A Randomized, Double-blind, Comparator-controlled Trial to Assess the Effect of 12-week Treatment With Dapagliflozin Versus Gliclazide on Renal Physiology and Biomarkers in Metformin-treated Patients With Type 2 Diabetes Mellitus
Phase: PHASE4
Conditions: Diabetes Mellitus, Type 2, Diabetic Nephropathies
Sponsor: M.H.H. Kramer

 
 
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RESEARCH PROTOCOL 
A phase 4, monocenter, randomized, double-blind, comparator-controlled, parallel-
group, mechanistic intervention trial to assess the effect of 12-week treatment with 
the sodium-glucose linked transporters (SGLT)-2 inhibitor dapagliflozin versus the 
sulfonylurea (SU) derivative gliclazide on renal physiology and biomarkers in 
metformin-treated patients with type 2 diabetes mellitus (T2DM) 
 
 
Short Title (Acronym):  
RED: Renoprotective Effects of Dapagliflozin in Type 2 Diabetes 
 
 
 
CONFIDENTIAL 
 
 
 
Investigators: 
M.H.H. Kramer (PI) 
D.H. van Raalte (PI) 
E. van Bommel 
M.H.A. Muskiet 
L. Tonneijck 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Diabetes Center 
PO Box 7057 
1007 MB Amsterdam 
The Netherlands 
Telephone: +31-(0)20-44440651 
Fax: +31-(0)20-4443349 
Sponsor Code number: DC 2015 RED 01 
 
Universal Trial number: U1111-1173-7074 
EudraCT number: 2015-003818-24 
 
 
CCMO number: NL54965.029.15 
 
NCT number: NCT02682563  

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Confidential 
 
 
 
 
 
 
 
 
 
           DC2015RED1 
Kramer, van Raalte 
                                                            Version 2.1 / 15-02-2016 
DOCUMENT INFORMATION 
 
Protocol ID 
DC2015RED1 
Full title 
A phase 4, monocenter, randomized, double-blind, comparator-controlled, 
parallel-group, mechanistic intervention trial to assess the effect of 12-week 
treatment with the sodium-glucose linked transporters (SGLT)-2 inhibitor 
dapagliflozin versus the sulfonylurea (SU) derivative gliclazide on renal 
physiology and biomarkers in metformin-treated patients with type 2 diabetes 
mellitus (T2DM) 
Short title (Acronym) 
RED: Renoprotective Effects of Dapagliflozin in Type 2 Diabetes 
Date 
04-03-2019 
Version 
5.0 
 
 
Contact information 
Principal Investigators 
Prof. M.H.H. Kramer, MD PhD 
Department of Internal Medicine 
De Boelelaan 1117, Room 4A34 
1081 HV Amsterdam 
Telephone: +31-(0)20-4443409  
(assistant: Mrs. Annelies Kabalt–Zoet) 
Email: m.kramer@vumc.nl 
D.H. van Raalte, MD PhD 
Department of Internal Medicine 
De Boelelaan 1117, Room ZH-4A58 
1081 HV Amsterdam 
Telephone: +31-(0)20-4440534/40533 
(secretary Mrs. Lilian van 't Hull)  
Email: d.vanraalte@vumc.nl 
Sponsor 
Prof. M.H.H. Kramer, MD PhD 
Department of Internal Medicine 
De Boelelaan 1117, Room 4A34 
1081 HV Amsterdam 
Telephone: +31-(0)20-4443409 (assistant: Mrs. Annelies Kabalt–Zoet) 
Email: m.kramer@vumc.nl 
Independent physician 
E.J.G. Peters, MD PhD  
Department of Internal Medicine 
De Boelelaan 1117, Room PK-1-Z108 
1081 HV Amsterdam 
Telephone: +31-(0)20-4440596 
Email: e.peters@vumc.nl  
 
  
 
 

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                                                            Version 2.1 / 15-02-2016 
TABLE OF CONTENTS 
 
DOCUMENT INFORMATION ......................................................................................................... 2 
TABLE OF CONTENTS ................................................................................................................. 3 
ABBREVIATIONS, DEFINITIONS AND TERMINOLOGY .............................................................. 4 
ABSTRACT .................................................................................................................................... 6 
1. 
INTRODUCTION ..................................................................................................................... 8 
2. 
STUDY OBJECTIVES ........................................................................................................... 10 
3. 
STUDY DESIGN .................................................................................................................... 12 
4. 
STUDY POPULATION .......................................................................................................... 13 
5. 
STUDY DRUGS ..................................................................................................................... 15 
6. 
STUDY OUTLINE .................................................................................................................. 17 
7. 
STUDY PROCEDURES ......................................................................................................... 20 
8. 
LABORATORY ASSESSMENTS .......................................................................................... 23 
9. 
EFFICACY MEASUREMENTS .............................................................................................. 24 
10. SAFETY (REPORTING) ........................................................................................................ 26 
11. DATA MANAGEMENT AND QUALITY ASSURANCE .......................................................... 29 
12. STATISTICS .......................................................................................................................... 30 
13. ETHICS .................................................................................................................................. 31 
14. ADMINISTRATIVE ASPECTS AND PUBLICATION ............................................................. 34 
 
REFERENCES ............................................................................................................................. 36 
 
APPENDIX A       (STUDY OUTLINE) .......................................................................................... 36 
APPENDIX B       (TEST MEDICATION) ...................................................................................... 41 
APPENDIX C1     (RENAL PROTOCOL) ...................................................................................... 43 
APPENDIX C2     (CLAMP PROTOCOL) ...................................................................................... 46 
APPENDIX D       (MICROVASCULAR FUNCTION ASSESSMENT) ............................................ 48 
APPENDIX E       (PULSE WAVE ANALYSIS) .............................................................................. 49 
APPENDIX F       (CARDIOVACULAR AUTONOMIC NERVOUS SYSTEM TESTS) .................... 50 
APPENDIX G       (FECAL TESTS) ............................................................................................... 52 
APPENDIX H       (MONITORING PROCEDURE) ........................................................................ 53 
 
 
 

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ABBREVIATIONS, DEFINITIONS AND TERMINOLOGY 
 
 
8-OHdG 
8-hydroxy-2' -deoxyguanosine 
ACR 
Albumin-to-Creatinine Ratio 
ADA 
American Diabetes Association 
AE 
Adverse Event 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANP 
Atrial Natriuretic Peptide 
ANS 
Autonomic Nervous System 
AR 
Adverse Reaction 
ARB 
Angiotensin II Receptor Blocker 
AST 
Aspartate aminotransferase 
BCM 
Body Cell Mass (variable derived with BIA) 
BIA 
Bio-Impedance Analysis 
BMI 
Body Mass Index 
BNP 
Brain Natriuretic Peptide 
CARTs 
Cardiovascular autonomic reflex tests 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: Centrale Commissie 
Mensgebonden Onderzoek 
CKD 
Chronic Kidney Disease 
CRF 
Case Report Form 
eCRF 
Electronic Case Report Form 
CRU 
Clinical Research Unit 
CNS 
Central Nervous System 
DCCT 
Diabetes Control and Complications Trial 
DKD 
Diabetic Kidney Disease 
DNA 
Deoxyrubonucleic acid 
EASD 
European Association for the Study of Diabetes 
ECG 
Electrocardiogram 
ECW 
Extracellular Water (variable derived with BIA) 
EMA 
European Medicines Agency 
ERB 
Ethical Review Board 
ERBF 
Effective Renal Blood Flow 
ERPF 
Effective Renal Plasma Flow 
ERVR 
Effective Renal Vascular Resistance 
ESRD 
End Stage Renal Disease 
EudraCT 
European Drug Regulatory Affairs Clinical Trials  
FDA 
Food and Drug Administration 
FE 
Fractional Excretion (measure of renal tubular function) 
FPG 
Fasting Plasma Glucose 
FSH 
Follicle-Stimulating Hormone 
GCP 
Good Clinical Practice 
GFR 
Glomerular Filtration Rate 
GGT 
Gamma-Glutamyl Transferase 
GMP 
Good Manufacturing Practice 
HbA1c 
Glycated hemoglobin (A1c) 
HDL-C 
High Density Lipoprotein - Cholesterol 
HF 
High Frequency (component of HRV) 
HRV 
Heart Rate Variability 
hsCRP 
high sensitivity C-Reactive Protein 
ICH 
International Conference on Harmonisation 
IFCC 
International Federation of Clinical Chemistry 
IIT 
Investigator Initiated Trial 
IL-6 
Interleukin 6 
IMP 
Investigational Medicinal Product (‘study drug’) 
IQR 
Interquartile Range 
KIM-1 
Kidney Injury Molecule-1 

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LDL-C 
Low Density Lipoprotein - Cholesterol 
LF 
Low Frequency (component of HRV) 
Long-term tests 
End-point assessments performed after 12-week study drug intervention 
MCP-1 
Monocyte Chemoattractant Protein-1 
MedDRA 
Medical Dictionary for Regulatory Activities 
MSD 
Merck Sharp & Dohme 
NGAL 
Neutrophil Gelatinase-Associated Lipocalin (a marker for renal tubular injury) 
NT-proBNP 
N-terminal pro-B-Type Natriuretic Peptide 
NVDO 
Dutch Association for the Study of Diabetes (in Dutch: Nederlandse Vereniging voor 
Diabetes Onderzoek) 
NYHA 
New York Heart Association 
OGTT 
Oral Glucose Tolerance Test 
PAH 
Para-aminohippuric acid 
PROactive 
PROspective pioglitAzone Clinical Trial In macroVascular Events 
PWA 
Pulse Wave Analysis 
RAS 
Renin-angiotensin-aldosterone system 
RCT 
Randomized Clinical Trial 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SGLT-2 
Sodium glucose linked transporter-2 
SMBG 
Self-Monitoring Blood Glucose 
SOP 
Standard Operating Procedure 
SPC 
Summary of Product Characteristics 
Sponsor 
The sponsor is the party that commissions the organization or performance of the research, 
for example a pharmaceutical company, academic hospital, scientific organization 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 subsidizing party 
sRAGE 
soluble receptor for advanced glycation end products  
SU 
Sulfonylurea derivative (antihyperglycemic agent) 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
T1DM 
Type 1 Diabetes Mellitus 
T2DM 
Type 2 Diabetes Mellitus 
TC 
Total Cholesterol 
TBW 
Total Body Water (variable derived with BIA) 
TG 
Triglycerides 
TGF- β1 
Transforming growth factor beta 1 
TNF-α 
Tumor necrosis factor alpha 
TSH 
Thyroid-Stimulating Hormone 
UAE 
Urinary Albumin Excretion 
VUMC 
VU University Medical Center 
WFI 
Water for injections 
WBP 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgegevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-wetenschappelijk 
Onderzoek met Mensen) 

                                         Renoprotective Effects of Dapagliflozin in Type 2 Diabetes 
 
 
 
 
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ABSTRACT 
Background: Worldwide, diabetic nephropathy or Diabetic Kidney Disease (DKD), is the most common cause 
of chronic and end-stage kidney disease. With the increasing rates of obesity and type 2 diabetes (T2DM), 
many more patients with DKD may be expected in the coming years. DKD is a multi-factorial condition, 
involving factors such as obesity, chronic hyperglycemia, advanced glycation end products, oxidative stress, 
pro-inflammatory cytokines, systemic- and glomerular hypertension. Large-sized prospective randomized 
clinical trials suggest that intensified glucose and blood pressure control, the latter especially by using agents 
that interfere with the renin-angiotensin-aldosterone system (RAS), may halt the progression of DKD, both in 
type 1 diabetes and T2DM. However, despite the wide use of angiotensin-converting enzyme inhibitors and 
angiotensin receptor blockers, a considerable amount of patients develop DKD during the course of diabetes, 
indicating an unmet need for renoprotective therapies. Sodium-glucose linked transporters (SGLT-2) inhibitors 
are novel glucose-lowering drugs for the treatment of T2DM. These agents seem to exert pleiotropic actions 
‘beyond glucose control’, including reduction of blood pressure and body weight. In addition, SGLT-2 inhibitors 
decrease proximal sodium reabsorption and decrease glomerular pressure and albuminuria in rodents and 
type 1 diabetes patients. In rodents, SGLT-2 inhibitors also improved histopathological abnormalities 
associated with DKD. To date, the potential renoprotective effects and mechanisms of these agents have not 
been sufficiently detailed in human type 2 diabetes. The current study aims to explore the clinical effects and 
mechanistics of SGLT-2 inhibitors on renal physiology and biomarkers in metformin-treated T2DM patients 
with normal kidney function.                      
Hypothesis: Treatment with the SGLT-2 inhibitor dapagliflozin, as compared to the sulfonylurea (SU) 
derivative gliclazide, may confer renoprotection by improving renal hemodynamics, and decreasing blood 
pressure and body weight in type 2 diabetes.  
We will test this hypothesis by addressing the following research objectives:  
Primary objective: what are the long-term effects (i.e. after 12-week drug exposure) of the SGLT-2 inhibitor 
dapagliflozin versus the SU gliclazide on renal hemodynamics (glomerular filtration rate (GFR) / effective renal 
plasma flow (ERPF)) in metformin-treated T2DM patients?  
Secondary objectives: renal tubular function, renal damage markers, blood pressure, heart rate, body 
anthropometrics, body fat, markers of inflammation, glycemic variables, lipid spectrum, microvascular function, 
arterial stiffness, systemic hemodynamics, cardiac autonomic nervous system function.  
Exploratory objectives: additional markers of renal function/damage, inflammation and (cardiovascular)-
biomarkers, deoxyribonucleic acid (DNA), gut microbiome, insulin sensitivity and measures of beta-cell 
function. 
 
Methods:  
Study Design: A phase 4, monocenter, randomized, double-blind, comparator-controlled, parallel-group, 
mechanistic intervention trial to assess the effect of 12-week treatment with the sodium-glucose linked 
transporters (SGLT)-2 inhibitor dapagliflozin versus the SU gliclazide on renal physiology and biomarkers in 
metformin-treated patients with type 2 diabetes mellitus (T2DM) 
Patients: Patients with T2DM (male and female; HbA1c 6.5-9%); aged 35-75 years; BMI ≥25 kg/m2 
Study Endpoints and methods: Renal hemodynamics, i.e. GFR and ERPF will be measured by the gold-
standard inulin- or iohexol and para-aminohippurate clearance methods, respectively, during both euglycemic- 
and hyperglycemic clamp; renal tubular function will be measured by 24-hour urine sodium, potassium, 
chloride, calcium, magnesium, phosphate and urea and glucose; markers of renal damage will include urinary 
albumin excretion, neutrophil gelatinase-associated lipocalin and kidney injury molecule-1; blood pressure will 
be measured using an automated oscillometric blood pressure device (Dinamap®); body anthropometrics, 
including body weight, height, body-mass index and waist circumference, and body fat contents (by bio-
impedance analysis) will be measured; blood samples will be obtained to determine glycemic variables, lipids 
and markers of inflammation; systemic hemodynamic variables (including stroke volume, cardiac output and 
total peripheral resistance) will be measured by continuous beat-to-beat hemodynamic monitor (NexFin®); 
heart rate will be determined by oscillometric blood pressure device; microvascular function will be measured 

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by capillary videomicroscopy and Laser Doppler; arterial stiffness will be assessed by applanation tonometry, 
(SphygmoCor®); additional urine, blood and feces will be collected for conditional determination of various 
markers of renal damage/function, inflammation, oxidative stress and (cardiovascular)-biomarkers and DNA; 
CANS will be measured by electrocardiography and NexFin®; insulin sensitivity will be measured by glucose 
infusion during the euglycemic clamp (M-value); beta-cell function will be measured by quantification of insulin 
secretion during the hyperglycemic clamp. 
 
Expected results: This active-comparator controlled mechanistic intervention study, using state-of-the-art 
methodology will expand our knowledge regarding the potential renoprotective effects of SGLT-2 inhititors in 
human T2DM. We hypothesize that SGLT-2 inhibitors may confer renoprotection by improving renal 
hemodynamics, and decreasing blood pressure and body weight, all important factors in the development of 
DKD. Ultimately, we expect that this study will contribute to guiding clinicians in their decision-making process 
of individualizing therapies for T2DM patients at risk for, or suffering from, DKD. 
 

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1. 
INTRODUCTION 
 
Diabetic Kidney Disease 
Globally, 382 million adults are living with diabetes (primarily type 2 diabetes mellitus; T2DM), and this estimate is 
projected to rise to 592 million by 2035 (1). Among the various diabetes-related microvascular and macrovascular 
complications, approximately 20−40% of patients with T2DM will ultimately develop diabetic kidney disease (DKD) 
(2). DKD is defined as kidney disease attributable to diabetes and the clinical diagnosis is usually made by combined 
finding of decreased glomerular filtration rate (GFR) and albuminuria (3). In parallel with the diabetes pandemic, 
DKD has now become the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) in 
developed countries (3). In the United States, the prevalence of DKD rose from 7.4% in 1988 to 9.6% in 2006, and 
this trend is forecast to continue (4). In addition, DKD results in high cardiovascular morbidity and mortality and 
decreased patients’ health-related quality of life (5,6). 
 
