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Efficacy measurement more detailed: 
 
1) Height. A height measuring board will be used to assess height. Patients will be 
asked to remove their footwear and headgear and to stand on the board with their 
feet together, heels against the back board and knees straight. The patient should be 
looking straight ahead, without tilting the head up, with eyes and ears at the same 
level. The measure arm will gently be moved down onto the head of the participant 
and height will be read in centimeters at the exact point. 
2) Weight. Body weight will be recorded by a digital calibrated weighing scale on a 
firm, flat surface. Patient will be asked to remove their footwear, extra layers of 
clothing, jeweler and any items in his/her pockets before standing still on the weighing 
scale with arms placed on the side. Measurement will be recorded on the Case 
Report Form (CRF) to the nearest kilogram. No deduction for clothing will be 
permitted. 
3) Body Mass Index (BMI). BMI will be derived from height and bodyweight by using 
the mathematical formula: BMI=[kg/[m]2. 
4) Waist circumference. A constant tension tape will assess waist circumference 
with the patient in a standing position with the arms relaxed at the sides and without 
clothing, that is, directly over the skin. Measurement will be performed at the midpoint 
between the lower margin of the last palpable rib and the top of the iliac crest at the 
end of a normal expiration. 
5) Body-impedance analysis. To measure body fat content we will use the 
tetrapolar Soft Tissue Analyzer® (STA, Akern, Florence, Italy), which can perform 
Bio-Impedance Analysis (BIA). This technique measures the resistance in the body 
tissues when a 50 kHz AC electrical current is given between four electrodes. 
Resistance can be measured by placing two electrodes on the dorsal hand and two 
on the foot at the unilateral side of the body (Lukaski & Bolonchuk, 1988). 
Extracellular water (ECW) as a percentage of total body water (TBW) and body cell 
mass (BCM) will be calculated independent of weight and height. 
6) ECG: A 12-leads ECG will be made in the fasting resting state during screening in 
order to assess cardiac conduction parameters. Heart rate on the ECG will be defined 
as the mean rate of 10 R-R intervals. PQ-time, QRS-time and QT-time will be 
calculated, QT-time will be corrected for heart rate by using the Bazett formula (C. 
Bazett, 2006). The cardiac vector and ECG morphology will be assessed. 
7) Blood pressure and heart rate (office): Blood pressure and heart rate will be 
measured using an automatic oscillometric device (Dinamap®, GE Healthcare) After 
an acclimatization period of >5 minutes, blood pressure will be measured three times 
at the non-dominant arm, with the subject in a semi-recumbent position, using an 
appropriate cuff-size. The mean of the last 2 measurements will be used as final 
value (Mengden et al., 2010; Pickering et al., 2005).  
8) GFR is measured by steady-state plasma iohexol concentrations measured at 
T=120 and T=150 as done in our previous approved protocols. 

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9) ERPF is measured by steady-state plasma PAH concentrations measured at 
T=120 and T=150 as done in our previous approved protocols. 
10) Filtration fraction (GFR/ERPF) and renal vascular resistance (ERPF/mean 
arterial pressure) are calculated.  
12) 24-hour blood pressure is derived from Mobil-O-graph devices (24 ambulatory 
blood pressure monitors, The Netherlands), which will be available at both sites. 
Daytime systolic blood pressure (SBP), daytime diastolic blood pressure (SBP), 
nighttime SBP, daytime DBP and nocturnal dipping pattern (in mmHg) are recorded. 
12) sodium sensitivity is calculated as following: changes in blood pressure due to the 
high-sodium diet versus diet at screening (estimated based on experience/previous 
trials at ˜165-170 mmol/day) and the effect on this parameter by ertugliflozin versus 
placebo. 
13) Multiparametric MRI will be carried out to measure additional kidney physiology 
parameters. The multiparametric MRI is performed on a tesla 3 machine. The MR 
examination will last 45 minutes maximum. During the scans, heart rate and blood 
pressure will be monitored. Patients get hearing protection in the form of ear-plugs 
and headphones. During the examination, they are in contact with the technicians via 
the intercom. Throughout the examination, patients will receive breathing instructions, 
for example to hold their breath for 20 seconds maximum, in order to produce 
accurate images.  The MRI exam includes BOLD MRI to measure oxygenation, but 
also ASL and phase contrast MRI to obtain renal artery blood flow. 
- BOLD-MRI: BOLD-MRI will be performed in order to measure renal tissue 
oxygenation. BOLD MRI exploits the difference in magnetic properties between 
oxygenated and deoxygenated hemoglobin (Pruijm et al., 2018). Presence of 
deoxygenated hemoglobin will result in an accelerated signal decay on the BOLD 
images. Measurement of this signal decay results in a measure of the amount of 
deoxygenated hemoglobin, the apparent relaxation rate R2*, which inversely relates 
to level of tissue oxygenation. 
-ASL: In ASL, the blood travelling through the aorta is magnetically labeled before 
acquiring an image (the “label” image) (Odudu et al., 2018). Other images are 
acquired without any label (the “control” image). The difference between the label and 
control image will only be around 2%, so this procedure is repeated several times. 
The average difference between those images can be used to obtain a perfusion map 
of the kidney. Phase-contrast MRI is done to obtain renal artery blood flow. 
14) serum biomarkers: hematocrit, serum albumin, plasma glucose and additional 
serum biomarkers are measured in the fasted state 
15) 24-hour urine albumin and glucose excretion is measured. Additional material 
for urinary biomarkers of interest such as markers of inflammation and electrolytes is 
stored. 
16) Safety and tolerability: recent history and focused physical examination is 
carried out to assess potential adverse effects of study medication. 
 

