The Renal Hemodynamic Effect of SGLT2 Inhibition in ... · DOI: 10.1161/CIRCULATIONAHA.113.005081 2...

DOI: 10.1161/CIRCULATIONAHA.113.005081

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The Renal Hemodynamic Effect of SGLT2 Inhibition in Patients with

Type 1 Diabetes

Running title: Cherney et al.; SGLT2 inhibition and renal hyperfiltration

David Z. I. Cherney, MD, PhD1*; Bruce A. Perkins, MD, MPH2*; Nima Soleymanlou, PhD3*;

Maria Maione, RN1; Vesta Lai, RN1; Alana Lee, RN1; Nora M. Fagan, MS4; Hans J. Woerle,

MD5; Odd Erik Johansen, MD, PhD5; Uli C. Broedl, MD5†; Maximilian von Eynatten, MD4†

1Dept of Medicine, Division of Nephrology; 2Dept of Medicine, Division of Endocrinology,

Toronto General Hospital, University of Toronto, Toronto, Canada; 3Boehringer Ingelheim

Canada Ltd./Ltée, Burlington, Canada; 4Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield,

CT; 5Boehringer Ingelheim Pharma GmbH & Co.KG, Ingelheim, Germany

*contributed equally as primary co-authors; †contributed equally as senior co-authors

Address for Correspondence:

David Cherney, MD, PhD, FRCP(C)

Toronto General Hospital

585 University Ave, 8N-845

Toronto, Ontario M5G 2N2

Canada

Tel: 416-340-4151

Fax: 416-340-4999

E-mail: [email protected]

Journal Subject Codes: Diabetes:[189] Type 1 diabetes, Treatment:[27] Other treatment, Vascular biology:[97] Other vascular biology, Hypertension:[193] Clinical studies

MD ; Odd Erik Johansen, MD, PhD ; Uli C. Broedl, MD †; Maximilian von Eyynanattttenen, , MDMD †

1Dept of Medicine, Division of Nephrology; 2Dept of Medicine, Division of Endocrinology,

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Abstract

Background—The primary objective of this mechanistic open-label, stratified clinical trial was

to determine the effect of 8 weeks’ sodium glucose cotransporter 2 (SGLT2) inhibition with

empagliflozin 25 mg QD on renal hyperfiltration in subjects with type 1 diabetes (T1D).

Methods and Results—Inulin (glomerular filtration rate; GFR) and paraaminohippurate

(effective renal plasma flow; ERPF) clearances were measured in individuals stratified based on

having hyperfiltration (T1D-H, GFR 135 ml/min/1.73m2, n=27) or normal GFR (T1D-N, GFR

90-134 ml/min/1.73m2, n=13) at baseline. Renal function and circulating levels of renin-

angiotensin-aldosterone system (RAAS) mediators and nitric oxide (NO) were measured under

clamped euglycemic (4-6 mmol/L) and hyperglycemic (9-11 mmol/L) conditions at baseline and

end of treatment. During clamped euglycemia, hyperfiltration was attenuated by -33

ml/min/1.73m2 with empagliflozin in T1D-H, (GFR 172±23 to 139±25 ml/min/1.73 m2, p<0.01).

This effect was accompanied by declines in plasma NO and ERPF and an increase in renal

vascular resistance (all p<0.01). Similar significant effects on GFR and renal function parameters

were observed during clamped hyperglycemia. In T1D-N, GFR, other renal function parameters,

and plasma NO were not altered by empagliflozin. Empagliflozin reduced HbA1c significantly

in both groups, despite lower insulin doses in each group (p 0.04).

Conclusions—In conclusion, short-term treatment with the SGLT2 inhibitor empagliflozin

attenuated renal hyperfiltration in subjects with T1D, likely by affecting tubular-glomerular

feedback mechanisms.

Clinical Trial Registration Information—www.clinicaltrials.gov. Identifier: NCT01392560.

Key words: Renal hyperfiltration, SGLT2 inhibition, tubuloglomerular feedback, diabetic nephropathy, nitric oxide, renal physiology, renin angiotensin system, tubular transport, hypertension, kidney, diabetes (kidney)

end of treatment. During clamped euglycemia, hyperfiltration was attenuated by -33333

ml/min/1.73m2 with empagliflozin in T1D-H, (GFR 172±23 to 139±25 ml/min/1.1.737373 mmm222,,, p<p<p<0.00.010101).).)

This effect was accompanied by declines in plasma NO and ERPF and an increase in renal

vascsculululararar rrresesesisisistat ncncceee (a( ll p<0.01). Similar significcananantt t effects on GFR annd d d rerer nal function parameters

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Introduction

Hyperfiltration is an early renal hemodynamic abnormality in experimental models of diabetes

and is thought to reflect increased intraglomerular pressure 1-3. In humans, the prevalence of

renal hyperfiltration in subjects with type 1 diabetes (T1D) has been reported to be as high as

60% and this condition is accompanied by a significantly increased risk for development of

diabetic nephropathy in many studies 4-6. The pathogenesis of hyperfiltration, however, is

complex and involves changes in both neurohormonal/vascular factors (the “vascular

hypothesis”) as well as ‘tubuloglomerular feedback’ mechanisms (TGF – the “tubular

hypothesis”) 7. Translational clinical studies aiming to attenuate renal hyperfiltration so far have

exclusively focused on the vascular hypothesis through the use of pharmacological interventions

that modulate renal arteriolar tone, such as cyclooxygenase-2 inhibitors, renin-angiotensin-

aldosterone system (RAAS) blockers, and nitric oxide (NO) synthase inhibitors such as NG

monomethyl L-arginine (L-NMMA) and investigational protein kinase C inhibitors 8-11.

However, targeting renal arterial tone via blockade of neurohormonal factors has thus far largely

neglected the role of TGF (Figure 1) in the pathogenesis of renal hyperfiltration in humans 8-11.