Current renoprotective treatment strategies 
Chronic hyperglycemia and related glomerular hyperfiltration are the main driving pathogenic forces of DKD in type 
1 diabetes (T1DM) patients. In contrast, in T2DM, the pathophysiology of DKD is more complex, since a cluster of 
cardiometabolic abnormalities (including obesity, systemic hypertension and dyslipidemia) may additionally 
contribute from early stages of the disease. In accordance with this complex etiology and based on landmark 
studies, clinical guidelines recommend a multifactorial treatment approach to improve renal outcome in T2DM, 
including lifestyle interventions (diet/exercise to achieve weight loss, smoking cessation) and pharmacological 
management of glucose, blood pressure and lipids are advocated (7–9). With respect to blood pressure control, 
angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) are especially 
recommended, since these inhibitors of the renin-angiotensin-aldosterone system (RAS) have demonstrated 
renoprotective effects beyond their ability to lower blood pressure. However, even targeting multiple risk factors 
simultaneously, residual risk for DKD development remains. Indeed, in the Steno-2 trial, although intensive 
multifactorial treatment of 160 T2DM patients with persistent microalbuminuria had sustained beneficial effects on 
renal complications, 25% of patients developed DKD nevertheless. Also, although rates of diabetes-related 
complications have declined substantially in the past two decades, the smallest decline was observed for ESRD 
(−28.3%; 95% CI: −34.6 to −21.6), leaving a tremendous burden of renal disease in this patient group (10). These 
numbers highlight the need for novel strategies or new therapeutic drugs to improve renal outcome in T2DM. 
 
New management options in T2DM: SGLT-2 inhibitors 
Sodium-glucose linked transporters (SGLT) are a main class of glucose transporters involved in glucose 
homeostasis. The SGLTs are located at the brush border membrane of the gut epithelium to enhance glucose 
absorption, and apical membrane of renal tubular cells to facilitate glucose reabsorption after glomerular filtration 
(11). Among the 12 SGLT family-members, the high-capacity low-affinity SGLT-2 is abundantly expressed in the 
S1 segment of the proximal renal tubule and accounts for the vast majority (90%) of filtered glucose reuptake, by 
coupling glucose transport to the electrochemical sodium gradient (12–14). The low-capacity high affinity SGLT-1 
is mainly responsible for glucose absorption from the small intestine, while contributing to about 10% of renal 
glucose reabsorption in the straight S3 segment of the proximal renal tubule (13). Healthy human kidney’s filter 
approximately 160-180 g of glucose daily, almost all of which is reabsorbed by SGLT-2 (90%) and SGLT-1 (10%) 
(14). The maximal capacity of the proximal tubule (Tm) for glucose-reabsorption varies among individuals and 
averages 375 mg/min for healthy subjects − reflecting a plasma glucose concentration of 10-11 mmol/l (14). In 
animal models of diabetes, the rate of renal glucose reabsorption in the proximal tubule is increased (15–18), 
whereas similar results were seen in humans with poorly controlled T1DM (19) and T2DM (20). On a molecular 
level, the increased renal glucose reabsorption may be explained by increased SGLT-2 and GLUT-2 expression 
and activity (15–17,21). Thus, in diabetes, a higher than normal amount of glucose is reabsorbed by the kidneys, 
which contributes to and sustains hyperglycemia (12). 
 
Since renal glucose hyperreabsorption has been considered a maladaptive response to ambient hyperglycemia, 
SGLT inhibition was regarded a therapeutic target for glycemic control in diabetes (14). Rossetti et al were the first 
to show that phlorizin − a beta-D-glucoside isolated from the bark of the apple tree − could function as a non-
selective SGLT-1/2 inhibitor (22). In a proof of concept study in partly pancreatectomised rats (a model for T2DM), 
subcutaneous injections of phlorizin induced glucosuria and normalized both fasting and postprandial glucose levels 
(22). Phlorizin has to be administered by injection and incites significant SGLT-1-mediated gastrointestinal side 

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effects, the compound has therefore not been developed as treatment for T2DM (12). However, results obtained 
with phlorizin have provided the basis for the development of specific SGLT-2 inhibitors. As a result, several SGLT-
2 inhibitors have been successfully introduced, which have proven to be efficacious in reducing HbA1c levels 
(0.66% reduction versus placebo; similar efficacy compared to active comparators), providing a new insulin-
independent approach for the treatment of T2DM (23), and potentially T1DM. Important side effects of SGLT-2 
inhibitors comprise a modest increase in urinary and genital tract infections (23). Hypoglycemia rates for this novel 
drug class are similar to placebo. Currently, three SGLT-2 inhibitors −dapagliflozin, canagliflozine and 
empagliflozin− were granted marketing authorization by the European Medicines Agency. 
Animal and human data indicate that SGLT-2 inhibitors may prevent the onset and progression of DKD. Treatment 
of db/db mice (a mouse model of obesity) with nonspecific (24) and specific SGLT-2 inhibitors (25), improved 
albuminuria and histological features of DKD. Similar findings were noted in streptozotocin induced diabetes (26–
28) and Akita mice (29) (both models of T1DM). Also, in BTBR ob/ob mice (30) and T2DN rats (31) (models for 
T2DM), treatment with a SGLT-2 inhibitor resulted in decreased albuminuria and histopathological improvements, 
indicating decreased renal injury. In human registration phase-III trials, SGLT-2 inhibitors were associated with 
reduced albuminuria in T2DM (32,33). These data were confirmed and expanded in a pooled analysis of four phase-
III trials of empagliflozin, which showed a significant reduction in microalbuminuria by 30% after 24 weeks of 
treatment (34). Larger renal outcome studies with SGLT-2 inhibitors are currently ongoing which focuses on 
albuminuria and estimated GFR. 
 
Mechanisms of SGLT-2 inhibitors 
The exact mechanism underlying the beneficial effects of SGLT-2 inhibitors on the renal system in T2DM remains 
to be elucidated. Phase-III trials demonstrate a reduction in body weight of about 1.80 kg −most likely due to osmotic 
diuresis and loss of calories− and systolic blood pressure (4.45 mmHg reduction versus active comparators) (23). 
In addition, preclinical studies (27–29,35) and human T1DM data indicate that SGLT-2 inhibitors reduce glomerular 
hyperfiltration (36). Hyperfiltration in diabetes may be explained by proximal sodium hyperreabsorption resulting in 
reduced sodium delivery at the distally located macula densa, leading to afferent vasodilation and increased 
glomerular pressure via tubuloglomerular feedback (37). Blockage of SGLT-2 may decrease proximal sodium 
reabsorption, thereby potentially restoring this maladaptive response (36,38). Interestingly, during the first weeks of 
SGLT-2 inhibitor treatment, the estimated GFR decreases in T2DM patients with moderate renal impairment, 
followed by long-term stabilization (32,38,39). Comparable changes in GFR have been observed for RAS inhibitors 
and were proven to be beneficial (38). Furthermore, since SGLT-2 inhibitors increase RAS activity comparable 
to other diuretic agents, it has been suggested that combined SGLT-2 and RAS inhibition yield additive 
renoprotective effects (36). SGLT-2 inhibitors may also diminish glucose influx of proximal renal tubular cells, 
thereby potentially reducing glucotoxicity and consequent inflammation and fibrosis in the diabetic kidney 
(38,40,41). Finally, SGLT-2 inhibitors increase the −urine measured− potential renoprotective enzyme ACE2 (42). 
 
Conclusion 
In conclusion, the global prevalence of DKD is increasing. Current pharmacological management to prevent renal 
disease in T2DM consists of antihyperglycemic agents, RAS-interfering agents and statins. However, these 
interventions do not prevent the development and progression of DKD in many patients, indicating an unmet need 
for additional renoprotective therapies. Based on preclinical and emerging clinical studies in patients with diabetes, 
SGLT-2 inhibitors are regarded a promising option to reduce the DKD burden. Conversely, to date, the potential 
renoprotective mechanisms underlying these beneficial effects have not been detailed in T2DM. 
 
 

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2. 
STUDY OBJECTIVES 
 
Hypothesis 
Treatment with the SGLT-2 inhibitor dapagliflozin, as compared to the sulfonylurea (SU) derivative gliclazide, 
may confer renoprotection by improving renal hemodynamics, and decreasing blood pressure and body weight 
in type 2 diabetes.  
Primary Objective 
 
To investigate the effects of 12-week treatment with the SGLT-2 inhibitor dapagliflozin (10 mg QD) versus the 
SU gliclazide (30 mg QD) on renal hemodynamics in metformin-treated T2DM patients, measured as: 
 
GFR (measured by the inulin or iohexol-clearance technique)  
 
Effective renal plasma flow (ERPF; measured by the para-aminohippurate acid (PAH) clearance 
technique) 
 
Secondary Objectives 
To investigate the effects of 12-week treatment with the SGLT-2 inhibitor dapagliflozin (10 mg QD) versus the 
SU gliclazide (30 mg QD) on 
 
Renal tubular function, measured as: 
o 24-h urine sodium-, potassium-, chloride-, calcium-, magnesium-, phosphate-, urea and glucose 
 
Renal damage, measured by urine biomarkers as: 
o UAE (Glomerular) 
o Neutrophil gelatinase-associated lipocalin (NGAL) and Kidney injury molecule-1 (KIM‐1) (Tubular) 
 
Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP)  
(measured by oscillometric blood pressure device)  
 
Heart rate (measured by oscillometric blood pressure device) 
 
Body anthropometrics: waist circumference, height, body weight and body mass index (BMI) (using 
the formula: weight (in kg) / height2 (in m)), measured by a tape measure and calibrated weighing 
scale respectively 
 
Body fat content, measured by bio-impedance analysis (BIA) 
 
Marker of inflammation: high sensitivity C-reactive protein (hs-CRP) 
 
Glycemic variables: Glycated hemoglobin (HbA1c) and fasting plasma glucose (FPG)  
 
Lipid spectrum (triglycerides (TG), total-cholesterol (TC), high density lipoprotein cholesterol (HDL-
C), low density lipoprotein cholesterol (LDL-C) and free fatty acids (FFA)) 
 
Microvascular function, measured by capillary videomicroscopy and Laser Doppler techniques 
 
Arterial stiffness (Pulse Wave Analysis), measured by applanation tonometry  
 
Systemic hemodynamic variables (SBP, DBP, MAP, heart rate (HR), stroke volume (SV), cardiac 
output (CO)/-index (CI), and total systemic vascular resistance (TPR)) derived from non-invasive 
beat-to-beat finger blood pressure measurements 
 
Cardiac autonomic nervous system (CANS) function, as derived from electrocardiographic (ECG) and 
non-invasive beat-to-beat finger blood pressure measurements (NexFin®) measured as: 
o Heart rate variability (HRV); Cardiovascular autonomic reflex tests (CARTs) 
 
Exploratory Objectives 
To investigate the effects of 12-week treatment with the SGLT-2 inhibitor dapagliflozin (10 mg QD) versus the 
SU gliclazide (30 mg QD) on: 
 
Complementary markers of renal function/damage (plasma cystatin C, fibroblast growth factor 23 
(FGF23), parathyroid hormone (PTH), soluble Klotho, urinary transforming growth factor-β1 (TGF-β1), 
collagen type IV, nephrin, podocin, microparticles, uric acid, 8-hydroxy-2' -deoxyguanosine (8-OHdG), 
Calcitriol, Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α) 
 
Additional markers of inflammation (interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1) 
 
(Cardiovascular)-biomarkers (N-terminal pro-B-Type Natriuretic Peptide (NT-proBNP), Brain 
Natriuretic Peptide (BNP), Atrial Natriuretic Peptide (ANP), urinary angiotensinogen, plasma renin 

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activity (PRA), angiotensin II, aldosterone, catecholamines, insulin, C-peptide, soluble receptor for 
advanced glycation end products (sRAGE) 
 
Deoxyribonucleic acid (DNA) (to study the influence of genetic factors on the parameters measured 
and the response to SGLT-2 inhibitor) 
 
Gut microbiome composition 
 
Insulin sensitivity as derived from the euglycemic clamp 
 
Beta-cell function measures as derived from the hyperglycemic clamp 
The extent of these complementary measurements is conditional to available budget. 

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3. 
STUDY DESIGN 
 
A phase 4, monocenter, randomized, double-blind, comparator-controlled, parallel-group, mechanistic 
intervention trial to assess the effect of 12-week treatment with the sodium-glucose linked transporters (SGLT)-
2 inhibitor dapagliflozin versus the sulfonylurea (SU) derivative gliclazide on renal physiology and biomarkers 
in metformin-treated patients with type 2 diabetes mellitus (T2DM) 
 
Figure 1: Study Outline 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
BL = Baseline tests performed to assess study objectives at baseline 
LT = Long-term follow-up tests performed to assess study objective at end-point (i.e. after 8 weeks of intervention)  
R = Randomization 
S 
= Screening visit and eligibility assessment 
Sa =  Safety visit and measurements  
 
 

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4. 
STUDY POPULATION 
 
Subjects 
Patients with previously diagnosed T2DM according to American Diabetes Association (ADA) criteria (43) . 
 
Inclusion criteria 
 
Caucasian*  
 
Both genders (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) 
 
Age: 35 - 75 years 
 
BMI: >25 kg/m2 
 
HbA1c: 6.5 – 9.0% Diabetes Control and Complications Trial (DCCT) or 48 - 86 mmol/mol International 
Federation of Clinical Chemistry (IFCC) 
 
Treatment with a stable dose of oral antihyperglycemic agents for at least 3 months prior to inclusion 
 
Metformin monotherapy 
 
Combination of metformin and low dose SU derivative** 
 
Hypertension should be controlled, i.e. ≤140/90 mmHg, and treated with an ACE-I or ARB (unless 
prevented by side effect) for at least 3 months.  
 
Albuminuria should be treated with a RAS-interfering agent (ACE-I or ARB) for at least 3 months. 
 
Written informed consent 
 
*   In order to increase homogeneity 
** In order to accelerate inclusion, patients using combined metformin/SU derivative will be considered. In these patients, a 12 week wash-out period of 
the SU derivative will be observed, only when combined use has led to a HbA1c <8% at screening. Subsequently, patients will be eligible to enter the 
study, now using metformin monotherapy, provided that HbA1c still meets inclusion criteria. 
 
 
Exclusion criteria 
 
History of unstable or rapidly progressing renal disease 
 
Macroalbuminuria; defined as ACR of 300mg/g. 
 
Estimated GFR <60 mL/min/1.73m2 (determined by the Modification of Diet in Renal Disease (CKD-
EPI) study equation) 
 
Current/chronic use of the following medication: TZD, SU derivative, GLP-1RA, DPP-4I, SGLT-2 
inhibitors, glucocorticoids, immune suppressants, antimicrobial agents, chemotherapeutics, 
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. 
 
Volume depleted patients. Patients at risk for volume depletion due to co-existing conditions or 
concomitant medications, such as loop diuretics should have careful monitoring of their volume status. 
 
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, head-ache or back 
ache). However, no such drugs can be taken within a time-frame of 2 weeks prior to renal-testing 
 
History of diabetic ketoacidosis (DKA) requiring medical intervention (eg, 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 
 
Complaints compatible with neurogenic bladder and/or incomplete bladder emptying (as determined 
by ultrasonic bladder scan) 
 
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 
 
 (Unstable) thyroid disease; defined as fT4 outside of laboratory reference values or change in 
treatment within 3 months prior to screening visit 
 
History of or actual malignancy (except basal cell carcinoma) 

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 
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 

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5. 
STUDY DRUGS 
This paragraph will briefly address the different agents. For more information we refer to the included Summary 
of Product Characteristics (SPCs). 
 
Study Medication 
In addition to their ongoing oral antihyperglycemic medication (metformin only) and drugs for cardiovascular 
risk management (e.g. RAS‐interfering agent and/or statin), subjects will be assigned to oral treatment with 
either encapsulated dapagliflozin or gliclazide (see Table 2). 
 
Table 2: Overview treatment allocation 
Group 
Treatment 
Number of subjects 
I 
Dapagliflozin 10 mg QD 
22 
II 
Gliclazide 30 mg QD 
22 
QD, once daily 
 
Dapagliflozin (Forxiga® by AstraZeneca) 
The SGLT-2 inhibitor Forxiga (dapagliflozin tablet à 10 mg) will be used, i.e. a yellow, biconvex, approximately 
1.1 x 0.8 cm diagonally diamond-shaped, film-coated tablets with “10” engraved on one side and “1428” 
engraved on the other side (44). The agent will be purchased commercially and masked by encapsulation (see 
below), such that this drug will be visually undistinguishable from encapsulated gliclazide.. Following 
randomization, participants will start using dapagliflozin 10 mg once daily (QD), titration is not necessary. 
Dapagliflozin will be taken orally, with or without food, preferably in the evening. 
 