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8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
any consequences. The investigator can decide to withdraw a subject from the study 
for urgent medical reasons. When participants decide to withdraw their participation, 
they will be asked to participate in ‘early-term assessments’.  
 
After subjects have entered the study, participation will be stopped in case of 
occurrence of any of the following: 
• 
Participants own decision 
• 
The occurrence of an AE or clinically significant laboratory change or 
abnormality that, in the judgment of the investigator, warrants withdrawal of the 
study 
• 
Subjects need medical procedures that are not allowed in the protocol 
• 
Subjects cannot undergo the procedures to investigate the research 
questions as outlined in this protocol 
• 
Non-compliance 
• 
In addition to these requirements for study drug discontinuation, the 
investigator should discontinue study participation for a given subject if, on balance, 
he thinks that continuation would be unbeneficial to the subjects well-being 
 
8.4.1 Specific criteria for withdrawal (if applicable) 
 
Not applicable 
 
8.5 Replacement of individual subjects after withdrawal 
 
Participants will be replaced to maintain sufficient power. 
 
8.6 Follow-up of subjects withdrawn from treatment 
If patients withdraw from the study, vital signs will be collected and recent history to 
confirm safe withdrawal from the study. There is no harm in suddenly stopping study 
medication, although glucose levels may increase mildly. 
 
8.7 Premature termination of the study 
I do not expect premature termination of the study. No interim analysis for efficacy is 
performed. Ertugliflozin is a drug with a well-established safety profile, and no new side 
effects are expected to be found in this study with 2 blocks of 10 days of active treatment. 
Obviously, if SAE are encountered that may be related to the study medication, a careful 
decision will be made to halt the study prematurely.  

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed.  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (Aes) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to [the investigational product / 
trial procedure/ the experimental intervention]. All adverse events reported 
spontaneously by the subject or observed by the investigator or his staff will be 
recorded. 
 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
 
Because this study is considered to be low-risk, as agreed by the Ethical Committee, 
SAE’s will be recorded and reported yearly through the web portal ToetsingOnline to 
the accredited METC that approved the protocol. 
 
 

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9.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational 
product related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of 
the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity 
of the adverse reaction are not in agreement with the product information as 
recorded in: 
- 
Summary of Product Characteristics (SPC) for an authorised medicinal 
product; 
- 
Investigator’s Brochure for an unauthorised medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline  to the METC  
 SUSARs that have arisen in the clinical trial that was assessed by the METC; 
 SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
 
The remaining SUSARs are recorded in an overview list (line-listing) that will be 
submitted once every half year to the METC. This line-listing provides an overview 
of all SUSARs from the study medicine, accompanied by a brief report highlighting 
the main points of concern.  
The expedited reporting of SUSARs through the web portal Eudravigilance or 
ToetsingOnline is sufficient as notification to the competent authority. 
 
The sponsor will report expedited all SUSARs to the competent authorities in other 
Member States, according to the requirements of the Member States.  
 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will 
be maximal 7 days for a preliminary report with another 8 days for completion of the 
report.  