The tubular hypothesis is based upon an increase in proximal tubular glucose delivery

due to chronic hyperglycemia in diabetes. This leads to a maladaptive increase in glucose

reabsorption along with sodium via the sodium-glucose cotransporter 2 (SGLT2) in the proximal

tubule. As a result, distal sodium chloride delivery to the macular densa is decreased 12. This

distal tubular condition is sensed as a low effective circulating volume stimulus at the level of the

juxtaglomerular apparatus, causing an afferent renal vasodilatory response (Figure 1b). The

consequence of this altered TGF results in supranormal GFR values into the hyperfiltration

range. Targeting TGF in renal hyperfiltration has shown promising results in experimental

exclusively focused on the vascular hypothesis through the use of pharmacologiciccalal iintnttereerveveventntntioiionsf

hat modulate renal arteriolar tone, such as cyclooxygenase-2 inhibitors, renin-angiotensin-

alldodoostststeererononne e e syysttememem (RAAS) blockers, and nitricc oooxxiide (NO) syntthahah se iiinhnhnhiibitors such as NG

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animal models by using phlorizin, a non-specific inhibitor of the renal tubular glucose

transporters SGLT1 and SGLT2 13, 14. The clinical relevance of these findings, however, could

not be conclusively studied in humans, because of the poor tolerability of phlorizin due to its low

selectivity for SGLT2, SGLT1 inhibition-related gastrointestinal side effects and very limited

oral bioavailability 14. Subsequent studies with selective SGLT2 inhibitors in animals have also

shown similar significant effects on renal hyperfiltration 15.

More recently, several highly selective SGLT2 inhibitors have been developed for use in

clinical trials in patients with type 2 diabetes (T2D) 16, 17. These compounds generally do not

affect SGLT1 at clinical doses and have pharmacological features that allow once daily oral

dosing. In T2D, this class of drugs is well-tolerated and has consistently improved glycemic

control, along with weight loss, and antihypertensive effects 17, 18. Available evidence for SGLT2

inhibitors in T1D is however limited, and is mainly derived from experimental animal models.

Only one clinical pilot study has been conducted, which demonstrated that a single dose of

remogliflozin improved postprandial glucose profiles 19. Based on previous findings with

phlorizin and other SGLT2 inhibitors in animals, the concept of altering renal hyperfiltration by

blocking renal glucose absorption with SGLT2 inhibitors is intriguing, since reducing this

surrogate marker of intraglomerular pressure is renal protective in experimental models of

diabetes 13, 20, 21. However, potential renal hemodynamic effects of these drugs in subjects with

diabetes including effects on renal hyperfiltration remain unknown.

Accordingly, the primary objective of this 8-week, open-label, stratified clinical trial

(NCT01392560) was to determine the impact of treatment with the oral and highly selective

SGLT2 inhibitor empagliflozin (Boehringer Ingelheim), 25 mg qd, on renal hyperfiltration in

subjects with T1D. Based on previous work in animals, we hypothesized that SGLT2 inhibition

dosing. In T2D, this class of drugs is well-tolerated and has consistently improveveeddd glglg ycycycemmemicicic

control, along with weight loss, and antihypertensive effects 17, 18. Available evidence for SGLT2

nnhihiibibibitototorsrs iiin nn TTT1DDD iisis however limited, and is mainininlyly derived from m exee peperiririmmmental animal models.

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with empagliflozin would reduce renal hyperfiltration in patients with uncomplicated T1D under

controlled conditions of clamped euglycemia and hyperglycemia. This is the first-in-class study

to translate experimental animal findings of renal SGLT2 inhibition into the clinical setting of

renal hyperfiltration in patients with T1D.

Materials and Methods

Study participants

The flow chart for participants is shown in Figure 2. Forty participants successfully completed

the study and had the primary GFR endpoint evaluated, including 13 normofiltering subjects

(defined by GFR values 90 – 134 ml/min/1.73m2; T1D-N) and 27 individuals with renal

hyperfiltration (defined by GFR 135 ml/min/1.73m2 (9); T1D-H) (Table 1). In more detail,

inclusion criteria at screening were: 1. Male or female subject diagnosed with T1D 12 months

prior to informed consent; 2. Age 18 years; 3. HbA1c of 6.5%-11.0%; 4. Body Mass Index

(BMI) 18.5-35.0 kg/m2; 5. Experienced insulin pump users ( 3 months of use prior to the study

and willing to use the same insulin pump during the study) or be on multiple daily injections; 6.

Ability to follow an established carbohydrate counting method and an insulin titration algorithm

based on investigator recommendations; 7. Stable glycemic status (latest HbA1c, measured 2-12

months prior to screening, within 1.5% of baseline value) and 8. Estimated GFR 60

ml/min/1.73m². Exclusion criteria at screening were: 1. Evidence of macroalbuminuria; 2.

Leukocyte and/or nitrite positive urinalysis; 3. Any concomitant medication known to interfere

with RAAS activity and/or renal function based on investigator judgement; 4. Severe

hypoglycaemia that required emergency hospital treatment within 3 months prior to screening; 5.

History of organ transplantation, cancer, liver disease, macrovascular disease, autonomic

defined by GFR values 90 – 134 ml/min/1.73m2; T1D-N) and 27 individuals wiwithtth rrrennnalala

hyperfiltration (defined by GFR 135 ml/min/1.73m2 (9); T1D-H) (Table 1). In more detail,

nnclcllusususioioionn crcrcriititeeriaaa aaatt screening were: 1. Male or fffemememaale subject diagagagnoseseedd d wiw th T1D 12 months

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neuropathy, proliferative retinopathy, brittle diabetes or hypoglycaemia unawareness based on

investigator judgement; 6. Total daily insulin requirement > 1.5 U/kg; 7. Bariatric surgery or

other gastrointestinal surgeries that induce chronic malabsorption within the past two years; 8.

Treatment with anti-obesity drugs, surgery or aggressive diet regimen leading to unstable body

weight three months prior to screening; 9. Treatment with systemic steroids; 10. Blood

dyscrasias or any disorders causing hemolysis or unstable red blood cells; 11. Pre-menopausal

women who were nursing, pregnant, or of child-bearing potential and not practising an

acceptable method of birth control; 12. Participation in another trial with an investigational drug

within 30 days prior to informed consent and 13. Alcohol or drug abuse within three months

prior to informed consent that would interfere with trial participation or any ongoing clinical

condition that would jeopardize subject safety or study compliance based on investigator

judgement. The local Research Ethics Board at the University Health Network (Toronto,

Canada) approved the protocol and all subjects gave informed consent prior to start of study

procedures. The study was conducted according to the International Conference on

Harmonization on Good Clinical Practice.