Gliclazide (Diamicron® by Servier) 
A generic SU derivative of gliclazide (tablet à 30 mg) will be used, i.e. a white oblong tablet of 10 mm x 5 mm 
x 2,5 mm, with "DIA 30" engraved on one side and 
on the other side (45). The agent will be purchased 
commercially and masked by encapsulation (see below), such that this drug will be visually undistinguishable 
from encapsulated dapagliflozin. Following randomization, participants will start using gliclazide 30 mg QD, 
titration is not necessary. Gliclazide will be taken orally, with or without food, preferably in the evening. 
 
Test agents 
Following the meal and during the renal-test procedures, three medicinal products will be used.  
 Renal test = inulin or iohexol/PAH-clearance. Inulin is approved by the Austrian drug authority, iohexol is 
produced by GE Healthcare and marketed under the brand name Omnipaque, and PAH is produced by 
Bachem in GMP-grade and the product will undergo quality assurance prior to administration (46). These 
agents are widely used for the determination of renal hemodynamics. 
 Short-acting insulin (Novorapid®, obtained from Novo Nordisk) will be infused during the euglycemic clamp. 
The drug is widely used in patients with diabetes and is FDA approved. 
 Arginine 10% HCl will be infused during the hyperglycemic clamp after which insulin response will be 
measured. It is produced by our in house pharmacy and widely used in trials to determine beta cell function. 
Detailed information regarding these study agents can be found in Appendix B and in corresponding SPCs. 
 
Randomization 
The blinded treatment assignment will be performed by the trial pharmacists of VU University Medical Center 
(who have ample experience with this process), in order to maintain blinding for investigator, site personnel 
and patients: 
 
Block randomization will be performed with 
o 
Allocation ratio of 1:1 
o 
Block-size of 4 
o 
Stratification for ACE-I or ARB (yes/no) therapy 
It must be noted that the ‘Apotheek A15’ will provide randomization lists for encapsulated dapagliflozin and 
gliclazide for this trial. 
 

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Labeling, Drug dispensation and Accountability 
All trial drugs and the Total Dispensing Unit lists will be received and handled by the trial pharmacist. Patients 
will receive a capsule containing either dapagliflozin or gliclazide . To  be undistinguishable and packed 
blinded, the Total Dispensing Unit list is needed for dispensing trial products. Dapagliflozin and gliclazide will 
be encapsulated by an external GMP (Good Manufacturing Practice)-certified organization (A15 pharmacy). 
The trial products will be provided in labeled and specific boxes packages with participant identifying 
information to our institutional trial pharmacist who, subsequently, will store the trial drugs 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 visit (visit 2, see Appendix 
A), combined with extensive instructions on use and administration of the study drugs. 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 day (visit 3; see Appendix A), 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. 
 
Drug storage 
Both dapagliflozin (44) and gliclazide (45) do not require any special storage conditions. Nevertheless, when 
not distributed to participants, drugs will be stored at the institutional trial pharmacy. Keeping a temperature 
log document, 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). Storage facilities will be 
checked frequently (temperature at least every working day). 
 
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 VU Medical Center. 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 VU University Medical Center (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 10. 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 data-base lock (which will only occur after 
medical/scientific review), the blind will be broken by the trial pharmacist. 

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6. 
STUDY OUTLINE 
 
Recruitment 
Subjects will be recruited by researcher physicians involved in this trial using methods that are established 
practice for all human studies in the Diabetes Center 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, we will identify potential 
participants who 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 VUMC or affiliated hospitals / pharmacies 
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 (visit 1 and T1) 
After giving extensive oral and written information, 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, self-
reported birth weight), complete physical examination, drawing blood for hematologic and biochemistry testing 
(Full blood count, glucose, HbA1c, creatinine, AST, ALT, gamma-glutamyl transferase (GGT), alkaline 
phosphatase (ALP) and thyroid-stimulating hormone (TSH)), urine screening, urine creatinine and albumin,  
and a 12-lead electrocardiogram (ECG). 
 
Table 3: Timetable of end-point assessments performed at baseline and 12-week drug intervention 
 
 
Visit 2 
Visit 3 
Visit 4 
0700 
 
 
 
Renal Protocol and 
Cardiovascular Tests  
 
 
 
Renal Protocol and 
Cardiovascular Tests  
 
0800 
Safety evaluation + measurements 
0900 
 
 
1000 
 
 
1100 
 
 
1200 
 
 
1300 
 
 
1400 
 
 
1500 
 
 
1600 
 
 
1700 
Randomization + Instructions 
 
 
 
 

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Postvoid residual urine volume will be determined by ultrasonic bladder scan. In females, the menopausal 
status will be verified by history taking (no menses > 1 year) and in case of doubt measurement of FSH-level 
(cut-off defined as >31 U/L). If subjects are eligible, the investigator will inform them within two weeks after 
screening (T1, see Appendix A). After inclusion, participants will enroll in a 4‐week run‐in period to allow for 
study effects. 
 
Endpoint visits: Visit 2 (baseline) and Visit 4 (long-term follow-up) 
During the baseline measurements (visit 2; Table 3 and Appendix A), various tests will be performed to obtain 
baseline values for the predefined endpoints. The baseline measurements of the renal system, as well as the 
cardiovascular assessments, will be performed during one test visit and serve as reference values. The 
endpoint assessments will be repeated during visit 4 (Table 3 and Appendix A).  
 
Visit 2 (baseline assessments) 
Seven  days prior to visit 2, subjects will adhere to a `normal-salt’ (9 - 12 grams or 150 - 200 mmol per day) 
and protein (1.5 - 2.0 g/kg per day) diets, in order to minimize variation in renal physiology due to salt and 
protein intake (47,48). Participants will abstain from alcohol (24 hours), caffeine (12 hours) and nicotine (12 
hours) and heavy exercise prior to and during visits 2 (49). One day prior to the testing day, subjects will collect 
24-hour urine. 
Following an overnight fast (10 hours prior to the start of investigation), participants will be instructed to drink 
500 mL of water, but to delay all morning-time medication (apart from metformin) until conclusion of the 
examination day. Subjects will arrive at the Clinical Research Unit (CRU) at 07.30 AM. After taking subjects 
history, current weight and blood pressure, intravenous catheters will be placed in both forearms after which 
blood and urine will be collected (see Figure 2). Then, subjects will assume a semi-recumbent position 
throughout the duration of the visit and after a resting period, an automatic blood pressure measurement is 
performed. Thereupon the combined renal and clamp protocol will commence (Chapter 7, sections A and B). 
During the first equilibration period, body fat content assessment and the cardiovascular tests are carried out. 
 
Figure 2: Schematic representation of measurements during visits 2 and 4 
 
Figure 2. During the renal testing days, renal hemodynamics will be evaluated in fasting state, during an 
euglycemic-hyperinsulinemic clamp and a hyperglycemic clamp. Plasma glucose levels will be kept at 5.0 
mmol/L and 15.0 mmol/L, respectively. Total duration of the visits: approximately 10 hours. 
 
Safety and tolerability: Telephone consultation 2 and Visit 3 (follow-up)  
The 12-week intervention study includes a telephone consultation (T2 at week 2) and a follow-up visit (Visit 3 
at week 6) (see Table 3 and Appendix A). During T2 and visit 3, participants will be asked about the occurrence 
of adverse events and the intake of study drugs will be checked. Furthermore, recent history will be taken, 
physical examination/anthropometric measurements performed and blood and urine samples collected.  
 

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Early-term assessments 
At any time during the course of the study, all subjects have the right to withdraw without providing a reason, 
as required by ICH-GCP and Dutch laws. When participants decide to withdraw their participation, they will be 
asked to participate in ‘early-term assessments’. During these assessments, some end-point parameters will 
be measured, which can possibly be used for final data analyses (see Appendix A). 
 
Withdrawal from study 
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 detrimental 
to the subjects well-being 
 
The study physician can be guided by the criteria above, but may discontinue participation at any time based 
on his/her clinical judgment.  
 
Study Completion  
The study will be considered completed for an individual participant when he/she completes the final tests (visit 
4, i.e. the last day of the 12-week end-point assessments). The study as a whole will be considered completed 
when all planned randomized subjects have completed the treatment phase, followed by the final 
measurements.

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7. 
STUDY PROCEDURES 
This paragraph provides short definitions of the study procedures used in this study. Detailed information or 
‘standard operation procedures’ (SOPs) for most of the procedures can be found in the respective appendices. 
 
A. 
Renal protocol (visit 2 and 4): 
During this protocol a combined inulin or iohexol/PAH-clearance technique will be performed to examine 
the GFR and ERPF respectively (36,50–52). In order to examine tubular function, sodium, potassium, 
chloride, calcium, magnesium, phosphate, urea and glucose will be measured in urine. In addition, urine 
osmolality will be determined. Moreover, biomarkers of renal damage (UAE, NGAL and KIM-1) will be 
measured in urine (see Appendix C1).  
 
B. 
Clamp protocol  
A combined hyperinsulinemic-euglycemic and hyperglycemic clamp will be performed. During the 
euglycemic clamp insulin will be infused at 40 mU/m2.min and glucose will be kept at 5 mmol/l by variable 
glucose 20% infusion. This technique allows for renal measurements during normoglycemia in all 
participants. After 3 hours of insulin infusion and an one hour rest period for exogenous insulin to be cleared, 
glucose will be infused in order to reach a plasma concentration of 15 mmol/l. In this matter, renal tests can 
be performed under stable hyperglycemia (see Appendix C2). From the euglycemic clamp, insulin sensitivity 
will be determined from the amount of glucose infused (M-value). Blood will be stored during hyperglycemia 
in order to be able to obtain measures of beta-cell function, including 1st phase insulin secretion, 2nd phase 
insulin secretion and arginine-induced insulin secretion. 
 
C. 
Blood pressure (visit 1, 2, 3 and 4):  
While the auscultatory method (sphygmomanometry) might be considered the gold standard, it is 
hampered by intra- and inter-observer variability. Because blood pressure measurements will be 
performed on multiple occasions, we will use an automatic oscillometric device (Dinamap®, GE 
Healthcare) during the entire study to reduce possible bias (53,54). 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 (53,54). 
Should the automated technique not be possible or yields erroneous results, the auscultatory technique 
will be used, using a sphygmomanometer according to methods of Riva-Rocci. SBP is defined as the 
appearance of vascular tones (Korotkoff sound 1), DBP as the disappearance (Korotkoff sound 5) (54). 
To assess the differences in automatic versus auscultatory techniques, we will perform the latter in the 
fasted state during the baseline- and long-term follow-up assessments.  
 
D. 
Body composition (visit 1, 2, 3 and 4): 
Waist circumference will be assessed by a constant tension tape 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 (55). 
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. 
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, jewelry 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. 
BMI will be derived from height and body weight by using the mathematical formula: BMI = [kg] / [m]2. 
 
 
E. 
Body fat content: (visit 2 and 4): 
To measure body fat content we will use the tetrapolar Soft Tissue Analyzer® (STA, Akern, Florence, 
Italy), which can perform BIA. This technique measures the resistance in the body tissues when a 50 

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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 (56). Extracellular 
water (ECW) as a percentage of total body water (TBW) and body cell mass (BCM) will be calculated 
independent of weight and height. 
 
F. 
Systemic hemodynamics (visit 2 and 4):  
To test drug-related changes on the cardiovascular system we will measure hemodynamic variables 
(including stroke volume, cardiac output, total peripheral resistance) non-invasively. For this, an automated, 
beat-to-beat blood pressure and ECG recording monitor (Nexfin®, BM Eye, Amsterdam, The Netherlands 
(57) will be used.   
 
G. 
Heart rate (visit 1, 2, 3 and 4):  
Heart rate will be measured using the automated oscillometric blood pressure device. Should the 
automated technique not be possible or yield erroneous results, heart rate will be assessed by counting 
the radial pulse rate during 30 seconds and multiplying the outcome by two. 
 
H. 
Microvascular function (visit 2 and 4): 
Microvascular function will be measured by nail fold skin capillary density on the middle finger, before 
and after 4 minutes of arterial occlusion with a digital cuff, yielding data on baseline capillary density and 
post-occlusive reactive hyperemia, respectively. Moreover, vasomotion analyses will be performed, in 
which skin blood flow will be measured using a Laser Doppler probe (58,59) (see Appendix D). 
 
I. 
Pulse Wave Analysis (PWA) (visit 2 and 4):  
Applanation tonometry using the SphygmoCor® system (Atcor Medical, West Ryde, Australia) will be 
performed to measure variables of arterial stiffness. For PWA, the tonometer will be applied to the radial 
artery of the non-dominant arm. The SphygmoCor® system will calculate the Augmentation index (AIx) 
from the pulse waves (see Appendix E) (60–62).  
 
J. 
CANS function (visit 2 and 4):  
To test drug-related changes in the autonomic nervous function and their impact on the cardiovascular 
system we will measure resting heart rate variability (HRV) (63) and perform CARTs, a modified battery of 
the Ewing tests (64). Using an automated, beat-to-beat blood pressure and ECG recording monitor (Nexfin®, 
BM Eye, Amsterdam, The Netherlands (57) heart rate and blood pressure will be recorded during: resting, 
taking deep breaths, exhalation into a mouth-piece with a measurable resistance (Valsalva maneuver) and 
standing up from a supine position. Information regarding changes in parasympathetic and sympathetic 
nervous system function can be derived from the measurements using dedicated software (65–67) (see 
Appendix F).  
 
K. 
ECG (visit 1): 
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 (68). The cardiac vector and any changes in ECG morphology will be assessed.  
 
L. 
Other (potential) analytes in blood/urine:  
In addition to the determination from blood samples as outlined for the above-mentioned procedures, 
additional blood and plasma samples will be collected and stored to determine relevant (emerging) 
biomarkers of renal / cardiovascular damage. The extent of these additional measurements are 
conditional to available budget: 
 
 
 
 
 
Renal damage 
Conditional: Plasma cystatin C, FGF23, PTH, calcitriol, soluble Klotho, 
urinary TGF-β1, collagen type IV, nephrin, podocin, microparticles, 
urinary 8-OHdG, urinary TNF-α 

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(Cardiovascular) 
biomarker 
Conditional: NT-proBNP, BNP, ANP, PRA, angiotensin II, angiotensin 
1-7, aldosterone, endothelin, catecholamines, insulin, C-peptide, GLP-
1, glucagon, GIP, sRAGE, urinary angiotensinogen, uric acid 
Inflammation 
hsCRP, Conditional: interleukin 6 (IL-6), PAI-1, urinary MCP-1 
Genetic sampling 
Conditional: DNA 
 
M. 
Fecal examinations:  
Each participant will collect a stool sample before the baseline tests and once before the long-term 
follow-up tests. At home, one feces sample must be stored at room temperature and one in the freezer 
and when arrived at the CRU, the feces will be stored at -20⁰C and -80⁰C, respectively. Stool samples 
will be stored for possible analysis of biomarkers, including calprotectin (69). Since it is becoming 
increasingly clear that the gut microbiome might be involved in the development of obesity and T2DM 
(70) and CKD disease (71,72), we will also store and analyze feces for microbial content (94). (see 
Appendix G). 
 

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8. 
LABORATORY ASSESSMENTS 
 
Sample collection and testing 
During the study blood and urine will be collected at various time points (see Appendix A). Blood sampling will 
be performed by drawing blood from intravenous catheters during the test days, or, when no catheter is placed 
(e.g. during the screening (V1) and safety/follow-up visit (V3), by venipuncture. 24-hour urine and spot urine 
will be collected using designated non-sterile containers.  
All procedures regarding biosample collection as well as the necessary administration will be in line with ICH-
Good Clinical Practice (GCP)/ Good Laboratory Practice (GLP). 
 
Screening and Safety Measurements 
During the screening visit (V1) hematology (hemoglobin (Hb), hematocrit (Ht), leucocytes and thrombocytes) 
and chemistry (glucose, HbA1c, AST, ALT, GGT, ALP, creatinine, TSH and FSH (in females only, in case of 
doubt)). In addition, urine will be screened using a urine test strip. These parameters will be used to assess 
eligibility. 
At visit 3, i.e. after 6 weeks of treatment blood samples will be obtained to assess hematology (Hb, Ht, 
leucocytes and thrombocytes) and chemistry (sodium, potassium, creatinine, BUN, AST, ALT, GGT, ALP) 
markers.  
 