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The site at Colorado University (PI Dr Bjornstad) will report all SAEs and SUSARs to the 
local competent authority according to the requirements of that Member State. In addition, 
they will inform the sponsor/coordinating investigator of the study (Dr. van Raalte) and 
MSD within a single day. 
 
Please describe also the method of breaking the code for SUSAR reporting: 
 
The pharmacy of each participating center performs the randomisation of subjects to the 
treatment categories. In the event of an emergency, the local pharmacy provides the local 
researcher with information concerning the treatment of the subject of the SUSAR. 
Unblinding is not to be performed for any reason other than an emergency where 
unblinding is required and should be reported in the CRF. The local investigator must also 
immediately inform the project leader and the principal investigator about the unblinding 
procedure. If it does not compromise the welfare of the participant, a close-out visit which 
includes drawing of blood samples for safety or pharmacodynamic assessments will be 
performed. 
9.3 Annual safety report 
 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, 
and competent authorities of the concerned Member States. 
This safety report consists of: 
 a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
 a report concerning the safety of the subjects, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
 
9.4 Follow-up of adverse events 
All Aes will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study within the Netherlands, as defined in the 
protocol. 

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10. STATISTICAL ANALYSIS 
 
Sample size calculations 
The primary endpoint of the study is to investigate the modifying effects of WHO-
recommended sodium intake (90 mmol per day) vs. high sodium intake (targeted at 250 
mmol per day) on the effect of ertugliflozin 15 mg daily, versus placebo, on 24-hour blood 
pressure in overweight/obese adults with type 2 diabetes. 
 
Based on previous studies in patients treated with SGLT2 inhibitors, we expect a change 
in systolic blood pressure of 6-8 mmHg during normal sodium intake (150 mmol/day) 
[PMID: 33318125; PMID: 23668478]. Based on our hypothesis, this effect is increased on 
a low-sodium intake and reduced during high-sodium intake. 
 
We expect a difference of 4 mmHg between the placebo-controlled effects of ertugliflozin 
on a low- versus high-sodium diet, with a conservative SD on this difference of 8 mmHg. 
 
A total of 34 patients completing the study provide 80% power at a two-sided alpha of 
0.05, to detect a 4 mmHg difference with the indicated SD of 8 mmHg. Drop-outs will be 
replaced. Based on the RED study and DAPASALT study, the power needed to detect 
changes in eGFR and hematocrit are of sufficient magnitude and therefore we expect 
sufficient power for these parameters as well. The calculation is based on a paired 
design. The sample size was calculated using Stata-Software (v. 11.2, College station, 
TX, USA).  
 
10.1 Primary study parameter(s) 
 
General considerations 
1. 
All tests of differences between treatments will be conducted at a two-sided 
significance level of <0.05. In general, the null hypothesis (H0: no difference exists 
between treatment with the study drug and placebo) intervention ) will be tested against 
the hypothesis H1: a difference exists between treatment with the study drug and 
placebo), with respect to the underlying parameter of interest.  
2. 
All variables will be expressed as mean ± SEM when normally distributed, 
or –in case of non-normal distribution- be expressed as median and interquartile range 
(IQR). Log transformation will be applied to obtain normal distribution as appropriate for 
the statistical tests. 

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3. 
In case of missing data, a multiple imputation technique will be applied and 
compared with complete case analysis.  
 
Analysis population 
The efficacy analysis population will be based on the per protocol (PP) principle – i.e. all 
subjects who completed the entire treatment period using the treatment as originally 
allocated. Safety analysis will be performed in all patients who received one or more 
doses of the study medication. 
 
Statistical analysis of study endpoints 
We will use a two-way ANOVA to evaluate the main effects of diet and treatment as well 
as an interaction between ertugliflozin vs. placebo during high and low-sodium intake on 
the main outcomes. Appropriate covariates may be added to the model. Because of the 
crossover design of our trial, we will assess the carryover effect by including the 
sequence allocation as a factor in the mixed model. If a carryover effect will be detected 
(p<0.1), only the first study period will be analysed (treating it as a parallel randomised 
controlled trial).  
 
For possible confounders, we will test whether there is an association with both the 
dependent and independent variable (p <0.1). If this is the case, this confounder will be 
added to the model as covariate. Since all outcome variables – primary, secondary and 
exploratory – are continuous, this statistical technique can be applied.   
 