Experimental Design

This clinical trial comprised 6 sequential phases (Figure 3): 1) a one-week screening period

(Visit 1 to Visit 2); 2) a two-week placebo run-in period (Visit 2 to Visit 3), 3) two back-to-back

days of baseline renal assessments conducted under controlled euglycemic (Visit 3) and

hyperglycaemic (Visit 4) clamp conditions; 4) an eight-week open-label treatment period with

empagliflozin 25 mg QD including telephone (Visits 5, 6, 7, 9, and 10) and in-hospital visits

(Visit 8 and Visit 11); 5) two back-to-back days of renal assessments under controlled

euglycemic (Visit 12) and hyperglycemic (Visit 13) clamp conditions; and finally 6) a two-week

prior to informed consent that would interfere with trial participation or any onggooioingngng ccclililinininicacacalll

condition that would jeopardize subject safety or study compliance based on investigator

uudgdgdgememmenenntt.t. ThTT ee lololocac l Research Ethics Board at tttheheh University HeHeeaalth h NeNeNetwork (Toronto,

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post-treatment observation period with 2 in-hospital visits (Visit 14 and Visit 15). At Visit 2,

participants were instructed to document their daily glucose levels, insulin and carbohydrate

intake for the remainder of the study using a patient diary. In order to avoid effective circulating

volume contraction and RAAS activation from sodium depletion, or hyperfiltration due to high

protein intake, participants were instructed to adhere to a high-sodium (>140 mmol/day),

moderate protein diet (<1.5 g/kg/day) for 7 days leading up to Visits 3-4 and Visits 12-13.

Treatment compliance was assessed based on tablet count of dispensed and returned medication

and was required to be 80% to 120% of the treatment dose.

On the day before the physiology experiments, HbA1c, 24-hour urinary albumin excretion

rate, and 24-hour urine sodium, urea and glucose were measured. For baseline and post-treatment

physiological experiments (Visit 3 and Visit 12), euglycemic (4-6 mmol/L) conditions were

maintained for approximately 4 hours preceding and during all investigations 10. On the

following days (Visit 4 and Visit 13), identical experiments were repeated under clamped

hyperglycemic conditions (9-11 mmol/L). During the clamp procedures, blood glucose was

maintained at a stable level as described previously 10.

Experiments were performed during clamped euglycemia and hyperglycemia for several

reasons. First, the definition of hyperfiltration in our previous work is GFR 135 ml/min/1.73m2

during clamped euglycemia 8-11, 22, 23. This definition based on ambient euglycemia is important

since acute induction of modest hyperglycemia can induce a hyperfiltration response, possibly

through neurohormonal activation, especially in normofiltering individuals 10, 24. Our second

reason for controlling ambient glycemic conditions was to separate the effects of systemic

hyperglycemia resulting in glucosuria (i.e. essentially uncontrolled diabetes) from increased

glucosuria with simultaneous euglycemia. The latter can be achieved experimentally by

ate, and 24-hour urine sodium, urea and glucose were measured. For baseline aanndnd pppossst-t-t-trtrtreaeaeatmtmt en

physiological experiments (Visit 3 and Visit 12), euglycemic (4-6 mmol/L) conditions were

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combining empagliflozin with a systemic euglycemic clamp. The establishment of a

physiological state of euglycemia allowed us to isolate the effects of modulating

tubuloglomerular feedback, while at the same time minimizing neurohormonal activation by

systemic hyperglycemia. Third, acute, modest hyperglycemia can raise blood pressure and

influence circulating neurohormonal mediators in patients with uncomplicated T1D 24, 25. Since

the effects of empagliflozin on blood pressure and circulating neurohormones were pre-defined

outcomes of this trial, we performed all studies under clamped euglycemic and hyperglycemic

conditions to reduce background variability in these factors. Finally, participants were also

studied under clamped hyperglycemic conditions to determine if the added renal glucose load

would result in enhanced TGF and renal hemodynamic effects. The inclusion of the

hyperglycemic day was also important to capture renal data during ambient conditions reflective

of clinical practice, which frequently includes episodes of hyperglycemia.

During steady state euglycemic or hyperglycemic clamp conditions, blood samples were

collected in order to assess the following parameters: inulin and paraaminohippurate (PAH),

plasma renin activity (PRA), angiotensin II, aldosterone. At corresponding time intervals, urine

samples were collected from which urinary NO, prostaglandin E2, D2, F1 and thromboxane B2

(all corrected for urinary creatinine concentration), were measured. Mean arterial pressure

(MAP) and heart rate were measured by an automated sphygmomanometer over the right

brachial artery (DINAMAP® sphygmomanometer, Critikon, Florida, USA) throughout the study.

For changes in insulin therapy after starting empagliflozin, study subjects were initially

instructed to reduce their prandial insulin by 30% at the start of the treatment phase one day

following V4. As an additional safety measure against nocturnal hypoglycemia, subjects were

also required to reduce basal insulin by 30%. All insulin dose adjustments were performed under

would result in enhanced TGF and renal hemodynamic effects. The inclusion of f thtthe ee

hyperglycemic day was also important to capture renal data during ambient conditions reflective

off ccclililinininicacalll prprp aaactiicecece, which frequently includes epipipisooodes of hyperglglglycy ememmiaiaia.

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the investigators’ guidance based on home blood glucose measurements.