Endpoint Measurements 
Throughout the study blood will be drawn for endpoint assessments at various time points. Many biochemical 
and hematological analyses will be performed directly. Blood for biomarkers will be centrifuged and 
plasma/serum will be stored at -80°C. An overview of the different endpoint laboratory measurements is 
merged with the efficacy measurements in Chapter 9. 
 
Blood volume 
The total amount of drawn blood is detailed in Table 4. 
 
Table 4: Description of estimated drawn blood volume 
 
*Storage samples for determination of additional (cardiovascular)biomarkers connected to the study objectives (depending on study 
budget) 
 
 
 
 
 
 
Test 
Screening 
-4 wk 
Baseline 
0 wk 
Safety 
6 wk 
Long-term  
12 wk 
Total 
Screening 
11  mL 
  
 
  
11 mL 
Control visit 
 
  
7  mL 
  
7 mL 
Chemistry/Hematology 
 
25 mL 
 
25 mL 
50 mL 
Renal protocol 
 
52 mL 
 
52 mL 
104 mL 
Clamp protocol 
 
90 mL 
 
90 mL 
180 mL 
Storage samples* 
 
71 mL 
 
71 mL 
142 mL 
Genetic sampling 
 
6 mL 
 
  
6 mL 
Total 
11 mL 
244 mL 
7 mL 
238 mL 
500 mL 

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9. 
EFFICACY MEASUREMENTS 
 
Primary Efficacy Measures 
Renal hemodynamics 
Inulin-, iohexol- and PAH- measurements will be performed in a fasting state and during euglycemic- and 
hyperglycemic clamps in a semi-recumbent position at visits 2 (baseline) and 4 (week 12). Both plasma and 
urine samples will be collected and stored for determination of inulin or iohexol/PAH levels, which will be used 
for the calculation of the GFR and ERPF. This can be corrected for hematocrit to calculate renal blood flow 
(RBF). Filtration fraction (FF) can be calculated by dividing GFR by ERPF. In addition, effective renal vascular 
resistance (ERVR) can be calculated (for specifics, see Appendix C1). 
 
Secondary Efficacy Measures 
Renal Tubular Function 
Prior to visits 2 (baseline) and 4 (week 12), subject will collect 24-hour urine for measurements of sodium, 
potassium, chloride, calcium, magnesium, phosphate, urea and glucose. Furthermore, urine osmolality is 
measured. These measurements are all markers for renal tubular function. 
 
Renal damage 
Prior to visits 2 (baseline) and 4 (week 12), subject will collect 24-hour urine for measurement of 
microalbuminuria. In addition, urine will be stored for subsequent measurements of NGAL and KIM-1 (tubular 
damage).  
 
Blood Pressure  
Oscillometric measurements for assessment of blood pressure will be performed at visits 2 (baseline), 3 and 
4 (week 12). The mean of the last 2 out of 3 measurements will be used as final outcome parameter. 
 
Heart rate 
Oscillometric measurements for assessment of heart rate will be performed at visits 2 (baseline), 3 (week 6) 
and 4 (week 12). The mean of the last 2 out of 3 measurements will be used as outcome parameter. 
 
Body anthropometrics and Body fat content 
Measurements for anthropometrics and body composition will be performed at visits 2 (baseline), 3 (week 6) 
and 4 (week 12). Weight will be recorded using a weighing scale, waist circumference will be measured using 
a tape measurement and body fat content will be assessed by BIA. The latter will produce different variables 
independent of weight or height (ECW, TBW and BCM). 
 
Glycemic variables, Lipid spectrum, Inflammation, Biochemistry and Hematology 
Blood will be drawn for direct determination of fasting glucose, HbA1c, FPG, TG, TC, HDL-C,LDL-C, FFA and 
hs-CRP at visits 2 (baseline), 3 (week 6) and 4 (week 12). Furthermore, different end-point assessments at 
visits 2 (baseline) and 4 (long-term; week 12) will serve as safety measurement at visit 3 (week 6). To this end, 
blood chemistry (sodium, potassium, creatinine, ALT, AST, GGT, ALP) and hematology (Hb, Ht, leukocytes, 
thrombocytes) will be determined.   
 
Microvascular function 
Measurements for assessing microvascular function will be performed at visits 2 (baseline) and 4 (week 12). 
Capillary videomicroscopy will be used to assess capillary density and capillary recruitment (after arterial 
occlusion). Laser Doppler will be applied to measure skin blood flow to define microvascular vasomotion (for 
specifics, see Appendix D). 
 
Arterial stiffness (PWA) 
Applanation tonometry will be performed at visits 2 (baseline) and 4 (week 12) in order to measure PWA (a 
collection of variables: augmentation pressure, augmentation index, ejection duration and subendocardial 
viability ratio; for specifics, see Appendix E). 
 
 
 
 

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Systemic hemodynamics 
Non-invasive beat-to-beat finger blood pressure measurements will be performed at visits 2 (baseline) and 4 
(week 12) for measurement of systemic hemodynamic variables (systolic and diastolic blood pressure, mean 
arterial pressure, heart rate, stroke volume, cardiac output/-index, systemic total peripheral resistance). 
 
CANS function 
ECG and non-invasive beat-to-beat finger blood pressure measurements will be performed at visit 2 (baseline) 
and 4 (week 12) during a time-period of 7 minutes following rest of at least 10 minutes in a semi-recumbent 
position, for determination of resting heart rate variability (HRV) defined by the High Frequency (HF) 
component, Low Frequency (LF) component and their ratio, as attained from Fourier analysis, in order to 
assess changes in the balance of the parasympathetic and sympathetic nervous system on resting heart rate 
due to study drugs. Identical measurements will be performed whilst performing the deep breathing, valsalva 
maneuver, and orthostasis in order to yield information on the cardiovascular autonomic reflexes (for specifics 
on cardiac ANS function, see Appendix F). 
 
Exploratory Efficacy Measures 
Biomarkers and gut microbiome 
During visits 2 (baseline assessment), 3 (safety follow-up; week 6) and 4 (long-term assessment; week 12), 
blood will be drawn in appropriate test tubes and serum/plasma will be stored to measure (emerging) 
biomarkers (conditional to available budget). Also urine is obtained and stored to measure (emerging) 
biomarkers (conditional to available budget). Possible biomarkers include: plasma NT-proBNP, BNP, ANP, 
plasma cystatin C, FGF23, PTH, soluble Klotho, PRA, angiotensin II, aldosterone, endothelin, catecholamines, 
plasma insulin, C-peptide, lipids, GLP-1, glucagon, GIP, sRAGE, hs-CRP, IL-6, PAI-1, urinary TGF- β1, 
angiotensinogen, endothelin, nephrin, podocin, microparticles. Feces will be stored for potential gut 
microbiome analysis and biomarkers, such as calprotectin. 
 
DNA 
During visit 2 (baseline), blood will be collected and stored for possible DNA-analyses. This will only be 
performed after specific and separate consent by the participant. DNA can be used to study the influence of 
genetic factors on the parameters and the response measured to the SGLT-2 inhibitor. 
 
Insulin sensitivity 
During visit 2 and 4, insulin sensitivity will be measured by gold-standard euglycemic clamp. The M-value 
(metabolic clearance of glucose) will be calculated and expressed as mg/kg.min.  
 
Beta-cell function 
During hyperglycemia, insulin secretion will be quantified as measure for beta-cell function. The following 
parameters are obtained: 1st phase insulin secretion (early secretion), 2nd phase insulin secretion and 
arginine-induced insulin secretion (the latter as measured of total secretory capacity). 

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10. SAFETY (REPORTING) 
In accordance with the guidelines of ICH-GCP, the European Clinical Trials Directive (2001/20/EC) and the 
local regulations, the investigator will inform the subjects and the reviewing Ethical Review Board (ERB) if 
anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly 
greater than was foreseen in the research proposal. 
 
Safety Evaluations 
The following safety measures will be collected during the study (as outlined in Appendix A): 
 
Occurrence of adverse events (as reported by the subject) 
 
Vital signs (pulse rate, blood pressure, temperature) 
 
Different end-point assessments that also serve as safety measurements: blood chemistry and 
hematology. 
Collection of adverse event information will begin at the signing of the informed consent and continues through 
30 days after administration of the last dose of study medication.  
 
Adverse Event 
AEs are defined as any undesirable experience occurring in a clinical investigation subject during the study 
and which does not necessarily have a causal relationship with the treatment. All AEs reported spontaneously 
by the subject or observed by the investigator or study site staff will be recorded on special CRF. The AE term 
will be recorded using standard medical terminology (Medical Dictionary for Regulatory Activities; MedDRA™). 
Every attempt will be made to describe the AE in terms of a diagnosis. Whenever the investigator is confident 
in making a unifying diagnosis, all related signs, symptoms and abnormal test results will be grouped together 
and recorded as a single AE. All adverse events will be evaluated for intensity and causal relationship with use 
of the study medication (using the definitions in Table 5). 
 
 
Table 5: Safety Definitions 
 
 
 
 
 
Severity 
Mild 
The event is transient and easily tolerated by the subject 
Moderate 
The event causes the subject discomfort and interrupts the subject’s usual activities 
Severe 
The event causes considerable interference with the subject’s usual activities 
Causality 
Definite 
An AE that follows a reasonable temporal sequence form administration of the drug (including the 
course after treatment withdrawal of the drug) AND that satisfies any of the following: 
 
Reappearance of similar reaction upon readministration (rechallenge) 
 
Positive results in a drug sensitivity test 
 
Toxic level of the drug revealed by measurement of drug concentrations 
Probable 
An AE that follows a reasonable temporal sequence form administration of the drug (including the 
course after treatment withdrawal of the drug) AND for which involvement of factors other than the drug 
– such as underlying diseases, complications, concomitant drugs, and concurrent treatment – can 
reasonably be excluded. 
Possible 
An AE that follows a reasonable temporal sequence form administration of the drug (including the 
course after treatment withdrawal of the drug) AND for which involvement of factors other than the drug 
– such as underlying diseases, complications, concomitant drugs, and concurrent treatment – may also 
be responsible 
Not related 
An AE that follows a reasonable temporal sequence form administration of the drug or that can be 
reasonably explained by other factors, such as underlying diseases, complications, concomitant drugs, 
and concurrent treatment. 

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Serious Adverse Event (SAE) 
A SAE is any untoward medical occurrence or effect that:  
 
Results in death 
 
Is life threatening (Note: the term “life-threatening” refers to an event/reaction in which the patient was 
at risk of death at the time of the event/reaction; it does not refer to an event/reaction which 
hypothetically might have caused death if it were more severe). 
 
Requires inpatient hospitalization or results in prolongation of existing hospitalization 
 
Results in persistent or significant disability or incapacity 
 
Is a congenital anomaly or birth defect 
 
Is a medically important event or reaction. Medical and scientific and scientific judgment should be 
exercised in deciding whether other situations should be considered serious, such as important 
medical events that might not be immediately life-threatening or result in death or hospitalization, but 
might jeopardize the patient or might require intervention to prevent one of the other outcomes listed 
in the definition above. 
 
It must be noted that the term “severe” is often used to describe the intensity (severity) of a specific event; the 
event itself, however, may be of relatively minor medical significance. By contrast, the term “serious” is used 
to describe an event based on a (possible) event outcome or actions usually associated with events that pose 
a threat to a patient’s life or functioning.  
 
Adverse Drug Reaction (ADR) 
There is a reasonable possibility according to the Sponsor-Investigator that the product may have caused the 
event. 
 
Suspected unexpected serious adverse reaction (SUSAR) 
An ADR, the nature or severity of which is not consistent with the applicable product information (e.g. SPC). 
An expected ADR with fatal outcome should be considered unexpected. 
 
AE of special interest (AESI) 
An AE (serious or non-serious) of scientific and medical concern, specific to the product or program of study 
drug supplier (AstraZeneca), for which ongoing monitoring and rapid communication by the investigator may 
be appropriate. Please refer to the site master file for the current list of topics. 
 
Reporting 
Each SAE will be investigated thoroughly and an assessment will be made whether the SAE is a SUSAR (i.e. 
unexpected and drug-related, as indicated in the most recent SPC provided by the drug supplier). Because 
this study is an investigator sponsored trial (IST), the investigator also acts as sponsor. Therefore, the 
investigator will be responsible for reporting the SAEs and SUSARs to the accredited ERB. This will be done 
within 15 days after first knowledge, using the specified web portal (‘ToetsingOnline’), which automatically 
leads to informing the EMA and the respective national health authorities. For fatal or life threatening SAEs 
and SUSARs this term will be 7 days for a preliminary report (‘expedited’) with another 8 days for completion 
of the report. The expedited reporting through the web portal ‘ToetsingOnline’ is sufficient as notification to the 
competent authority. Since this study is a monocenter study, no other sites have to be informed.  
 
The involved pharmaceutical company (AstraZeneca) will be notified of all SAEs, pregnancy, clinical relevant 
overdosing and AESIs, if any. These events must be transmitted within 1 working day of the Investigator’s 
awareness or identification of the event, using standardized forms. Reports will consist of patient identification 
(subject number, initials, sex, age), event (diagnosis), drug reporter identification (name, initials), causality and 
outcome and should be written in English. Results of any relevant complementary exams performed to obtain 
the final diagnosis of any SAE (e.g., hospital discharge summary, autopsy, consultation) will be made available 
to AstraZeneca entity upon request. 
 
Contact details for safety reporting pharmaceutical company: 
AEMailboxClinicalTrialTCS@AstraZeneca.com  

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Annual safety report 
In addition to the expedited reporting of SAEs, the investigator will include a safety section containing an 
overview of all study SAEs in the periodic report, which will be submitted to the accredited ERB and the 
competent authorities once a year throughout the clinical trial.  
 
Follow-up of AEs 
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. The follow-up information should describe whether the event has resolved 
or continues, if and how it was treated, and whether the patient continued or discontinued study participation. 

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11. DATA MANAGEMENT AND QUALITY ASSURANCE 
 
Handling and storage of data and documents 
In accordance with the Dutch law ‘Wet Bescherming Persoonsgegevens’ (WBP; Personal Data Protection Act) 
and GCP all patient data will be handled confidentially and anonymously. Therefore, all participants will get a 
study code (‘RED-xxx’). 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 authorized investigator (i.e. research physician) 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 15 years. Subject 
data, both anonymous (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.  
 
Data Management 
To ensure correct entrance of data from the CRF to the database which will be used for statistical analyses, a 
special database program called OpenClinica© (Community Edition, v3.1.2, OpenClinica©, LLC, Waltham, MA, 
USA) will be used. A study environment will be build, containing the specific visits and necessary data entry 
fields. Electronic-CRFs (eCRF) will be made, similar to the hardcopy CRFs. These eCRFs will be enhanced 
by safeguards and queries to warrant correct entry of data. The investigator, or his/her designated 
representative, will enter all data from the hardcopy printed CRF on the eCRFs, which automatically stores the 
data in the database. All changes made to the database will be logged (‘audit trail’), which is required by GCP. 
On a regular basis all data from the OpenClinica©-database will be extracted to an Excel-file for backup 
purposes.  
 
Monitoring 
As required by GCP, a monitoring procedure will be performed in order to oversee the progress of the clinical 
trial and ensure that it is conducted, recorded and reported in accordance with the protocol, SOPs, GCP and 
the applicable regulatory requirements. Because the risk classification of the current study is estimated to be 
“moderate risk” (see Chapter 13), 2-3 monitor visits is required per year (73).  
Monitoring will be performed by a GCP-certified assistant of the Clinical Research Bureau VUMC. The monitor 
will verify inclusion of patients, the use of the investigational medicinal products (e.g. drug accountability), 
source documents and reporting of adverse events according to a pre-defined format (for specifics, see 
Appendix H). The findings will be reported to the investigator. 

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12. STATISTICS 
 
Sample Size Calculations 
The primary end-point is change in GFR/ERPF after 12 weeks of treatment with dapagliflozin or gliclazide, as 
derived from the inulin or iohexol/PAH clearance test. Based on literature we used the following calculation to 
define our sample size: to detect a change in GFR from 89 mL/min to 75 mL/min (16%), SD 13.0 mL/min, 
assuming α = 0.05 (2-sided testing), a power (1-β) of 90% (36,39,74,75), we need 19 subjects per group. 
However, taking into account a maximum dropout percentage of 15%, we have chosen to increase the amount 
of subjects per group to N=22. This sample size was calculated using Stata-software (v.11.2, College Station, 
TX, USA).  
 