The large amount of secondary/exploratory endpoints potentially justifies the use of 
corrections for multiple tests. However, there is an increasing debate on whether this 
should be done, because of the increased risk of type 2 errors which could lead to 
prematurely discarding of potential useful observations (Rothman, 1990). As recently 
pointed out (Streiner & Norman, 2011), in a hypothesis-generating study – such as the 
current study – correction for multiple outcomes can be detrimental.  
 
10.2 Secondary study parameter(s)  
The secondary endpoint is analysed by comparing the effects of high-sodium on blood 
pressure increments during placebo and ertugliflozin treatment. An ANOVA analysis will 
be carried out to test significance between different conditions. 

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10.3 Other study parameters 
All exploratory variables are analysed as described under 10.1. For exploratory 
parameters, no correction for multiple testing will be conducted and the exploratory nature 
of the data will be stressed when the data are reported in a manuscript as our group has 
done previously. 
 
10.4 Interim analysis (if applicable) 
No interim analysis will be performed 

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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki 
(Fortaleza, 2013), in accordance with the Medical Research Involving Human Subjects 
Act (WMO), and in accordance to the European Clinical Trial Regulation (ECTR). The 
investigator will ensure that all aspects of the institutional ERB review are conducted in 
accordance with current institutional, local and national regulations. 
 
11.2 Recruitment and consent 
 
Patients will be enrolled to the trial from centers in the Netherlands (1 center) and the United 
States (1 center). Potential participants will be approached through (a) the diabetescenter 
database, which has registered participants that have given consent to be approached for 
new studies by their informed consent form of a former stud, (b) adds online or in local 
newspapers, or (c) through their own physician. 
 
(a) Former participcants of former studies, that gave their consent to be approached for 
new studies, can be contacted by the research physician. In case of a positive respons, 
the participant information letter wil be send out. After a minimum of a week, in 
which the possible participant can read the information and formulate questions, the 
research physician will contact the possible participant again and answer any 
questions. If the possible participant wishes to participate, an appointment will be 
made for the screening visit. During this visit there again is room for explanation of 
the study and questions. Than, before the commencement of any study procedures, the 
informed consent form will be signed in twofold by the participant and the research 
physician. 
(b) People who have read the add and have interest in participation of the study can 
contact the research physician by mail or by phone. After the inital contact has been 
made, the procedure is similar tot he procedure described above. The research 
physician will explain the details of the study and the PIF will be send. After at least a 
week, there will be a second contact moment and room for questions. In case of a 
positive response, the possible participant will be invited to the hospital for a 
screening visit. During this visit there again is room for explanation of the study and 
questions. Than, before the commencement of any study procedures, the informed 
consent form will be signed in twofold by the participant and the research physician. 
(c) A doctor that is familiar with current study, can ask his/her own patients if they have 
interest in participation in the study and if the research physician can contact them. In 
casse of a positive respons, the procedure is similar tot he procedure described above. 
The research physician will contact the possible participant and explain the details of 
the study and the PIF will be send. After at least a week, there will be a second contact 
moment and room for questions. If the possible participant wishes to participate he/she 
will be invited to the hospital for a screening visit. During this visit there again is room 
for explanation of the study and questions. Than, before the commencement of any 
study procedures, the informed consent form will be signed in twofold by the 

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participant and the research physician. 
 
 
11.3 Benefits and risks assessment, group relatedness 
All participants will receive ertugliflozin, an SGLT-2 inhibitor which is approved for blood-
glucose lowering treatment in T2DM patients. Based on currently available data from 
thousands of patient years, the drug has shown to be safe. In addition to glucose-
lowering, SGLT-2 inhibitors have other beneficial effects, by inducing a decrease in blood 
pressure and body weight for instance. Clinically, SGLT2 inhibitors prevent kidney 
function decline, hospitalization for heart failure and cardiovascular events after longer-
term use.  
SGLT2 inhibitors have side effects. The most common side effect are genital mycotic 
infections, which can be treated with a topical antimycotic crème. Other side effects 
include polyuria, frequent voiding and nycturia.  
 