Assessment of Renal Hemodynamic Function

After ambient clamp euglycemia or hyperglycemia was stabilized, inulin and PAH were infused

via a third intravenous line. The infusion was primed with 25% inulin (60 mg/kg) and 20% PAH

(8 mg/kg), which were infused continuously at a rate calculated to maintain plasma

concentrations at 20 mg/dl and 1.5 mg/dl, respectively. After a 90-minute equilibration period,

blood samples were collected for inulin, PAH and hematocrit (HCT) with additional blood being

drawn 30 minutes later. GFR and ERPF were estimated under steady state conditions of infusing

inulin and PAH, respectively 10. Two independent clearance periods were used to calculate mean

baseline GFR and ERPF values at baseline and after treatment with empagliflozin. Renal blood

flow (RBF) was derived using ERPF / (1-HCT). Renal vascular resistance (RVR) was derived by

dividing MAP by RBF. All renal hemodynamic measurements were adjusted for body surface

area.

Sample Collection and Analytical Methods

Inulin and PAH: Blood samples were immediately centrifuged at 3000 rpm for 10 minutes at 4°

C. Plasma was separated, placed on ice, and stored at -70° C before assay. Inulin and PAH were

measured in serum by colorimetric assays using anthrone and N- (1-naphthy) ethylenediamine,

respectively.

Angiotensin II: Blood samples were collected into pre-chilled tubes containing EDTA

and angiotensinase inhibitor (0.1 ml Bestatin Solution, Buhlmann Laboratories, Switzerland).

After centrifugation, plasma was stored at -70oC until analysis. On the day of analysis, plasma

samples were extracted on phenylsilysilica columns followed by a competitive angiotensin II

radioimmunoassay kit supplied by Buhlmann Laboratories AG (Switzerland). Aldosterone was

baseline GFR and ERPF values at baseline and after treatment with empagliflozizinnn. RRRenennalalal bbblololoodod mm

flow (RBF) was derived using ERPF / (1-HCT). Renal vascular resistance (RVR) was derived by

diiviviidididinngng MMMAPAPAP bbyyy RRRBF. All renal hemodynamic mememeasurements wwerere e aadjdjdjuususted for body surface

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measured by radioimmunoassay (Coat-A-Count system, Siemens). Renin Activity: PRA was

measured with a radioimmunoassay kit (GammaCoat® Plasma Renin Activity 125I RIA Kit, CA-

1533, Diasorin, Stillwater, Minnesota, USA). PRA determination involves an initial incubation

of plasma to generate angiotensin I, followed by quantitation of angiotensin I by

radioimmunoassay. In the GammaCoat® PRA 125I RIA Kit, the antibody is immobilized onto the

lower inner wall of the GammaCoat tube. After incubation of standards, unknown samples and

125I angiotensin I in the GammaCoat tube, the reaction mixture is removed by aspiration and the

bound tracer counted in a gamma counter. A standard curve is constructed and the concentration

of angiotensin I of the unknown sample obtained by interpolation.

NO: To assess NO formation 26, plasma and urine NO levels were measured at baseline

and after 8 weeks of empagliflozin during clamped euglycemia and hyperglycemia. For NO

levels (NO, R&D Systems, Minneapolis, MN -total NO/Nitrite/Nitrate assay kit, Cat

No.KGE001) the assay determines NO concentration based on the enzymatic conversion of

nitrate to nitrite by nitrate reductase. The reaction is followed by colorimetric detection of nitrite

as an azo dye product of the Griess Reaction. The Griess Reaction is based on the two-step

diazotation reaction in which acidified nitrite produces a nitrosating agent which reacts with

sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-

naphthyl)ethylenediamine to form the chromophoric azo-derivative which absorbs light at 540-

570 nm. Urine NO concentrations were also measured accordingly and were corrected for

urinary creatinine concentrations. To assess possible influences of renal prostanoids on

hemodynamic function, urinary prostaglandin E2, D2, F1 and thromboxane B2 levels

(corrected for urinary creatinine) were measured as described previously 8.

Urinary albumin excretion rate was determined by immunoturbidimetry. 24-hour urine

NO: To assess NO formation 26, plasma and urine NO levels were measururrededd att t bababaseseselililinene

and after 8 weeks of empagliflozin during clamped euglycemia and hyperglycemia. For NO

eeveveelslsls (((NONONO,,, R&RR DDD SySyS stems, Minneapolis, MN -totoottat lll NO/Nitrite/NNititi ratetee aaassssay kit, Cat

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ninitrtrtratatateee toto nnnitititrirritete bbby y y ninittrtratatte e reredudud ccctatatasesese. TThThe ee rerereacaca tititiononon iss s fofoollllowowowededd bbbyy y cocoololol rririmmemetrtrricici dddetetececectttiononn ooof f niniitrritite

as an azo dyyee prprprododducucuct ofoff thehehe GGGririiesesesss ReReR acacactititiononon. ThThT e ee GrGrGriei ssssss RRReaeaeactctctioioion isisis bbbasasasededed oon n n ththhe e e twtwtwo-o step



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sodium, urea and glucose were assessed by standard laboratory methods. HbA1c was measured

by high-performance liquid chromatography. Other endpoints related to glucose-lowering effects

of empagliflozin in patients with T1D will be presented elsewhere.

Statistical analyses

The primary endpoint of this study was change from baseline in GFR after treatment with

empagliflozin 25mg QD for 8 weeks under stable euglycemic and hyperglycemic clamp

conditions. The primary hypothesis of interest and related primary analysis was to evaluate

change in GFR within T1D subjects with hyperfiltration. The selected sample size was chosen to

evaluate both within-group and between-group differences in GFR, where the two groups were

T1D subjects with (T1D-H) and without (T1D-N) renal hyperfiltration. Within the T1D-H group:

Assuming a standard deviation of 5 ml/min/1.73m2, a paired t-test, with a two-sided significance

level of 0.05 and a sample size of 4 subjects, would provide 80% power to detect a mean

difference in GFR of 11 ml/min/1.73m2 from baseline to 8 weeks. Between groups: Assuming a

standard deviation of 5 ml/min/1.73m2, a two-group t-test, with a two-sided significance level of

0.05 and a sample size of 12 subjects per group, would provide 80% power to detect a mean

difference in GFR of 6 ml/min/1.73m2 from baseline to 8 weeks. To have an adequate sample

size for both within-group and between-group comparisons, we therefore planned to include 12

participants per group. The effect sizes and standard deviations were estimated based on prior

experiments 10, 11. Paired t-tests were performed to evaluate within-group differences in GFR

after treatment with empagliflozin. Comparisons between the groups were performed using an

analysis of variance (ANOVA). Similar statistical analyses were performed on other renal

hemodynamic parameters, and on blood and urinary biochemical parameters.