Statistical Analysis Plan 
General considerations 
 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 the treatment groups) will be tested against 
the hypothesis H1: a difference exists between treatment groups, with respect to the underlying parameter 
of interest.  
 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. 
 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 
In order to compare the effects between the two study arms we will perform t-tests and/or multivariable linear 
regression models. The latter technique allows us to use continuous outcome variables and both continuous 
and dichotomous independent variables. The efficacy endpoint of interest will be added to the model as 
dependent variable. Treatment group will be added as independent categorical variable. Appropriate 
covariates may be added to the model. Baseline measurements of specific variables (e.g. age, gender) will be 
added to the regression models as covariates should differences in the baseline values of these variables 
occur between treatment groups. Moreover, corrections will be performed when relevant for the specific 
endpoint. The possible covariates will be elaborated prior to conducting the statistics. 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.   
Our primary outcomes will be the most important outcomes for testing the hypothesis that SGLT-2 inhibitor 
dapagliflozin versus the SU gliclazide causes an improvement in renal hemodynamics (GFR and ERPF). 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 (76). As recently pointed 
out (77), in a hypothesis-generating study – such as the current study – correction for multiple outcomes can 
be detrimental. A final decision will be made in the detailed Statistical Analysis Plan (SAP) prior to the 
conduction of the analysis. 

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13. ETHICS 
The trial will be conducted in accordance with the Declaration of Helsinki (64th WMA General Assembly, 
Fortaleza, Brazil, October 2013) for biomedical research involving human subjects and in accordance with 
ICH-GCP. The investigator will ensure that all aspects of the institutional ERB review are conducted in 
accordance with current institutional, local and national regulations.  
 
Ethical review 
Any member of the ERB who is directly affiliated with this study as an investigator or as 
site personnel must abstain from the ERB vote on the approval of the protocol. 
Recruitment 
Subjects will be recruited by researcher physicians involved in this trial using methods that are established 
practice for all human studies in the Diabetes Center 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, we will identify potential 
participants who 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 VUMC or affiliated hospitals / pharmacies 
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. 
 
Informed consent form for trial subjects 
Prior to any trial related activity, the investigator will give oral and written information about the trial in a form 
that the subject can read and understand. It is the investigators responsibility to ensure the trial subject is fully 
informed about the aims of the trial, procedures, potential risks, any discomforts and expected benefits. The 
investigator must ensure the subject is informed and agrees that VUMC personnel, their representatives and 
possibly health authority (national or other) personnel can require access to the subject’s data. It must be 
emphasized that participation is voluntary and that the subject has the right to withdraw from the trial at any 
time without prejudice. 
A voluntary, signed and dated ‘Informed Consent’ will be obtained from the subject prior to any trial related 
procedure. The written informed consent must be signed by the person who conducted the informed consent. 
If information becomes available that may be relevant to the subject’s willingness to continue participating in 
the trial, the investigator must inform the subject in a timely manner and a revised written informed consent 
must be obtained.  
Unexpected findings will be reported to the participant and to his general practitioner. If the participant does 
not want to be informed, he/she cannot participate in the trial. 
A physician who is not involved nor has any interest in the performance of the trial will be available; this person 
has knowledge about the protocol and can be consulted by subjects potentially interested in participation.  
 
Financial Compensation  
All subjects will receive a financial compensation for their participation (based on the minimum wages in The 
Netherlands). We have determined this to be €300 when the participant completes the entire study (as defined 
in Chapter 6). Moreover, reimbursement for travelling expenses will be given based on current prices of public 
transportation. 
 
Insurance 
In case of possible damage as a result of participating in this research project, the loss is, in agreement with 
legal demands, covered by insurance. This is in accordance with article 7, subsection 6 of the Medical 

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Research Involving Human Subjects Act; in Dutch: Wet Medisch-wetenschappelijk Onderzoek met Mensen 
(WMO).  
 
The sponsor (also) has an insurance which is in accordance with the legal requirements in The Netherlands 
(Article 7 WMO and the Measure regarding Compulsory Insurance for Clinical Research in Humans of 23th 
June 2003). This insurance provides cover for damage to research subjects through injury or death caused by 
the study. 
1. 
€ 650.000,-- (i.e. six hundred and fifty thousand Euro) for death or injury for each subject who 
participates in the Research; 
2. 
€ 5.000.000,-- (i.e. five million Euro) for death or injury for all subjects who participate in the Research;  
3. 
€ 7.500.000,-- (i.e. seven million and five hundred thousand Euro) for the total damage incurred by the 
organization for all damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the 
meaning of said Act in each year of insurance coverage. 
 
 
 
The insurance applies to the damage that becomes apparent during the study or within 4 years after the end 
of the study. The insurance is covered by: 
 
 
 
Onderlinge Waarborgmaatschappij Centramed B.A.  
Postbus 191 
2270 AD Voorburg  
The Netherlands  
 
Benefits and risk assessment 
Study Medication 
Participants will be randomized for a 12 week treatment with two different active study agents. Consequently, 
independent of treatment allocation, beneficial effects can be expected, as both SGLT-2 inhibitors and SU 
derivatives improve glycemic control. Both study medications have been approved for blood-glucose lowering 
treatment in T2DM patients and, based on currently available data, are considered to be safe. Furthermore, 
SGLT-2 inhibitors in general may decrease blood pressure and body weight compared to gliclazide.  
The most common side effects for dapagliflozine are hypoglycemia (especially when used in combination with 
a SU derivative or insulin) and genitcal mycotic- and urinary tract infections. These side effects are usually 
mild and transient. In addition dyslipidemia and increased hematocrit have been described. Long-term (side) 
effects of SGLT-2 inhibitors are currently being evaluated in large-scaled outcome trials. For gliclazide, 
hypoglycemia and blurred vision in the initiation phase of treatment (due to changes in blood glucose levels) 
are the most common side effects. Also gastrointestinal side-effects (nausea, vomiting, diarrhea or 
constipation) have been reported. Of note, the gliclazide in this study will be used at a relatively low dose (i.e. 
30 mg per day) for a relatively short treatment duration (12 weeks). Therefore, also given the inclusion- and 
exclusion criteria of the study participants, the overall risk of hypoglycemia is believed to be low. In the unlikely 
case of hypoglycemia symptoms, patients will be provided with a self-monitoring blood glucose (SMBG) device 
and instructed to perform ambulatory blood glucose checks in case of symptoms, during the course of the 
study. Also, they will receive standardized diabetes education, including information regarding carbohydrate 
use and self-management / resolution of hypoglycemia. As in all drug intervention trials, in this study, we will 
closely monitor patients for adverse drug and study events during the follow-up visit and by telephone 
consultation according to GCP (see Appendix A). Participants can contact the research staff 24 hours a day. 
 
Possible inconvenience for participants 
Over the last 10 years, we have gained ample experience with similarly demanding mechanistic drug 
intervention studies in T2DM patients. Based on the positive feedback from our participants, the low drop-out 
rate (max 5-10%) and the large proportion of participants that returns to participate in yet another (similarly 
demanding) study, we are confident that the burden on participants is perceived as not being too high. Indeed, 
we have built in different ways to alleviate the burden for participants, including clear, repeated communication, 
frequent contacting, intensified (diabetes) care, 24-hour availability of research staff, study and travel 
reimbursement, enabling participants to receive the newest study medication (that for most of them would not 

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be reimbursed in daily practice) and offering follow-up care in our out-patient clinic. During test-days, we 
provide meals and allow participants to read or watch TV/DVDs when possible. Finally, it should be noted that 
several tests are similar to currently or previously performed in patient care for diagnostic purposes (e.g. inulin 
or iohexol/PAH-clearance). 
We are aware of the fact that in the current study participants will undergo multiple tests that demand a 
considerable time investment from their end. The total duration of visits is 1-2 (screening- and control visit) and 
9 hours (visits 2 and 4). In addition, the renal/cardiovascular test-days during euglycemic- and hyperglycemic 
clamp may be perceived as demanding that amongst others involves frequent blood and urine collection, 
infusions, blood pressure, heart rate and microvascular measurements. During the cardiac autonomous 
nervous system function tests participants may experience transient dizziness or lightheadedness. As 
mentioned above, all possible measures will be taken to minimize the discomfort for the participants during the 
tests (e.g. comfortable beds are available which allow a semi-recumbent position).  
 
 
Safety issues 
The study examinations/tests are considered to be safe. No invasive procedures (besides intravenous 
peripheral catheters) are involved. During the study tests, two ‘diagnostic agents’ (i.e. inulin or iohexol and 
PAH) need to be administered; both agents are inert and have no side effects. As defined in Chapter 8, the 
total amount of drawn blood will be 500 mL during a total period of 16 weeks. Side effects are not expected 
because the blood volume taken per visit is relatively small, especially in comparison with regular blood 
donation, which amounts 500 mL per donation (and is allowed 5 times a year for men and 3 times for women). 
 
Risk classification 
The Dutch Federation of University Medical Centers (in Dutch: ‘Nederlandse Federatie van Universitair 
Medische Centra’) proposed a guideline for determination of the risk classification of a clinical trial (73). As 
stated above, the study medication and examinations/test are generally considered to be safe, with a small 
increased risk for hypoglycemia. For the latter, appropriate measures will be taken to minimize risk. Within the 
T2DM population, our study population can be considered as relatively low risk. However, sporadic serious 
side effects have been described (dehydration, hypervolemia, severe hypoglycemia), although most patients 
recover quickly. Therefore, while there is a low risk, the possible consequences might be ‘moderate’ to 
‘serious’. Therefore, we feel that the current study has a classification of ‘moderate risk’. 
 

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14. ADMINISTRATIVE ASPECTS AND PUBLICATION 
 
Amendments  
Amendments are changes made to the research after a favorable opinion by the accredited ERB has been 
given. A ‘substantial amendment’ is defined as an amendment to the terms of the ERB 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 
 
The quality or safety of any intervention used in the trial 
 
All substantial amendments will be notified to the ERB, the competent authority and subsidizing party/study 
(AstraZeneca). Non-substantial amendments will not be notified to ERB and competent authority, but will be 
recorded and filed by the sponsor/investigator and reported to AstraZeneca. 
 
Study time lines 
(Please note that the time lines stated below are estimations as they may vary according to, among others, study drug 
availability, approval by the ERB and inclusion rates).  
 
Date 
Description 
01-06-2015 
Study initiation: familiarizing with literature; attending courses for GCP / 
methodology; writing study full-protocol/SOPs; consulting statisticians/ 
methodologists; writing deliverables for pharma-companies for drug requests; 
exploring and arranging logistics/infrastructural collaborations; familiarizing 
with and validating of methods / training personnel; preparing data base/CRF 
01-09-2015 
Submission research committee 
01-10-2015 
Submission ERB 
01-01-2016 
Start Study (First patient in) 
01-08-2017 
Last drug dispensation (Last patient in) 
01-11-2017 
Collecting data (Last patient out) 
01-01-2018 
Database Lock 
01-01-2018 – 01-10-2018 
Data analysis 
01-06-2018 – 01-12-2018 
Manuscript submissions 
01-05-2018 
Final study report (ERB / Competent Authority) 
 
Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited ERB 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.  
 
End of study report 
The sponsor will notify the accredited ERB 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.  
In case the study is ended prematurely, the sponsor will notify the accredited ERB 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 ERB and the Competent 
Authority.  
 
Publication policy 
AstraZeneca will be provided will all intended publication(s) and abstract(s), preceding submission at least 30 
days and 7 days, respectively, AstraZeneca can make suggestions to change the wording of any  
 

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planned publication or abstract in relation to the study and/or the results. The sponsor-investigator is under no 
obligation to implement such changes, unless local regulatory requirements specify otherwise.  
The study will be part of a PhD project for one MD PhD student. A PhD-project has to fulfill the requirements 
of the Medical Schools at VU University, Amsterdam, the Netherlands. In general, a PhD-thesis has to contain 
at least 5 chapters, consisting of manuscripts published in international peer-reviewed journals. We anticipate 
that this study will yield sufficient data to meet these criteria. Papers will be written at the end of the study, i.e. 
second half of 2018, and will be submitted to different relevant international peer-reviewed journals (Diabetes, 
Diabetes Care, etc.). Authorship of the papers will be determined using the Vancouver criteria for authorship. 
 
Data will be presented at relevant national and international scientific meetings, including those from the Dutch 
Association for the Study of Diabetes (NVDO), European Association for the Study of Diabetes (EASD), 
American Diabetes Association (ADA), etc. Since the competition in this field is considerable, the usual caution 
should be entertained when presenting abstracts, which usually contain preliminary results.

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APPENDIX A 
TEST MEDICATIONS 
 
Contact / Visit 
V1 
T1 
V2 
T2 
V3 
V4 
ET* 
Weeks relative to 1 test visit 
-4 
-4 
0 
2 
6 
12 
 
Informed consent 
X 
 
 
 
 
 
 
Screening (in-/exclusion) 
X 
 
 
 
 
 
 
Inform about eligibility 
 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
Recent history / Adverse event review 
X 
 
X 
X 
X 
X 
X 
Physical examination / anthropometrics 
X 
 
 
 
(X) 
 
 
Vital Signs / Body weight 
X 
 
X 
 
X 
X 
X 
Bio-Impedance Analysis 
 
 
X 
 
 
X 
X 
Laboratory sampling 
XA 
 
XB,C 
 
XC,D 
XB,C 
XC,E 
Renal ProtocolF 
 
 
X 
 
 
X 
(X) 
Clamp ProtocolG 
 
 
X 
 
 
X 
(X) 
Cardiovascular testsH 
 
 
X 
 
 
X 
(X) 
ECG 
X 
 
 
 
 
 
 
DNA collection 
 
 
X 
 
 
 
 
Randomization 
 
 
X 
 
 
 
 
Intervention period 
 
 
start 
 
 
 
 
Study medication dispensed 
 
 
X 
 
X 
 
 
Study drug accountability 
 
 
 
 
X 
X 
X 
Time spent in CRU (hours) 
1.5 
phone 
9 
phone 
1.0 
9 
1.5 
 
 
 
CRU=Clinical research unit; ET=Early-Term assessments; T=Telephone consultation; V=Visit 
 
 
 
A Measurements: Blood (Hematology (Hb, Ht, leucocytes and thrombocytes), glucose, HbA1c, AST, ALT, GGT, ALP, sodium, potassium, 
creatinine, BUN, TG, TSH and FSH (in females only, in case of doub) and urine (screening, ACR) 
B Measurements: Hematology (Hb, Ht, leukocytes and thrombocytes), fasting glucose, HbA1c, TG, TC, HDL-, LDL-C, creatinine, BUN, 
AST, ALT, GGT, ALP.  
C Blood/urine sampled and stored for various (cardiovascular- & inflammatory)-biomarker analyses (cystatin C, FGF23, , PTH, soluble 
Klotho, NT-proBNP, BNP, ANP, PRA, angiotensin II, aldosterone, catecholamines, insulin, C-peptide, lipids, GLP-1, glucagon, GIP, 
sRAGE, hsCRP, IL-6, PAI-1, TGF-β1, collagen type IV, nephrin, podocin, angiotensinogen, mRNA, microparticles) 
D Safety (and efficacy) lab measurements: Hematology (Hb, Ht, leucocytes and thrombocytes) and chemistry (sodium, potassium, 
creatinine, BUN, AST, ALT, GGT, ALP) 
E Measurements: Blood (Hematology, HbA1c, TG, TC, HDL-, LDL-cholesterol, creatinine, Na, BUN, albumin, AST, ALT, GGT, ALP, 
lipase, amylase) and urine (sodium, creatinine, BUN) 
F Renal protocol consists of: inulin or iohexol/PAH-clearance, tubular function tests and renal damage measurements 
G Cardiovascular test-battery consists of oscillometric blood pressure measurements, applanation tonometry, capillary 
videomicroscopy, ECG and beat-to-beat finger blood pressure measurements 
H
      Clamp protocol consists of: insulin and glucose infusion; measurements of insulin sensitivity and beta-cell function will be obtained. 
 
 
 
 
 
 
 
 
 
* When a subject wants to discontinue participation prior to the end of the study protocol, all possible effort will be made to counsel the 
participant to undergo as much endpoint measurements as possible. The procedures mentioned in the ET-column should at least be 
performed. 
 
 
 

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APPENDIX B 
TEST MEDICATIONS 
 
Inulin (Inutest® by Fresenius Kabi Austria GmbH) (1) 
Supplied as: 20 mL ampoules with a clear, colorless to slight yellow solution containing 25% sinistrin. 
Approved agent: Approved by the Austrian drug authority (Bundesamt für Sicherheit im Gesundheitswesen).  
Action: Sinistrin is a naturally occurring sugar polymer which, like inulin, belongs to the fructan group. While 
traditionally inulin has been used for determination of GFR (hence the so called ‘inulin-clearance’), sinistrin 
shows a better solubility in water and improved alkaline stability, while the clearance ratios of inulin and sinistrin 
(inutest) are virtually identical. The laboratory determination of inulin and sinistrin is identical. Like inulin, 
sinistrin is inert and therefore has no effects on human physiology. While we will use sinistrin, for clarity and 
recognition, throughout the protocol the term inulin is used.  
Side effects: Side effects are rare, but extremely large doses may cause osmotic diuresis. Allergic and 
anaphylactic reactions have been reported, but are very uncommon. 
Dosing: For the steady-state inulin-clearance test, an initial bolus dose is given, followed by continuous 
infusion with a dose calculated to maintain the plasma concentration between 200mg/L and 250mg/L. 
Storage: Inutest can be stored up to 3 years below 25°C. 
 