As in all drug intervention trials, in this study, we will closely monitor patients for adverse 
drug and study events. Participants can contact the research staff 24 hours a day. It 
should be noted that most of these side effects rarely occur in such a short treatment 
duration as in the current study. There is no risk of a short-term change in sodium diet on 
long-term health outcomes. In addition, effects of high-sodium diet [e.g. on blood 
pressure] are closely monitored.  
11.4 Compensation for injury 
 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of 
the WMO. The sponsor (also) has an insurance which is in accordance with the legal 
requirements in the Netherlands (Article 7 WMO). This insurance provides cover for 
damage to research subjects through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.5 Incentives  
Patients will receive restitution of all costs for transportation and a honorarium of 500 euro 
for participating. In case of premature termination, the reimbursement of the participant 
will be based on the actual duration of participation in proportion to the planned duration 
of participation. 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
 
In accordance with the Dutch law ‘Algemene verordening gegevensbescherming’ (AVG; General 
Privacy Law) and GCP all patient data will be handled confidentially. Therefore, all participants at 
Vumc will get a study code (‘DES-0xx’). The code list, with both identifier data and study number, will 
be stored securely on the server of the VUMC, protected by passwords only known by the 
responsible research physicians and principal investigator.  
 
All subject data will be recorded on printed CRF by the local  investigator and/or personnel that is 
instructed in performing these administrative tasks and study procedures. In the latter case, data will 
always be reviewed by the investigator. All subject related data will be stored for the legally required 
period of 25 years. With regard to the body material, data will be stored for a maximum of 5 years 
and will only be used for the research questions within the scope of current research. Subject data, 
both encoded (e.g. CRFs) or not (e.g. source documents), will always be stored securely, in a locked 
cabinet in the Diabetes Center VUMC or on password secured computers.  
 
For the site in Colorado, the same principles are maintained as indicated here. Data from the US site 
will be sent to Amsterdam UMC, in an anonymized matter (using a study code DES-1xx).  This has 
been described in the study contract between sites. Vice versa, data will not be send from the Dutch 
site to the US. 
 
Monitoring and Quality Assurance  
The study will be monitored by an appointed monitor of the VU ethical review board. The 
monitor will conduct ( a frequency of) quality assessments in accordance to the guidelines 
of the institution. Monitoring at Colorado University is carried along the same principles 
with a local monitor. 
 
12.2 Amendments  
 
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to 
affect to a significant degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the METC and to the competent authority. 
 

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Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
 
12.3 Annual progress report 
The investigator will submit a summary of the progress of the trial to the accredited METC 
once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, 
serious adverse events/ serious adverse reactions, other problems, and amendments.  
 
12.4 Temporary halt and (prematurely) end of study report 
 
The sponsor will notify the accredited METC and the competent authority of the end of 
the study within a period of 90 days. The end of the study is defined as the last patient’s 
last visit.  
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action. 
In case the study is ended prematurely, the sponsor will notify the accredited METC and 
the competent authority within 15 days, including the reasons for the premature 
termination. 
 Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC and the Competent Authority.  
 
 
12.5 Public disclosure and publication policy 
Publication will be in accordance with the basic principels of Central Committee on 
Research involving Human Subjects (CCMO) statement on publication policy. 
As described in the contract with MSD, the sponsor has full rights to publish the data 
unreservedly. This concerns both abstracts that are sent for presentation at conferences 
and manuscript for publication in high-impact peer-reviewed journals. The Amsterdam and 
Denver site will co-publish the data under the strong collaboration between PI van Raalte  
PI Bjornstad. Please refer to the contract with MSD for details. 
 
 

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13. STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
 
Study medication 
Participants will receive ertugliflozin 15 mg, an SGLT-2 inhibitor which is approved for blood-
glucose lowering treatment in T2DM patients, and based on currently available data, is 
considered to be safe. Furthermore, SGLT-2 inhibitors may have other beneficial effects in 
general and are associated with a decrease in blood pressure and body weight. In large-
sized cardiovascular outcome study, ertugliflozin was shown to have beneficial effects on 
cardiovascular outcomes and chronic kidney disease.  
The most common adverse effects for ertugliflozin are genital mycotic infections and to a 
lesser extent pruritus, polyuria, frequent voiding and nycturia. However, genital mycotic 
infections usually occur after prolonged treatment, at least weeks. For canagliflozin, not 
ertugliflozin, increased amputations were reported in one study, but not in people with kidney 
disease, mostly in people that had previous amputations. 
Hypoglycemia: SGLT2 inhibitors do not cause hypoglycemia as they do not increase insulin 
secretion. In people without diabetes, SGLT2 inhibitors do not cause hypoglycemia. When 
combined with drugs as insulin and sulfonylurea, SGLT2 inhibitors may cause hypoglycemia, 
particularly in people with normal eGFR. 
Keto-acidosis: this has been reported in people with type 1 diabetes,28 but is rare in people 
with type 2 diabetes with only a few cases reported worldwide.28 
 