T1D subjects with (T1D-H) and without (T1D-N) renal hyperfiltration. Within tthehehe TTT1DDD-H-HH grgrgrououpp

Assuming a standard deviation of 5 ml/min/1.73m2, a paired t-test, with a two-sided significance

eeveveelll ofofof 000.0.00555 aand dd aaa ssample size of 4 subjects, wouououlddd provide 80%% pppowwererer tto detect a mean

ddidiffffere ence in GFGFFR R offf 1111 mlmlml/m/m/miinin/1/1.7.7.73m3m3m2 ffrommm bbaseeeliine totoo 88 wweeeekskks. BeBettwtweeeeennn grggrououppps:: AAsAssuuumimimingngg a

ttananndadadardrdr ddeveveviiaiatitiononon oof f 55 mmlml/m/mminini /1/1/1 7.7.73m3m3 222,, a aa twtwtwo---grgrg oououpp p t---teteeststst, , wiwiiththth aaa tttwwwo-o-ssiddeded dd sisiigngngnififficiccannccece llevevvelel oof

0.05 and a samammplplple e e sisisizezee ooof 121212 sssububbjejejectctts ss pepeper r grgrgrouououp,p,p wwwouououldld ppprororovivividedede 8880%%% pppowowowererer ttoo dededetetetectctct aa mean



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Results

Baseline clinical and anthropometric characteristics

The study population comprised 27 patients in the T1D-H group (GFR 172±23 ml/min/1.73m2)

and 13 patients in the T1D-N group (GFR 117±11 ml/min/1.73m2). Overall, clinical and

anthropometric baseline characteristics and daily insulin doses were similar between the groups,

while carbohydrate intake tended to be higher in T1D-H (Table 1) 27. In T1D-N, all patients had

a diabetes duration of >5 years except for 1 individual. Similarly, in T1D-H, all patients had a

diabetes duration of >5 years except for 3 individuals. Mean diabetes duration values for each

group are reported in Table 1. HbA1c levels were similar in the two groups at baseline (Table 1).

Body weight was comparable between the groups (Table 1). Mean systolic and diastolic blood

pressure values were similar and within the normotensive range in each group (Table 2).

Effect of empagliflozin on renal function and blood pressure

Euglycemic clamp conditions

After 8 weeks of treatment, empagliflozin significantly lowered GFR by 33 ml/min/1.73m2 in

T1D-H under euglycemic conditions (baseline: 172±23, 8 weeks: 139±25 ml/min/1.73m2;

p<0.01; Figure 4 panel A). This GFR effect in T1D-H was accompanied by significant declines

in ERPF and RBF, a significant rise in RVR (p<0.01 for all) and a significant decline in systolic

blood pressure (p<0.05) (Table 2). In contrast, treatment with empagliflozin did not significantly

alter GFR or other renal parameters in T1D-N under euglycemic conditions (baseline: 117±11, 8

weeks: 126±15, p=0.15, Figure 4a). Consistent with these findings, the between-group

differences in change from baseline for GFR, ERPF, RBF and RVR for T1D subjects with and

without hyperfiltration during euglycemia were significant (p<0.01).

Hyperglycemic clamp conditions

Body weight was comparable between the groups (Table 1). Mean systolic and dddiaiiaststs olollicicic bbblololooodod

pressure values were similar and within the normotensive range in each group (Table 2).

EfEffefefectctct oofff ememempppaglglglifififlol zin on renal function andd bbblooood pressure

EEugglglycemic cclalampmpmp cconoondididitititionononss

AfAffteteter rr 888 weweekekekss ofoff trrereatatmemeentnt, eemempapapaglglgliififlolooziziinn n sisisignnnififi iciccaanntllyy y lololowwwererered d GFGFFR R R bybyby 3333 mlmlml/m/mmininin//1.7.7733mm22 iinn

T1D-H undeer r eueueuglglglycycycememmici cccononondidiititit onononsss (b(bbasasaselelelininine:e:e 11727272±2±2± 3,3,, 88 wwweeeeeeksksks: : 1313139±9±9±252525 mmml/l/mimimin/n/n 1.1.1 73737 m222;



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The significant effect of empagliflozin on GFR in subjects with T1D and hyperfiltration was

confirmed under hyperglycemic clamp conditions. GFR dropped significantly by 44

ml/min/1.73m2 (baseline: 186±33; 8 weeks: 142±29 ml/min/1.73m2; p<0.01; Figure 4 panel B).

This was again accompanied by significant changes in ERPF, RBF and RVR (p<0.01 for all) but

not by significant changes in blood pressure (Table 3). Consistent with euglycemic conditions,

empagliflozin had no effect on renal parameters in T1D-N in the hyperglycemic setting (GFR

baseline: 136±27, 8 weeks: 134±18, p=0.76, Figure 4b). Finally, the between-group differences

in change from baseline for GFR, ERPF, RBF and RVR for T1D subjects with and without

hyperfiltration were also significant during clamped hyperglycemia (p<0.01).

Effect of empagliflozin on RAAS mediators, NO and urinary prostanoids

During baseline clamped euglycemia, circulating levels of angiotensin II, aldosterone, PRA and

NO were similar between the groups (Table 2). After treatment with empagliflozin, angiotensin

II and aldosterone levels increased significantly in the T1D-H group, whereas only aldosterone

levels rose significantly in T1D-N. In addition, empagliflozin significantly lowered plasma NO

in T1D-H, but not in T1D-N (Table 2). Changes in circulating RAAS mediators and NO after

empagliflozin under euglycemia were similar to those observed during clamped hyperglycemia

(Table 3). Empagliflozin did not influence urinary NO or prostanoid excretion, aside from a

modest but significant rise in prostaglandin F1 in the T1D-H group, which was only observed

during clamped hyperglycemia (Table 3).