Iohexol (Omnipaque® by GE Healthcare B.V.) 
Supplied as: 200 mL ampoules with a clear, colorless solution containing 302 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. 
 
PAH (4-aminohippuric acid as sodium salt by Bachem Distribution Services GmbH.) (2) 
Supplied as: 10 mL vials containing sterile 20% (2 g/10 mL) of 4-Aminohippuric acid dissolved in sodium 
hydroxide solution and water for injections (WFI) 
Approved agent: PAH by Bachem has no EMA or FDA approval. However, PAH is widely used in clinical 
trials for the assessment of ERPF. Bachem produces PAH in GMP-grade and the product will undergo quality 
assurance prior to administration (document verification, chemical/physical/ microbiological properties and 
stability testing). A QP-release (release by a qualified person) will be given prior to use of PAH. 
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 defecate or urinate during or shortly following initiation of infusion. Allergic and 
anaphylactic reactions have been reported, but are very uncommon.  
Dosing: During one PAH-clearance test, an initial bolus dose 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 (excursions between 15 - 25°C are allowed) protected from 
light.  
 
Insulin aspart (NovoRapid® by Novo Nordisk). (3) 

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Supplied as: 100 units/ml solution for injection, containing a clear, colorless and aqueous solution 
Approved agent: EMA-approved 
Action: Rapid-acting insulin analogue 
Side effects: The most frequently reported adverse reaction during treatment is hypoglycemia. At the 
beginning of the insulin treatment, refraction anomalies and edema may occur. These reactions are usually of 
transitory nature. Fast improvement in blood glucose control may be associated with acute painful neuropathy, 
which is usually reversible. 
Dosing: Each insulin infusion will be primed with an intravenous (IV) insulin bolus of 0.1 x kg body weight x 
desired elevation of plasma insulin level of 100 mU/L. The insulin infusion rate will be maintained at 40 
mU/min/m2 body surface area during the 3-hr euglycemic clamp. 
Shelf life: Before opening: 30 months. During use or when carried as a spare: The product must be stored for 
a maximum of 4 weeks. Store below 30°C. 
Storage: Before opening: Store in a refrigerator (2°C - 8°C). Do not freeze. During use or when carried as a 
spare: Store below 30°C. Do not refrigerate. Do not freeze. 
 
Arginine 10% HCl (50g=500ml) (VUmc pharmacy). 
Supplied as: 500 ml infusion fluid 
Approved agent: Arginine by the VUmc pharmacy has no EMA or FDA approval. However, it has been widely 
used in clinical trials (including at our department) to measure beta cell function during hyperglycemic clamp. 
The VUmc pharmacy has been producing arginine for many years and has ample experience with the process. 
Action: beta cell stimulation 
Side effects: side effects are rare. Dysgeusia, headaches, hot flashes have been reported.   
Dosing: At the end of the hyperglycemic clamp, 5 mg arginine (50ml)  will be administered. 
Shelf life: Arginine can be stored for 3 years  
Storage: At room temperature (below 25 degrees) and in the dark 
 
 
 
References 
(1) Fresenius 
Kabi. 
Summary 
of 
Product 
Characteristics 
- 
Inutest. 
2011. 
Available 
from: 
URL: 
http://fresenius-
kabi.at/en/files/4.2.3.Inutest_SPC_2011.pdf 
(2) Bachem (Christoph Hörth, Pharmacist). Documentation on 4-aminohippuric acid solution 20% (2g/10ml). 6 May 2015 
(3) Novo 
Nordisk 
A/S. 
Summary 
of 
Product 
Characteristics 
– 
NovoRapid. 
30-04-2009. 
Available 
from: 
URL: 
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/000258/WC500030372.pdf  
 
(4) Arginine HCl 10% infusievloeistof, farmacotherapeutische plaatsbepaling apotheek VUmc 
 
(5) SPC Arginine HCl 50 g= 500 ml, Apotheek haagse ziekenhuizen 
 
(6) SPC Omnipaque  
 
 

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APPENDIX C1 
RENAL PROTOCOL 
The ‘renal protocol’ consists of the measurements of the GFR, ERPF, tubular function and renal damage 
parameters. The GFR and ERPF will be assessed using the gold standard inulin or iohexol/PAH-clearance (1-
4). 
 
Preparation 
Seven days prior to the renal examination, the subject will adhere to a ‘normal-salt’ (9-12 grams or 150-200 
mmol per day) and -protein (1.5-2 g/kg per day) diet, in order to minimize variation due to salt and protein 
intake (5;6). Moreover, participants will abstain from alcohol (24 hours), caffeine (12 hours) and nicotine (12 
hours) and heavy exercise prior to and during the visits, as these have been shown to affect renal physiology 
(7-10). Participants will be instructed to abstain from taking any kind of medication on the morning after an 
overnight fast (at least 8 hr) of the renal tests, except for metformin. Prior to coming to the clinical research 
unit, the participant is asked to drink 500 mL of water. 
  
The participant will assume a semi-recumbent position, in a temperature controlled room (22-23⁰C). A 
peripheral 18 gauge venous cannula will be placed in both forearms. One of the cannulas will be used for 
infusion of the test substances, while the other will be used for venous blood sampling. The cannulas will be 
kept patent using 0.9% saline (no heparin will be used).  
 
Procedure 
Baseline measurements 
Prior to the start of the infusion of inulin or iohexol/PAH, urine will be collected by spontaneous voiding and a 
blood sample will be taken. This blood and urine will be used to determine the reference levels of inulin or 
iohexol and PAH for the clearance tests. Moreover, tubular function and renal damage will be determined using 
these samples.  
 
Start of Inulin or iohexol/PAH-protocol 
In order to measure GFR and ERPF, respectively inulin (Inutest®, Fresenius Kabi, Austria) (11) or iohexol and 
para-aminohippurate (PAH sodium salt, Bachem, Germany) (12) need to be administered. In order to yield 
steady state concentrations quickly, a priming dose will be given. Then, a continuous infusion will be started 
to maintain the plasma concentration at a level sufficient for the determination of the GFR and ERPF. The 
specific dosages will be derived from the SPCs. 
 
Because frequent voiding is necessary, a constant water intake is advocated to maintain an adequate urine 
output. During the equilibration period, the participant needs to drink 10 mL/kg body weight (with a maximum 
of 1000 mL). During the collection periods, subjects need to drink 200 mL/hour. 
 
Collection periods 
After an equilibration period of 90 minutes and and 15 minutes later blood samples will be drawn to measure 
PAH and inulin or iohexol to estimate GFR and ERPF. After 90 minutes during the euglycemic clamp (see 
figure), the participant is asked to void and blood will be drawn. This urine is of no value and will be discarded. 
From then on, urine will be collected by spontaneous voiding every 45 minutes for 2 periods of time. At the 
beginning and end of each collection period blood will be sampled from the venous cannula which is not used 
for the Inutest/PAH-infusion. Both urine and blood will be analyzed for inulin or iohexol and PAH.  
 
Sample handling and measurements 

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All blood and urine will be collected and processed according to standard operating procedures. In short, urine 
will be collected at all times using a urine chair or urinal. The volume of urine will be recorded and the urine 
will be transferred to a plastic container, which will be stored at -20⁰C or -80⁰C until assay.  
Blood will be drawn at set times. The different analyses will be performed as descripted in the table. 
 
In order to measure tubular function, blood and urine will be analyzed for sodium, potassium, chloride, calcium, 
magnesium, phosphate and urea and fractional excretions will be calculated (see Table). Urine osmolality will 
also be tested and plasma osmolality calculated. In order to calculate the fractional excretion, the blood and 
urine level of creatinine need to be measured. 
 
For renal damage the urine will be analyzed for 24-hour albumin, and NGAL and KIM-1 in spot urine.  
Serum and urine samples will be stored for possible (emerging) analyses depending on budget and when 
deemed interesting (e.g. cystatin C, FGF23, PTH, soluble Klotho, TGF-β1, collagen type IV, nephrin, podocin, 
microparticles). 
 
Table: schedule for renal protocol 
Test Time 
(min) 
Procedure 
Collection + Analyses 
Blood volume 
(mL) 
Inulin or iohexol / PAH 
0 
Start bolus Inutest/PAH 
Fasting state 
Blood + urine 
20 
10 
Start Continuous infusion Inutest/PAH  
 
 
100 
Measure fasting serum Inutest/PAH 
Blood 
15 
115 
Measure fasting serum Inutest/PAH 
Start euglycemic clamp 
Blood 
15 
205 
Voiding 
(measure urine volume + discard urine) 
Blood 
15 
255 
Measure urine volume 
Blood + urine 
15 
295 
Measure urine volume 
Blood + urine 
15 
355 
Start hyperglycemic clamp 
 
 
415 
Voiding 
(measure urine volume + discard urine) 
Blood 
15 
460 
Measure urine volume 
Blood + urine 
15 
505 
Measure urine volume 
Blood + urine 
15 
 
Calculations 
Different calculations will be performed, using the formulas in the box. During fasting state GFR and ERPF will 
be calculated without collecting urine, assuming that a steady state has been reached and infusion rate equals 
excretion rate. During the clamps a mean will be calculated for plasma levels of inulin or iohexol and PAH, 
using the blood collected at the beginning and end of each collection period. This mean will be used in the 
formulas. The two different values (two collection periods) will be combined to calculate the mean GFR, ERPF, 
FF, ERBF and ERVR. The mean value will be the reference value (the baseline value for the rest of the study). 
 

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References 
(1)  Donnelly SM, Miller JA. Losartan may modulate erythropoietin production. J Renin Angiotensin Aldosterone Syst 
2001 Dec;2(4):255-60. 
(2)  Goddard J, Johnston NR, Hand MF, Cumming AD, Rabelink TJ, Rankin AJ, et al. Endothelin-A receptor antagonism 
reduces blood pressure and increases renal blood flow in hypertensive patients with chronic renal failure: a 
comparison of selective and combined endothelin receptor blockade. Circulation 2004 Mar 9;109(9):1186-93. 
(3)  van der Zander K, Houben AJ, Hofstra L, Kroon AA, de Leeuw PW. Hemodynamic and renal effects of low-dose brain 
natriuretic peptide infusion in humans: a randomized, placebo-controlled crossover study. Am J Physiol Heart Circ 
Physiol 2003 Sep;285(3):H1206-H1212. 
(4)    Cherney DZI, Perkins B a, Soleymanlou N, Maione M, Lai V, Lee A, et al. The Renal Hemodynamic Effect of SGLT2 
Inhibition in Patients with Type 1 Diabetes. Circulation 2014 Feb;129:587-97  
(5)  Frank H, Graf J, Amann-Gassner U, Bratke R, Daniel H, Heemann U, et al. Effect of short-term high-protein compared 
with normal-protein diets on renal hemodynamics and associated variables in healthy young men. Am J Clin Nutr 
2009 Dec;90(6):1509-16. 
(6)  Miller JA. Renal responses to sodium restriction in patients with early diabetes mellitus. J Am Soc Nephrol 1997 
May;8(5):749-55. 
(7)  Pawlik WW, Jacobson ED, Banks RO. Actions of nicotine on renal function in dogs. Proc Soc Exp Biol Med 1985 
Apr;178(4):585-90. 
(8)  Sargent WQ, Simpson JR, Beard JD. The effect of acute and chronic alcohol administration on renal hemodynamics 
and monovalent ion excretion. J Pharmacol Exp Ther 1974 Feb;188(2):461-71. 
(9)  Leonard TK, Watson RR, Mohs ME. The effects of caffeine on various body systems: a review. J Am Diet Assoc 1987 
Aug;87(8):1048-53. 
(10)  Poortmans JR. Exercise and renal function. Sports Med 1984 Mar;1(2):125-53. 
(11) Fresenius Kabi. Summary of Product Characteristics - Inutest. 2011. Available from: URL: http://fresenius-
kabi.at/en/files/4.2.3.Inutest_SPC_2011.pdf 
(12) Bachem (Christoph Hörth, Pharmacist). Documentation on 4-aminohippuric acid solution 20% (2g/10ml). 6 May 2015
 
 
GFR:  
𝑈𝑟𝑖𝑛𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑖𝑛𝑢𝑙𝑖𝑛 ∗ 𝑢𝑟𝑖𝑛𝑒 𝑓𝑙𝑜𝑤
𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑖𝑛𝑢𝑙𝑖𝑛
 or  
𝐼𝑛𝑓𝑢𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑖𝑛𝑢𝑙𝑖𝑛
𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑖𝑛𝑢𝑙𝑖𝑛 
 
ERPF: 
𝑈𝑟𝑖𝑛𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑃𝐴𝐻 ∗ 𝑢𝑟𝑖𝑛𝑒 𝑓𝑙𝑜𝑤
𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑃𝐴𝐻
 or  
𝐼𝑛𝑓𝑢𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑃𝐴𝐻
𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑃𝐴𝐻 
 
Filtration Fraction: 
𝐺𝐹𝑅
𝐸𝑅𝑃𝐹 
 
Effective Renal Blood Flow:   
𝐸𝑅𝑃𝐹
1−ℎ𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 
 
Effective Renal Vascular Resistance:   
𝑀𝑒𝑎𝑛 𝐴𝑟𝑡𝑒𝑟𝑖𝑎𝑙 𝑃𝑟𝑒𝑠𝑠𝑢𝑟𝑒
𝐸𝑅𝐵𝐹
 
 
Urinary solute excretion: 𝑢𝑟𝑖𝑛𝑎𝑟𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 𝑢𝑟𝑖𝑛𝑒 𝑓𝑙𝑜𝑤 
 
FE-solutes: 
𝑈𝑟𝑖𝑛𝑎𝑟𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
𝑃𝑙𝑎𝑠𝑚𝑎 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 
𝑃𝑙𝑎𝑠𝑚𝑎 𝑖𝑛𝑢𝑙𝑖𝑛/𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛
𝑈𝑟𝑖𝑛𝑎𝑟𝑦 𝑖𝑛𝑢𝑙𝑖𝑛/𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛 𝑥 100 

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APPENDIX C2 
CLAMP PROTOCOL 
 
Preparation of subjects. The subjects will arrive at the clinical research unit in the morning after an overnight 
fast (at least 8 hr). All subjects will remain fasted throughout the clamp. At the evening prior to the clamp 
procedure, subjects will be instructed to take their usual medication. They should refrain from taking any drugs 
in the morning of the clamp day, except for metformin.  
Subjects will be placed in a semirecumbent position, and a cannula will be inserted into the non-dominant (if 
available) antecubital fossa for infusion of saline, glucose, and insulin. A contralateral dorsal hand vein will be 
cannulated in a retrograde manner and maintained at 55º C to permit sampling of arterialized venous blood at 
5 min intervals throughout the clamp protocol. The cannulae will be kept patent using 0.9% saline. After both 
cannulae have been inserted, the veins will be allowed at least 15 min of rest time prior to starting blood draws 
for the clamp. Glucose (20%) will be infused at a variable rate to maintain glucose at the desired concentration. 
The glucose infusion rate will be adjusted according to bedside measurement of blood glucose (BG) every 5 
min based on a predefined algorithm. Blood samples will be taken before and during the tests as described in 
the following table. In all subjects, a euglycemic insulin clamp will be performed during the first 3-hr followed 
by an interval (“rest period”) of 1 hour during which insulin infusion will be discontinued and glucose 
concentration maintained by the clamp technique, and a subsequent hyperglycemic clamp procedure with a 
standard 80-min square-wave hyperglycemia of 15 mmol/L. During the last 30 min of the hyperglycemic clamp, 
an arginine bolus will be administered. The duration of the total procedure is about 450 min, during which 
approximately 90 mL blood will be collected. 
 