Sodium diet: 
Participants will undergo two periods of increased sodium intake (of 20 days each). While 
higher sodium intake, may increase blood pressure, it is usually not associated with adverse 
effects. Of note, sodium intake in the Netherlands is really high, therefore, for some people it 
may be their normal intake. Lowering sodium intake is not related to any health risk. 
 
a. Level of knowledge about mechanism of action 
 
Regarding side effects, they have a well-defined mechanism of action: 
1. 
Genitourinary mycotic infections: due to SGLT2-induced glucosuria, urine 
provides a better culture media for mycotic infections.  
2. 
Hypoglycaemia: higher risk in people with concomitant insulin therapy 
and/or sulfonylurea. Due to glucosuria, the amount of circulation can be inappropriate 
leading to hypoglycaemia. 
3. 
Keto-acidosis: due to lowering of insulin concentrations due to glucosuria, 
and due to SGLT2-induced increment in glucagon concentrations, ketogenesis is 
enhanced. The nature of this mechanism facilitates clear actions to mitigate this effect as 
detailed below. 
 

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b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
 
Please refer to the IB 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
 
Please refer to the IB 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
 
Please refer to the IB 
 
e. Analysis of potential effect 
 
The dosage of 10 mg has good safety profile. Please refer to the IB. 
 
 
f. Pharmacokinetic considerations 
 
The dosage of 10 mg has good safety profile. Please refer to the IB. 
 
g. Study population 
 
The included participants are people with type 2 diabetes that are otherwise healthy. They 
are similar to the patients included in the ertugliflozin trials, making the effects on our 
population predictable.   
 
h. Interaction with other products 
 
Please refer to the IB 
 
i. Predictability of effect 
 
Please refer to the IB 
 
j. Can effects be managed? 
 
The most common side effect of SGLT2 inhibition therapy is genital mycotic infections. They 
occur usually after more chronic treatment. The incidence is increased 2.47-fold compared to 
placebo. However, a recent study in people with heart failure indicated that this may be 
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(incidence rate per 100 patient years 0.5) when proper instructions with respect to hygiene 
are provided, as will be done in our study. In addition, it is easily treated with a topical drug. 
The most feared side effect is euglycemic diabetic ketoacidosis (DKA). Due to lowering of 
endogenous or exogenous insulin levels, people with type 1 diabetes showed marked 
increases in euglycemic DKA despite controlled trial setting. For this reason, SGLT2 
inhibitors are only in exceptional situations given to people with type 1 diabetes. In people 
with type 2 diabetes, euglycemic DKA is extremely rare, approximately 1 case per 1000 
patient years. This likely concerns misclassified type 2 diabetes patients in whom insulin was 
stopped or reduced. To mitigate euglycemic DKA risk in our study, type 1 diabetes patients 
are excluded in our study, as well as individuals with diabetes and BMI <25 kg/m2 as they 
phenotypically resemble type 1 diabetes patients. To prevent hypoglycemia, insulin dosages 
are lowered by the study team prior to initiation of the study drug if indicated. The study team 
consists of lead experts in the field of diabetology with ample experience in dosing glucose-
lowering drugs. For the sodium diet we will monitor blood pressure when complaints are 
expressed by the study participant.  
 
13.2 Synthesis 
 
Although there will be no medical benefit for participants of this study, we feel that the risk of 
the study protocol is moderate. The drug is well-tolerated in thousands and thousands of 
patient years with side effects being predictable, preventable and treatable. The effects of the 
sodium diet also do not pose a risk. In addition, the participants may receive education how 
to lower their sodium intake following study participation. The risk of the study procedures 
are low with placement of IV cannula as the most invasive study activity. Our previous 
protocols have demonstrated safety with respect to the imaging and kidney function tests. 
 
 
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