Effects of empagliflozin on glucose control, body weight and laboratory parameters

Significant declines in HbA1c in conjunction with significant decreases in total daily insulin

doses were observed within each group at end of treatment compared to baseline and these

changes were similar in the two groups (Table 1). Carbohydrate intake increased in both groups

Effect of empagliflozin on RAAS mediators, NO and urinary prostanoids

During baseline clamped euglycemia, circulating levels of angiotensin II, aldosterone, PRA and

NONOO wwweereree sisisimimim lar rr bbebetwt een the groups (Table 2). AAAfftter treatment wwwiti h ememmpppagliflozin, angiotensin

II aannnd aldosteroronnne levevele ss s ininincrcrreaeeasesed dd sisis ggnniifficaannttlly innn tthe TTT1D1DD-HH H grgrrouup,p wwwhheerereeasass oonlnlly yy alalaldodd stststererononone e

eevevevelslss rrrososee sisisigngnififficccananttlly y inin TT111D-D-D NN.N. IIIn n adadaddididitititiononn,, ememmppapaglglglififflololozzzin nn sisis ggngnififificici aaanttltly y lololowwewerereed d d plplaaasmmama NNNOO O

n T1D-H, buutt t nonon t t t ininin TTT1D1D1 -N-NN (((TaTaTablblb e e e 22).).). CCChahahangngngesese iin nn cicic rcccululu atatatinining g g RARAR ASASAS mmmededediaiai totoorsrsrs aaandndnd NNO after



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by similar amounts, and based on the larger sample size in T1D-H, within-group changes

reached significance in this group. Despite increased carbohydrate intake, body weight and body

mass index decreased to a similar extent in both groups (Table 1). 24-hour urinary glucose

excretion increased significantly in both groups after empagliflozin treatment (within-group

changes, p<0.01) with greater changes in glucose excretion in T1D-H vs. T1D-N (Table 1). 24-

hour urine volume did not increase significantly versus baseline in T1D-N (+13.6%; p=0.23).

However, there was a significant 56% rise in T1D-H (p<0.01). 24-hour urinary sodium excretion

rates were not significantly altered by empagliflozin in either group. Urinary albumin to

creatinine ratio was within the normal range at baseline and did not change at week 8 (Table 1).

After treatment with empagliflozin, there were no clinically relevant changes in the

biochemical and hematological parameters assessed, including serum sodium, potassium,

calcium, magnesium, chloride, phosphate, bicarbonate, blood count, erythropoietin, liver

enzymes, lactate dehydrogenase, creatine kinase, lipase, lipid profiles and uric acid. When

subjects from both groups were pooled there were, however, small but significant increases in

hematocrit (0.373 0.041 to 0.386 0.038; p<0.01) and blood urea (4.6 1.6 to 5.4 1.5 mmol/L;

p<0.01). These findings were also evident within each of the groups (Table 1).

Adverse events

Few adverse events related to renal function were reported. Pollakuria was reported by 33

(78.6%) patients, thirst by 31 (73.8%) patients, and 1 patient (2.4%) reported each of nocturia,

dysuria, and increase in urine output. No cases of acute renal failure or tubular necrosis occurred.

Two cases of diabetic ketoacidosis leading to discontinuation occurred during the study: one in

the context of insulin pump failure and the other in the setting of acute gastroenteritis. These

patients were withdrawn within the first 3 days of drug exposure and were not included in the

After treatment with empagliflozin, there were no clinically relevant chananngegees s ininin ttthehehe

biochemical and hematological parameters assessed, including serum sodium, potassium,

caalclcciuiuiumm,m, mmmagagagnesisisiuumum, chloride, phosphate, bicarrbbobonnnate, blood counununt, rr ererrytytythrh opoietin, liver

ennzyyzymes, lactaatete ddehehhydydy rororogegegenananassese,, crcrc eeaeattiinne kiinnaaase, liippassee,, , lilipppiddd prprofoffilileses aanandd uururicicc aacicicid.d.d WWWheeenn

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hematocrit ((0.0.37373733 0.0.0 04040 11 tooo 000.3. 868686 0.0.0 03338;8;8; ppp<0<0<0.0.001)1)1) aaandndnd bbblololoododod uuurererea aa (4(44.6.66 1.1.1.6 6 6 totot 555.4.44 1.1.1 555 mmol/L;



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analysis of the remaining 40 subjects. The overall incidence of reported adverse effects for the 42

patients who took study drug is reported in Table 4.

Discussion

Animal studies have demonstrated that acute and chronic SGLT2 blockade significantly reduced

hyperfiltration, confirming the crucial contribution of TGF in early renal hemodynamic

abnormalities related to diabetes 13, 15, 20. This experimental evidence was so far only explored in

one human study performed almost 8 decades ago. In this landmark study, Shannon et al

demonstrated that phlorizin, a non-selective inhibitor of SGLT1 and SGLT2, acutely reduced

GFR in five healthy humans 28. Unfortunately, due to poor tolerability of phlorizin, the role of

TGF in humans subsequently remained unstudied for the last 75 years due to a lack of

appropriate physiological probes to modulate TGF. The recent development of selective oral

SGLT2 inhibitors for the treatment of diabetes has led to the opportunity to study the tubular

component of diabetes-related renal hyperfiltration, providing a unique approach to connect

novel pharmaceutical interventions with the work by Shannon et al 28.