The euglycemic clamp procedure. Insulin infusion will be be maintained at 40 mU/min/m2 body surface area 
during the 3-hr euglycemic clamp. The blood glucose concentration will be maintained at 5.0 mmol/L by 
performing manual glucose clamp for the remaining duration of the euglycemic clamp procedure. The glucose 
infusion rates will be adjusted according to the equation originally described by DeFronzo et al. (1979) with 
supplementary glucose infusion when BG decreases by > 0.2 mmol/L in 5 min.  
Resting period. During the subsequent resting period of 60 min the subjects will receive an infusion of saline 
or glucose to maintain euglycemic levels at around 5 mmol/L (or at the blood glucose concentration reached 
at 90 min of the euglycemic clamp).  Insulin infusion will be discontinued during this period.  
The hyperglycemic clamp procedure combined with arginine stimulation. The hyperglycemic clamp 
procedure will be started with a body weight-adjusted IV bolus of 20% glucose administered over 1 min 
followed by a continuous infusion to achieve target blood glucose concentration of approximately 15 mmol/L 
(27 x bodyweight [kg] x desired increase in blood glucose [mmol/L] = bolus dose [mg]). The glucose infusion 
will be adjusted to maintain BG at 15 mmol/L. After 150 min of hyperglycemia, an IV bolus of 5 g arginine 
(dissolved in 50 mL) will be injected over 45 s while the glucose level will be maintained at 15 mmol/L.   
Blood collections and sample handling. Blood glucose concentrations will be determined by the glucose 
oxidase method with a Yellow Springs glucose analyzer (Yellow Springs, OH, US) at 5 min intervals, except 
for the first 10 min of the hyperglycemic clamp (at 3 min, 5 min, 8 min, and 10 min) and the first 5 min after the 
arginine bolus (at 2 min, 3 min, 4 min, and 5 min). Sampling for plasma insulin and C-peptide will be performed 
as in the following table. All samples will be processed according to standard operating procedures within 1 hr 
and stored at -80°C until assay. The amount of infused glucose and saline will  be recorded.  
 
 
 
 

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Euglycemic and Hyperglycemic Clamp Procedures 
 
Test time 
(min) 
Real time 
(min) 
Procedures 
Period 
Analytes 
Blood Volume 
(mL) 
Insulin 
C-Peptide 
-30 
0 
 
 
X 
X 
1.5 
-15 
15 
 
X 
X 
1.5 
0 
30 
Begin insulin IV 
Euglycemic Clamp 
 
 
 
10 
40 
 
 
 
 
60 
90 
 
 
 
 
90 
120 
Reach glucose clamp 
target (5 mM) 
 
 
 
100 
130 
Start period 1 
X 
X 
1.5 
145 
175 
Start period 2 
X 
X 
1.5 
190 
220 
End period 2 
X 
X 
1.5 
190 
220 
End insulin IV 
Rest Period 
 
 
1.5 
-10 (=50) 
270 
 
X 
X 
1.5 
-5 (=55) 
275 
 
X 
X 
1.5 
0 (=60) 
280 
Begin glucose IV 
(target 15 mM) 
Hyperglycemic Clamp 
 
 
 
1 
 
 
X 
X 
1.5 
2 
 
 
X 
X 
1.5 
3 
 
 
X 
X 
1.5 
4 
 
 
X 
X 
1.5 
5 
 
 
X 
X 
1.5 
6 
 
 
X 
X 
1.5 
7 
 
 
X 
X 
1.5 
8 
 
 
X 
X 
1.5 
9 
 
 
X 
X 
1.5 
10 
290 
 
X 
X 
1.5 
15 
295 
 
X 
X 
1.5 
30 
310 
 
X 
X 
1.5 
60 
340 
Start period 1 
X 
X 
1.5 
70 
350 
 
X 
X 
1.5 
105 
385 
Start period 2 
X 
X 
1.5 
150 
430 
End period 2 
X 
X 
1.5 
150 
 
I.V. Arginine 
Bolus (45s) 
Arginine Challenge 
X 
X 
1.5 
151 
 
 
X 
X 
1.5 
152 
 
 
X 
X 
1.5 
153 
 
 
X 
X 
1.5 
154 
 
 
X 
X 
1.5 
155 
 
 
X 
X 
1.5 
156 
 
 
X 
X 
1.5 
157 
 
 
X 
X 
1.5 
158 
 
 
X 
X 
1.5 
159 
 
 
X 
X 
1.5 
160 
440 
 
X 
X 
1.5 
170 
450 
 
X 
X 
1.5 
180 
460 
End of Clamp 
Recovery Period 
X 
X 
1.5 
190 
470 
End Recovery 
 
 
 
Subtotal Blood Volume (mL) 
55.5 
Blood Volume for Glucose Analysis 
44.5 
Total Blood Volume (mL) 
90.0 
BG, blood glucose; YS, Yellow Springs glucose analyzer 
 
 

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APPENDIX D 
MICROVASCULAR FUNCTION ASSESSMENTS 
 
In order to measure the microvascular function we will perform capillary videomicroscopy and Laser Doppler 
methods (1-3).  
 
Preparation 
Subjects will be in a fasting state (for at least 10 hours) and will be asked to refrain from alcohol, caffeine and 
nicotine for at least 12 hours. Participants will be instructed to abstain from taking any kind of medication on 
the morning of the tests, except for metformin.  
The participant will assume a semi-recumbent position, with the non-dominant hand at heart level. 
Measurements will be conducted after 30 minutes of acclimatization in a quiet, temperature controlled room 
(22-23 ⁰C).  
 
Procedure 
Capillary Videomicroscopy 
Microvascular function will be measured by assessing skin capillary density and capillary recruitment after 
arterial occlusion by video-microscopy of the nail fold capillaries in the fasting state, according to a previously 
described protocol (2;3).  
In short, perfused nail fold capillaries in the dorsal skin of the third finger of the non-dominant hand will be 
visualized by a capillary microscope (Carl Zeiss) linked to a television camera, a video recorder and a monitor. 
To visualize the capillaries, a 3.2X objective (Zeiss 3.2/0.07) is used, with a total system magnification of 99X.  
Two separate visual fields of 1 mm2 will be recorded before and after 4 minutes of arterial occlusion with a 
digital cuff (300 mmHg). The images will be stored on videotape for off-line analysis. Capillary density is defined 
as the number of erythrocyte-perfused capillaries per mm2 of nail fold skin. Percentage capillary recruitment 
during post-occlusive reactive hyperemia is assessed by dividing the capillary density during post-occlusive 
reactive hyperemia by the number of capillaries at baseline.  
Although performed in the original protocol (as described in (2;3)), data by van Genugten et al (SPHINX-study) 
suggests that venous occlusion does not lead to additional information (4). Therefore, in this study, no venous 
occlusion will be performed.  
 
Laser Doppler 
To perform vasomotion analyses, skin blood flow will be measured with a Laser Doppler probe (PF 457; 
Perimed, Stockholm, Sweden) at the dorsal side of the third finger of the non-dominant arm. Signals will be 
recorded for 30 minutes in the fasting state. Fast-Fourier transform analysis will be performed by means of 
Perisoft dedicated software (PSW version 2.5, Perimed) to determine the contribution of the five frequency 
components of the variability of the Laser Doppler signal (i.e. endothelial: 0.01 – 0.02 Hz; neurogenic: 0.02 – 
0.06 Hz; myogenic: 0.06 – 0.15 Hz; respiratory: 0.15 – 0.40 Hz; and heart beat: 0.40 – 1.60 Hz). 
 
 
 
 
 
References 
 (1)  Antonios TF, Rattray FE, Singer DR, Markandu ND, Mortimer PS, MacGregor GA. Maximization of skin capillaries 
during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive 
hyperaemia and core heat load tests. Clin Sci (Lond) 1999 Oct;97(4):523-8. 
 (2)  Serne EH, Stehouwer CD, ter Maaten JC, ter Wee PM, Rauwerda JA, Donker AJ, et al. Microvascular function 
relates to insulin sensitivity and blood pressure in normal subjects. Circulation 1999 Feb 23;99(7):896-902. 
 (3)  Serne EH, Gans RO, ter Maaten JC, Tangelder GJ, Donker AJ, Stehouwer CD. Impaired skin capillary recruitment 
in essential hypertension is caused by both functional and structural capillary rarefaction. Hypertension 2001 
Aug;38(2):238-42. 
   (4)    van Genugten RE, Serné EH, Heymans MW, van Raalte DH, Diamant M. Postprandial microvascular function 
deteriorates in parallel with gradual worsening of insulin sensitivity and glucose tolerance in men with the metabolic 
syndrome or type 2 diabetes. Diabetologia. 2013 Mar;56(3):583-7. 

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APPENDIX E 
PULSE WAVE ANALYSIS 
 
Pulse Wave Analysis (PWA) will be assessed using the SphygmoCor® System (Atcor Medical, West Ryde, 
Australia), a non-invasive system using applanation tonometry (1-3).  
 
Preparation 
The subject will assume a supine position in a temperature-controlled room (22-23⁰C) for 10-30 minutes prior 
to taking any recordings. Participants will be in a fasting state, and will be asked to refrain from alcohol, caffeine 
and nicotine for at least 12 hours. Participants will be instructed to abstain from taking any kind of medication 
on the morning of the test, except for metformin. 
 
Procedure 
Pulse Wave Analysis 
The SphygmoCor® PWA-setting will be used to derive a calibrated blood pressure waveform at the ascending 
aorta from a peripheral pressure waveform recorded at the radial artery of the non-dominant arm. To realize 
this, the radial artery will be sought by palpation. The tonometer will be placed in a 90⁰ angle on top of the 
artery. Once a consistent pressure waveform has been found, a minimum of 12 seconds / 3 full waveforms 
should be recorded. The SphygmoCor® system will derive an Augmentation Pressure (AP), Augmentation 
Index (AIx), Augmentation Index corrected for heart rate (AIx@HR75), Ejection Duration (ED) and 
Subendocardial Viability Ratio (SEVR) (1).  
 
Quality control 
In order to use valid measurements, the ‘Quality Control Section’ of SphygmoCor® will be used. If any of the 
parameters for PWA (average pulse height, pulse height variation, diastolic variation or shape deviation) is 
outside of the predefined range, the measurement will be repeated. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
References 
 (1)  Davies JI, Struthers AD. Pulse wave analysis and pulse wave velocity: a critical review of their strengths and 
weaknesses. J Hypertens 2003 Mar;21(3):463-72. 
 (2)  Laurent S, Cockcroft J, Van BL, Boutouyrie P, Giannattasio C, Hayoz D, et al. Expert consensus document on 
arterial stiffness: methodological issues and clinical applications. Eur Heart J 2006 Nov;27(21):2588-605. 
 (3)  Wilkinson IB, Fuchs SA, Jansen IM, Spratt JC, Murray GD, Cockcroft JR, et al. Reproducibility of pulse wave velocity 
and augmentation index measured by pulse wave analysis. J Hypertens 1998 Dec;16(12 Pt 2):2079-84.

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APPENDIX F 
CARDIOVASCULAR AUTONOMIC NERVOUS SYSTEM FUNCTION TESTS 
 
A non-invasive, automated, beat-to-beat blood pressure and ECG recording monitor (Nexfin®, BM Eye, 
Amsterdam, the Netherlands) (1) will be used to non-invasively measure heart rate and blood pressure.  
 
Preparation 
Participants will be in a fasting state and have restrained from drinking caffeine and smoking for at least 12 
hours. Participants will be instructed to abstain from taking any kind of medication on the morning of the tests, 
except for metformin. Before starting the tests, subjects will assume a supine position for at least 10 minutes 
in a temperature controlled room (22-23 ⁰C).  
  
Procedures 
Throughout the years many different tests have been developed and used in order to test cardiovascular ANS 
function (2;3), however the following tests were recommended by a joint panel (4): 
 
Resting HRV (heart rate variability) 
Using the Nexfin® monitor, the resting heart rate will be recorded during 7 minutes. With the use of dedicated 
software, different time domain and power spectral analyses of heart rate fluctuations can be performed in 
order to quantitatively evaluate beat-to-beat cardiovascular control (5;6). Different possible measures and their 
definition are explained in the table. Resting heart rate variability reflects the balance between the sympathetic 
nervous system and parasympathetic. Also, the baroreflex sensitivity can be assessed, reflecting the sensitivity 
of spontaneous baroreflex control of the heart rate (see table). 
 
CARTs  
Deep breathing: In a supine position, subjects will be asked to breathe standardized frequency during 1 minute. 
They will be asked to take 10 seconds for every in- and expiration (5 seconds in, and 5 seconds out), resulting 
in 6 completed breathings per minute. Heart rate is recorded throughout the period of deep breathing and a 
marker will be used to indicate the start of the in- and expiration. The Expiration-Inspiration difference (E-I-
Difference) will be calculated (see table), reflecting cardiac parasympathetic activity.  
 
Valsalva maneuver: In a supine position, subjects will be asked to blow into a mouth piece connected to an 
anaeroid manometer. They will be asked to hold it at a pressure of 40 mmHg during 15 seconds. During this 
performance, heart rate is recorded. This maneuver will be performed 3 times. From this maneuver the 
Valsalva ratio can be calculated (see table), reflecting both cardiac sympathetic and parasympathetic function. 
 
Orthostasis: Subjects will be asked to stand up from a supine position. Prior to this action and up to 2 minutes 
afterwards, heart rate and blood pressure will be recorded. A characteristic immediate increase in heart rate 
is observed around the 15th beat after standing and a compensatory overshoot bradycardia is seen around the 
30th beat. This physiologic response is used to calculate the “30:15-ratio” or R-R interval Max/Min (see table) 
and is a marker for cardiac parasympathetic function. 
In response to standing, blood pressure will usually fall and slowly rise again. The fall in blood pressure will be 
reported as the difference between the positions in systolic blood pressure and reflects cardiac sympathetic 
function. 
 
Each separate test will be started with a resting period of 1 minute between the tests, to prevent influences by 
previous test conditions. It must be emphasized that throughout all procedures the participant must remain 
silent.  
 
Screening 
During the screening visit, Ewing tests will be performed as stated above (deep breathing, Valsalva and 
orthostasis). For this, cut-off values for ‘normal’, ‘borderline’ and ‘abnormal’ will be used as described by Ewing 
(3).  
 
 

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References 
 (1)  Eeftinck Schattenkerk DW, van Lieshout JJ, van den Meiracker AH, Wesseling KR, Blanc S, Wieling W, et al. Nexfin 
noninvasive continuous blood pressure validated against Riva-Rocci/Korotkoff. Am J Hypertens 2009 
Apr;22(4):378-83. 
 (2)  Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Diabetes Care 2003 May;26(5):1553-
79. 
 (3)  Ewing DJ, Martyn CN, Young RJ, Clarke BF. The value of cardiovascular autonomic function tests: 10 years 
experience in diabetes. Diabetes Care 1985 Sep;8(5):491-8. 
 (4)  Kahn R. Proceedings of a consensus development conference on standardized measures in diabetic neuropathy. 
Autonomic nervous system testing. Diabetes Care 1992 Aug;15(8):1095-103. 
 (5)  Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the 
European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 1996 
Mar 1;93(5):1043-65. 
 (6)  Akselrod S, Gordon D, Ubel FA, Shannon DC, Berger AC, Cohen RJ. Power spectrum analysis of heart rate 
fluctuation: a quantitative probe of beat-to-beat cardiovascular control. Science 1981 Jul 10;213(4504):220-2.
 
Table: Frequently used measures of cardiovascular autonomous nervous system function 
Measure 
Unit 
Definition 
During spontaneous breathing in a supine position    
Mean NN 
ms 
The mean of all normal to normal, i.e. sinus rhythm, RR intervals 
SDNN 
ms 
The standard deviation of all normal to normal, i.e. sinus rhythm, RR 
intervals 
LF Power 
ms2 
Low frequency power, in absolute units: energy in the power spectrum 
between 0.004 and 0.12 Hz 
HF Power 
ms2 
High frequency power, in absolute units: energy in the power spectrum 
between 0.12 and 0.40 Hz 
LF/HF 
 
The ratio of low frequency power to high frequency power 
LF/(LF+HF) 
 
The ratio of low frequency power to the sum of the low and high 
frequency power 
HF/(LF+HF) 
 
The ratio of high frequency power to the sum of the low and high 
frequency power 
Baroreflex 
sensitivity 
(BRS) 
ms mmHg-1 
A measure of baroreflex sensitivity, computed as gain, i.e. ratio of the 
energy in the cross-spectrum of systolic blood pressure and RR intervals 
and the energy in the power spectrum of the RR interval; all between 
0.05 and 0.15 Hz and with a squared coherence (γ2) of 0.5 or higher 
(BRS can also be calculated during deep breathing). 
During Valsalva maneuvre 
Valsalva ratio 
 
The longest RR interval divided by the shortest RR interval within 45 
seconds of peak heart rate, averaged over the three consecutive 
maneuvers 
During deep breathing  
E-I-Difference 
ms 
The difference in maximum and minimum RR interval duration during 
expiration and inspiration, averaged over the six consecutive breaths 
During an active change in position from lying to standing 
RR Max 
ms 
The difference between the mean RR interval during 1 min of rest prior 
to standing up and the minimum RR interval within 15 s after standing 
up 
RR Max/Min  
(or “30:15”) 
 
Maximum RR interval between 15 and 30 s after standing up divided by 
minimum RR interval within 15 s after standing up 
SBP difference 
mmHg 
Systolic blood pressure after standing up (mean of 1.5–2 min after 
standing) minus systolic blood pressure in supine position 
 
 
 
Abbreviations: BRS: baroreflex sensitivity; HF: High Frequency; LF: Low Frequency; mmHg: millimeter mercury; ms: 
millisecond NN: normal-to-normal; RR: time span between two pulse beats; SBP: systolic blood pressure; SDNN: 
standard deviation of normal-to-normal. 
It must be noted that this table summarizes the frequently used measures. However, for the analysis, measures not 
listed in this table can be used. 