Our first major novel observation was that among T1D subjects with renal

hyperfiltration, treatment with empagliflozin for 8 weeks led to a significant reduction in

hyperfiltration during clamped euglycemic and hyperglycemic conditions. This, to our

knowledge, is the first study to demonstrate that pharmacological inhibition of SGLT2 attenuates

hyperfiltration in patients with diabetes. Corrections of elevated GFR in T1D-H during clamped

euglycemia resulted in a mean end-of-study GFR value that was close to the threshold for

normal. Attenuation of hyperfiltration by SGLT2 inhibition in our study was likely mediated via

effects on ERPF, RBF and RVR. This particular pattern of renal hemodynamic function change

GFR in five healthy humans 28. Unfortunately, due to poor tolerability of phloriziziin,n, tthehehe rrololole e e oofof

TGF in humans subsequently remained unstudied for the last 75 years due to a lack of

appprprpropopopriririatatatee e phphp ysssioioiolological probes to modulate TGGFGF.. The recent deevevv loopmpmpmene t of selective oral

SSSGLLLT2 inhibiitotorrrs fforor tthehee tttrrereatatatmmemenntnt ooof f didiabeettess haas lledd ttooo ththhe opopppoportrtununiiityyy ttoo ststtududy y ththhe e ttutubububulaalarr

coompmpmponono enentt t ofofof ddiiaabbebetetess-rrerelalateeedd rrrenenenalalal hhyypyperererfififilltrararatitit onoon, , , prprrovvvidididiining g g d aa ununniqiqiqueuee aaappppp rroroaacach h tototo coononnnenectctt

novel pharmamacececeututticicicalala iiintntn errrveveventnn ioioionsnsn wwwittth hh thththe e e wowoworkrkk bbby y y Shhhananannononon n n etetet al l l 282828..



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is compatible with pre-glomerular vasoconstriction. In contrast, renal hemodynamic parameters

remained unchanged in T1D subjects with normal renal function, suggesting that TGF does not

contribute significantly to the regulation of renal function in these individuals. Since changes in

weight, glycemic control, metabolic parameters and dietary factors after treatment with

empagliflozin were similar between the T1D-H and T1D-N groups, our results support the

concept that baseline differences in renal hemodynamic function were mainly due to increased

proximal tubular sodium-glucose co-transport and that SGLT2 inhibition resulted in an enhanced

physiological effect in T1D subjects with hyperfiltration. The pronounced impact of

empagliflozin on urine volume and urinary glucose excretion parameters in T1D-H further

suggest that greater renal hemodynamic responses in this group were related to increased sodium

chloride delivery to the distal tubule, which occurs as a result of decreased NaCl reabsorption

along with glucose in the proximal tubule. It is also noteworthy that the magnitude of GFR

reduction (-33 ml/min/1.73 m2) during clamped euglycemia in this group is similar to changes in

hyperfiltration associated with ACE inhibition in young patients with uncomplicated T1D,

highlighting that the effect size of this drug class on a surrogate marker of intraglomerular

pressure in humans is in the same range expected with pharmacological RAAS blockade 11.

The neurohormonal factors that may have contributed to pre-glomerular vasoconstriction

after empagliflozin merit some additional consideration. Animal work has suggested that NO is

an important mediator of TGF. NO is released during activation of TGF, thereby blunting the

vasoconstrictor response, resulting in decreased TGF sensitivity 29, 30. The interaction between

angiotensin II and NO also modulates TGF, since intrarenal RAAS activation exaggerates TGF-

mediated vasoconstriction through inhibition of neuronal NO synthase 31. In addition to

interactions with the intrarenal RAAS, NO blunts the effect of another major vasoconstrictor

uggest that greater renal hemodynamic responses in this group were related to ininncrrreaeaseses d d d sososoddidiumu

chloride delivery to the distal tubule, which occurs as a result of decreased NaCl reabsorption

allononng g g wiwiwiththth ggglulucooosesese iin the proximal tubule. It is aaalslsl oo noteworthy tthahah t thhhe ee mmmagnitude of GFR

eeduuuctc ion (-333 mmml//mimimin/n/n 1.1..737373 mmm222) ) duduuririingngg cclammmpeeed eeuguguglycececemimiaaa inini tthhhisss ggroroupupp iis s sisisimimilalalar totot ccchahahangnggesss in

hyhypepeperfrfrfilili trtratattioioionn asasssooociciaaateedd wwititthh AAACECECE iinnhnhibibibitititiioion nn inini yooounngng pppatatatieeentntn ss wiwiwiththth uuunnccomommplplplicicatatateeded TTT1DDD,,

highlightingg tthahahat t thththeee efefeffefef cttt sssizizize ofofof tthihih s drdrd ugugug clcllasasasss onono aa ssurururrororogagagatetete mmmarararkkkererer ooof f f ininintrtrragagagloloomememerur lar



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17

involved in TGF, adenosine 32. Under conditions of chronic hyperglycemia and DM, the role of

NO in TGF physiology may be of particular importance since blockade of NO augments renal

vasoconstriction in DM animals vs. controls 33. Despite what has been shown in experimental

models of diabetes, pre-glomerular vasodilators (urine NO and prostanoids) did not decline

significantly after SGLT2 inhibition, suggesting that TGF-mediated renal hemodynamic changes

were independent of these neurohormonal mediators 8, 10. Nevertheless, our results do not rule

out paracrine changes in NO bioactivity after SGLT2 inhibition. Furthermore, since we have

previously shown that NO synthase inhibition alone can partially reduce hyperfiltration in

humans with T1D, future studies should combine NO synthase inhibitors with SGLT2 inhibition

to determine if a further correction of hyperfiltration can be achieved 8

Consistent with previous clinical trials in patients with T2D, SGLT2 inhibition in our

study was associated with a modest but significant blood pressure lowering effect 17.

Interestingly this was accompanied by a significant increase in circulating RAAS mediators,

predominantly in T1D subjects with renal hyperfiltration. Our results are consistent with studies

involving patients with T2D that demonstrated that SGLT2 inhibition significantly lowered

systolic and diastolic blood pressure partly by inducing effective circulating volume contraction

via a mild diuretic effect 17, 34-36. This is further reflected by mild but significant increases in

hematocrit and plasma urea within the normal range 18. Our study is the first to demonstrate that

similar to familial renal glucosuria, pharmacological SGLT2 inhibition-induced effective

circulating volume contraction is accompanied by an increase in circulating RAAS mediators 37.

This observation has important clinical implications. Firstly, activation of the RAAS serves as

further evidence for an effective circulating volume contraction after SGLT2 inhibition.