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APPENDIX G 
FECAL TESTS 
 
During this study, participants will collect multiple feces samples. The gut microbiome will be sequenced in 
order to gain insight in the association of the measured renal parameters with the intestinal flora (1-3). Also, 
the possible effect of GLP-1 receptor agonist therapy on the gut microbiome will be examined (1-3). Additional 
feces samples will be used to measure other explorative markers, i.e. calprotectin (marker for gut inflammation) 
(4). 
 
Collection 
Participants will receive a collection kit containing sample tubes, gloves and tools to transfer feces into the 
tubes. One tubes need to be frozen and stored immediately (at -20 ⁰C) in order to reduce bacterial growth. 
One tube can be stored in a dark and cool place. In order to prevent thawing of the frozen tubes, a container 
with ice packs will be provided.  
 
Sample handling 
After arrival at the clinical research unit, the research personnel will immediately store the frozen samples 
(meant for analysis of the gut microbiome)  at -80 ⁰C. The non-frozen sample will be homogenized and 
transferred to multiple smaller cups, which will also be stored at -20 ⁰C.  
 
Analyzes 
Microbiota analyses: using a multi-level system of increasing sophisticated measurements: 
1) 
All samples will be characterized using LCR/PCR analysis, a fast ‘screening’ technique, providing an 
insight in the 25 major phylogenetic bacterial groups and methanogens.  
2) 
A selection of 60 samples (n=10 in each treatment arm, at baseline and after long-term treatment) will 
be analyzed in detail using the ‘HITChip’ (Human Intestinal Tract phylogenetic microarray). The HITChip 
recognizes over 4800 specific 16S rRNA probes of intestinal microbes, identifying 1200 phylotypes (5;6).  
3) 
A subselection of 30 samples will be made based on the above analysis that will be targeted for 16S 
rRNA metagenomic sequencing (using 454 Titanium or other sequencing systems) as previously 
reported (6). These techniques allow deep sequencing, thereby able to map the entire microbiome.  
4) 
In order to provide a first hypothesis between diversity and function we will target 30 samples selected 
for metaproteomics analysis using advanced Fourier Transformation MS/MS analysis. We will analyze 
the generated sequences for specific diagnostic signatures that will be analyzed further by advanced 
functional search analysis. The selection of these subjects will take place at the end of the project when 
all clinical parameters and microbiota analysis have been completed.  
Calprotectin: this inflammatory markers will be assessed using an EliA Phadia-method. 
 
 
 
 
References 
 (1)  Burcelin R, Serino M, Chabo C, Blasco-Baque V, Amar J. Gut microbiota and diabetes: from pathogenesis to 
therapeutic perspective. Acta Diabetol. 2011 Dec;48(4):257–73.  
 (2)  Ramezani A, Raj DS. The gut microbiome, kidney disease, and targeted interventions. J Am Soc Nephrol. 2014 
Apr;25(4):657–70.  
 (3)  Sabatino A, Regolisti G, Brusasco I, Cabassi A, Morabito S, Fiaccadori E. Alterations of intestinal barrier and 
microbiota in chronic kidney disease. Nephrol Dial Transplant. 2014 Sep 4;  
 (4)   Judd TA, Day AS, Lemberg DA, Turner D, Leach ST. Update of fecal markers of inflammation in inflammatory bowel 
disease. J Gastroenterol Hepatol 2011 Oct;26(10):1493-9. 
 (5)  Rajilic-Stojanovic M, Heilig HG, Molenaar D, Kajander K, Surakka A, Smidt H, et al. Development and application of 
the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the 
abundant microbiota of young and elderly adults. Environ Microbiol 2009 Jul;11(7):1736-51. 
 (6)  Claesson MJ, O'Sullivan O, Wang Q, Nikkila J, Marchesi JR, Smidt H, et al. Comparative analysis of pyrosequencing 
and a phylogenetic microarray for exploring microbial community structures in the human distal intestine. PLoS One 
2009;4(8):e6669. 
 
 
 

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APPENDIX H 
MONITORING PROCEDURE 
 
Monitor 
The monitoring procedure will be performed by an independent monitor from the Clinical Research Bureau 
VUMC. 
 
Frequency 
Because of the anticipated moderate risk of this study, monitoring has to take place 2-3 times a year.  
 
Procedure 
General  
- 
Report of the amount of screened patients, inclusion rate, screen-failures 
- 
Verification of GLP-certification for laboratories 
 
Source Data Verification 
- 
100% verification for presence and completeness (names, dates, signatures) of the informed consent 
forms. Informed consent must be obtained prior to participation in the trial. 
- 
100% verification for in- and exclusion criteria for the first 5 participants, followed by 10% of the 
remaining subjects. When participants are wrongly included all dossiers may be verified. 
- 
10% verification of CRFs with source documentation 
- 
100% verification of reported SAEs and SUSARs. Report appropriately? Timely? Correct parties?  
- 
10% of participants will be checked for possible missed SAEs and SUSARs 
 
Investigational Medicinal Products (IMP) 
- 
Verification of GMP-certification for the involved pharmacies  
- 
Verification of storage times, conditions, sufficient supplies and whether IMPs are supplied only to 
those who are eligible to receive it 
- 
Verification that subjects are provided with instructions on the proper use of the IMPs 
- 
100% verification of receipt, use and return of the IMPs for Drug Accountability 
- 
Ensure that the most recent version of the SPC is present (for each IMP) 
 
Protocol 
- 
Verification that investigator(s) follow the approved protocol and amendment(s) 
- 
Verification of the presence of Standardized Operating Procedures for all study tests and whether 
study site personnel is adequately trained. 
- 
Determination of the maintenance of the essential documents (GCP, Chapter 8) 
 
Report 
The monitor will summarize his/her findings in an official report which will be handed to the PI. The ‘monitor 
visiting report’ will include the date, name of the monitor and the name of the investigator/individual(s) 
contacted. It should contain a summary of what the monitor reviewed and the monitor’s statements concerning 
the significant findings/facts, deviations and deficiencies, conclusions, actions taken or to be taken and/or 
actions recommended to secure compliance.  
 
 
 

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APPENDIX I 
RED substudy - RECOLAR 
 
Title: Renoprotective Effects of Combined dapagliflOzin and LosARtan (RED- RECOLAR) 
Background: This substudy elaborates on the primary results of the RED study, which seriously challenge 
our current understanding of the effects of SGLT-2 inhibitors on renal hemodynamics. From our results it 
seems that, in contrast to the current consensus based on results from animal studies and type 1 diabetes 
studies (1-3), the hypothesized protective hemodynamic mechanism in type 2 diabetes – a drop in glomerular 
pressure, indivated by an acute drop in eGFR and subsequent stabilization over time – is not due to afferent 
vasocontriction, but due to efferent vasodilatation. As is known, RAS inhibitors also act on this efferent 
arteriole, causing a protective vasodilative effect (1). The population of the RED study exists of participants 
treated with a RAS inhibitor (participants with hypertension were only included if treated with a RAS inhibitor) 
and non-hypertensive participants without RAS inhibition. Moreover, RAS inhibition using angiotensin-
converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) was both allowed. This 
hetergenity in RAS inhibition use hampers clinical interpretation of the results. This substudy will assess and 
untangle the acute hemodynamic effects of RAS inhibition and SGLT-2 inhibition using a simplified version of 
the renal protocol used in the RED study both in combination- and mono therapy in a T2DM population with 
identical in- and exclusion criteria used in the RED study.  
Hypothesis: Mono- and combination therapy with SGLT-2 inhibitor dapagliflozin and RAS inhibitor losartan 
may confer renoprotection by improving renal hemodynamics in T2DM.  
We will test this hypothesis by addressing the following research objectives:  
Primary objective: what are the acute effects (i.e. after 3 days of drug exposure) of mono- and combination 
therapy with the SGLT-2 inhibitor dapagliflozin and RAS inhibitor losartan on renal hemodynamics (glomerular 
filtration rate (GFR) / effective renal plasma flow (ERPF)) in metformin-treated T2DM patients?  
Secondary objectives: renal tubular function, renal damage markers, blood pressure, heart rate, body 
anthropometrics, markers of inflammation and (cardiovascular-)biomarkers.  
 
Methods:  
Design: An randomized, cross-over, mechanistic substudy to assess the effect of 3 days of mono- and 
combination therapy with the SGLT-2 inhibitor dapagliflozin 10 mg QD and RAS inhibitor losartan 50 mg QD 
on renal hemodynamics (glomerular filtration rate (GFR) / effective renal plasma flow (ERPF)) in metformin-
treated T2DM patients. 
Patients: 12 patients with T2DM (male and female; HbA1c 6.5-9%); aged 35-75 years; BMI ≥25 kg/m2. Same 
in- and exclusion criteria as the RED study except for antihypertensive agent use. Only use of alpha blockers 
is allowed as background therapy. Patients using an antihypertensive agent will be considered if this agent 
can be stopped (i.e. blood pressure adequate to stop at screening) or replaced by an alpha blocker. In these 
patients, a 4 week wash-out/run-in period will be observed prior to visit 2.  
Study Endpoints and methods: Renal hemodynamics, i.e. GFR and ERPF will be measured by the gold-
standard fasting iohexol and para-aminohippurate continue infusion methods, respectively; renal tubular 
function will be measured by 24-hour urine sodium, potassium, chloride, calcium, magnesium, phosphate and 
urea and glucose; markers of renal damage will include 24-hour urinary creatinine and albumin excretion, 
neutrophil gelatinase-associated lipocalin and kidney injury molecule-1; blood pressure and heart rate will be 
measured using an automated oscillometric blood pressure device (Dinamap®); body anthropometrics, 
including body weight, height, body-mass index and body fluid contents (by bio-impedance spectroscopy) will 
be measured; additional urine and blood samples will be obtained to determine markers of inflammation and 
(cardiovascular)-biomarkers. 
 
Expected results: This cross-over mechanistic substudy will expand our knowledge regarding the potential 
renoprotective effects of mono- and combination therapy with SGLT-2 inhititors and RAS in human T2DM. We 
hypothesize that SGLT-2 inhibitors and ARBs may confer renoprotection by improving renal hemodynamics. 
Moreover, combined use might lead to a additive or even synergistic effect, since we know from the RED study 

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that both agents act on the efferent arteriole. Ultimately, we expect that this study will contribute to guiding 
clinicians in their decision-making process of individualizing therapies for T2DM patients at risk for, or suffering 
from, DKD. 
Figure. Test vistit with simplified renal protocol.  
 
 
(1)  van Bommel EJ, Muskiet MH, Tonneijck L, Kramer MH, Nieuwdorp M, van Raalte DH. SGLT2 Inhibition in the Diabetic 
Kidney-From Mechanisms to Clinical Outcome. Clin J Am Soc Nephrol. 2017 Apr 3;12(4):700-710  
 (2)  Cherney DZ, Perkins BA, Soleymanlou N, Maione M, Lai V, Lee A, Fagan NM, Woerle HJ, Johansen OE, Broedl UC, 
von Eynatten M. Renal Hemodynamic Effect of Sodium-Glucose Cotransporter 2 Inhibition in Patients With Type 1 
Diabetes Mellitus. Circulation. 2014 Feb 4;129(5):587-97. 
 (3)  Kidokoro K1, Cherney DZI2, Bozovic A3, Nagasu H1, Satoh M4, Kanda E5, Sasaki T1, Kashihara N4. Evaluation of 
Glomerular Hemodynamic Function by Empagliflozin in Diabetic Mice using In Vivo Imaging. Circulation. 2019 Feb 
18 (4)   Judd TA, Day AS, Lemberg DA, Turner D, Leach ST. Update of fecal markers of inflammation in inflammatory 
bowel disease. J Gastroenterol Hepatol 2011 Oct;26(10):1493-9. 
 
 

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APPENDIX J 
RED substudy - REGROUP 
 
Title: Renoprotective Effects of dapaGliflozin in vaRisOUs Populations (RED-REGROUP) 
Background: This substudy elaborates on the primary results of the RED study, which led us to the research 
question whether the effects that we observe are dependent of hyperglycemia and whether they are preserved 
in patients with diabetic kidney disease. From our results it is clear that dapagliflozin affects renal 
hemodynamics in type 2 diabetes patients with adequate kidney function. However, if these effects remain the 
same in other populations, such as T2DM patients with impaired renal function, non-diabetic patients with 
impaired renal function and non-diabetic patients without renal impairment is currently unknown. This substudy 
will assess the acute hemodynamic effects of dapaglifozin in four different populations using a simplified 
version of the renal protocol used in the RED study.  
Hypothesis: Treatment with SGLT-2 inhibitor dapagliflozin may confer renoprotection by improving renal 
hemodynamics regardless of renal function or the presence of diabetes.  
We will test this hypothesis by addressing the following research objectives:  
Primary objective: what are the acute effects (i.e. after 3 days of drug exposure) of treatment with SGLT-2 
inhibitor dapagliflozin on renal hemodynamics (glomerular filtration rate (GFR) / effective renal plasma flow 
(ERPF)) in either metformin-treated T2DM patients with normal kidney function, or metformine-treated T2DM 
patients with impaired kidney function, or non-diabetic individuals with impaired kidney function, or non-diabetic 
patients without impaired renal function?  
 
Secondary objectives: renal tubular function, renal damage markers, blood pressure, heart rate, body 
anthropometrics, markers of inflammation and (cardiovascular-)biomarkers.  
 
Methods:  
Design: An open-label, parallel-group, mechanistic substudy to assess the effect of 3 days of treatment with 
SGLT-2 inhibitor dapagliflozin on renal hemodynamics (glomerular filtration rate (GFR) / effective renal plasma 
flow (ERPF)) in T2DM patients with normal kidney function, T2DM patients with impaired renal function, non-
diabetic patients with impaired renal function and non-diabetic patients without renal impairment but with 
hypertension.  
Patients: Hypertensive patients (male and female); aged 35-75 years; BMI ≥25 kg/m2; using RAS inhibition  
 
T2DM with an eGFR (CKD-EPI) above 75 mL/min/1.73m2 at the 
Screening Visit or 
 
T2DM with an eGFR (CKD-EPI) between 25 and 50 mL/min/1.73m2 at the 
Screening Visit or 
 
Non-diabetic with an eGFR (CKD-EPI) between 25 and 50 mL/min/1.73m2 at 
the Screening Visit or 
 
Non-diabetic with an eGFR (CKD-EPI) above 75 mL/min/1.73m2 at 
the Screening Visit. 
 
Diagnosis of T2DM according to RED study protocol. Other in- and exclusion criteria are the same as in the 
RED study. 
 
Study Endpoints and methods: Renal hemodynamics, i.e. GFR and ERPF will be measured by the gold-
standard fasting iohexol and para-aminohippurate continue infusion methods, respectively; renal tubular 
function will be measured by 24-hour urine sodium, potassium, chloride, calcium, magnesium, phosphate and 
urea and glucose; markers of renal damage will include 24-hour urinary creatinine and albumin excretion, 
neutrophil gelatinase-associated lipocalin and kidney injury molecule-1; blood pressure and heart rate will be 
measured using an automated oscillometric blood pressure device (Dinamap®); body anthropometrics, 
including body weight, height, body-mass index and body fluid contents (by bio-impedance spectroscopy) will 

                                         Renoprotective Effects of Dapagliflozin in Type 2 Diabetes 
 
 
 
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Kramer, van Raalte 
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be measured; additional urine and blood samples will be obtained to determine markers of inflammation and 
(cardiovascular)-biomarkers;  
 
Expected results: This parallel-group, mechanistic substudy will expand our knowledge regarding the 
potential renoprotective effects of treatment with SGLT-2 inhititor dapagliflozin in various populations. We 
hypothesize that SGLT-2 inhibitors may confer renoprotection by improving renal hemodynamics not only in 
the diabetic population with normal kidney function, but also in the population without diabetes or with impaired 
kidney function. We expect that this study will contribute to guiding clinicians in their decision-making process 
of individualizing therapies for T2DM patients at risk for, or suffering from, DKD. 
Figure. Test visit with simplified renal protocol.