Secondly, a rise in circulating RAAS mediators may be of particular importance in patients

o determine if a further correction of hyperfiltration can be achieved 8

Consistent with previous clinical trials in patients with T2D, SGLT2 inhibition in our

ttududdyy y wwawasss asasa sssociciiatatateded with a modest but significananant bblood pressureee lowwerereriining effect 17.

nnteeerer stingly ththisisis wwwasas aaccccccomomompapapaniniededed bbyy aa signnnifficaantntnt incccrereeasasee inin cccirircuculalatitt nngng RRRAAAAASSS mmemedidid atttoorors,s,

prpredededomomomininananntltltly y ininn TTT1D1DD ssububu jejectctc s s wiwiwiththth rreenenalalal hhhypypyperere fifiilttrrratatioioi n.n.n OOOururur rreeesululultstst aaareee cconononsisisiststenenentt t wiwiithhh sstutuudiiieses

nvolving paatitiienenentstss wwwitith hh T2T2D D D ththt atatat dddemememonononststtrararateteted d d ththhatatat SSSGLGLLT2T2T2 iiinhnhnhibibibitioioion n n sisisigngngnififificicananantltltly y y loloowew red



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18

concomitantly treated with RAAS inhibitors. It is well known that the use of RAAS blockers in

combination with other agents that induce diuresis leads to additive antihypertensive and anti-

proteinuric effects 38, 39. Importantly, RAAS blockade with SGLT2 inhibition in animals has

recently been shown to lead to additive renoprotective effects compared to either drug alone 21.

Hence, it is tempting to speculate that combined use of SGLT2 and RAAS inhibitors may lead to

similar synergistic effects through combined blockade of neurohormonal and tubular factors in

humans. Future studies should examine whether a combined strategy of dual RAAS and SGLT2

inhibition has the potential to exert long-term renal and systemic vascular protection, in part

through normalizing hyperfiltration.

Treatment with the SGLT2 inhibitor empagliflozin was associated with a significant

change in body weight in subjects with T1D. This is an important consideration for the overall

blood pressure effects of empagliflozin, since even small changes in weight are known to be

associated with significant antihypertensive effects 40. Thus, our results suggest that several

mechanisms may underlie the antihypertensive effects of SGLT2 inhibitors in humans. These

may not be limited to but certainly include i) improved glycemic control, ii) an effective

circulating volume contraction, and iii) weight loss 24. Although we cannot determine which of

these factors is dominant for lowering systemic blood pressure, it is fair to conclude that these

effects outweigh the increase in circulating RAAS mediators and decline in plasma NO, since

both of these effects would be expected to increase systemic vascular tone and blood pressure.

The glucose-lowering effect of empagliflozin is interesting from the perspectives of blood

pressure lowering and renal protection, particularly since the drug was generally well tolerated

aside from the development of diabetic ketoacidosis in the context of obvious clinical

precipitants in two participants. The risk of ketosis should therefore be carefully evaluated in

Treatment with the SGLT2 inhibitor empagliflozin was associated with aa sigiggniniififificacacantntnt

change in body weight in subjects with T1D. This is an important consideration for the overall

blloooood d d pprpresesssusus rrre eeffffffeecects of empagliflozin, since evvvenene small changesss in weweweiigight are known to be

assooocic ated witth hh siss ggngnifififici aananttt ananntititihyhypepepertrteennssive effffectsts 40. ThTThususs, ouour r reeesuultltltss ssusuggggggeesest t ththhatatt sseevvererer lalal

memeechchchanananisismsmsms mmayayy uuundndererlliliee thhhee e ananantititihyhyhypepeertttenenensssiveveve eeffffeecctsss ooofff SSGSGLLTLT222 inininhihih bbbitotoorsrs iiin n huhuh mamamanss.. TThThesesse

may not be llimimmitititededd ttto o bububut cececertrtrtaiainlnln y y y ininincllludududeee i)i)i) imimimprprp ovovoveded ggglylylycececemimimic c c cooontntntrororol,l,, iiii)i)i) aan nn efefeffefefectctctivi e



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future trials.

While the effect of SGLT2 inhibition on long-term renal protection is not yet known, it is

important to consider the clinical context in which this class of agents could optimally be used to

prevent the development of diabetic nephropathy for future clinical trials. Assuming that renal

protective effects of SGLT2 inhibitors are mainly mediated through reduced intraglomerular

pressure akin to the effect of RAAS inhibitors, then these agents should be effective in patients

with sufficient renal function that permits glucosuric and hence TGF effects, including patients

with moderate and even severe renal function impairment. This is supported by data

demonstrating that canagliflozin reduces estimated GFR and proteinuria within three weeks in

patients with T2D and estimated GFR values between 30-50 ml/min/1.73m2 41. Similar subacute

changes in GFR occur with empagliflozin in patients with stages 3 and 4 chronic kidney disease,

and are maintained at 52 weeks 42, 43. Perhaps most importantly, the decline in GFR was fully

reversible after a 3-week wash-out period 42, 43. Together, these observations strongly suggest

that renal hemodynamic functional effects of SGLT2 inhibition are due to a reduction in

intraglomerular pressure, similar to what would be expected with traditional renal protective

therapies such as RAAS inhibition, or intensified glycemic control 44. However, in contrast with

RAAS inhibitors which do not improve renal outcomes in normoalbuminuric patients with T1D

and normal renal function 45, our results suggest that SGLT2 inhibition reduces hyperfiltration 4,

which has been implicated in the initiation and progression of diabetic nephropathy, and also

improve glycemic control. Therefore, in addition to examining the effect of these agents in

patients with established renal disease, future studies should consider SGLT2 inhibition as a

primary prevention strategy in T1D. This is of particular importance in light of the glucose

lowering and antihypertensive effects achieved with SGLT2 inhibition, which we have now

patients with T2D and estimated GFR values between 30-50 ml/min/1.73m2 41. SiSiSimimim laaar r sususubababacucute

changes in GFR occur with empagliflozin in patients with stages 3 and 4 chronic kidney disease,

annd d d ararareee mamamaininintttainnnededed aat 52 weeks 42, 43. Perhaps momomostst importantly, ththhe dedeclclclinini e in GFR was fully

eeveeersr ible afterer aaa 33-wweeeek k k wawashshsh-ooututut pppererrioiood 4242,2, 433. Tooogggethhhererr, , ththheessee oobobseervvaattiiononnsss sststroronngnglylyly sssuggggegestss

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shown for the first time in patients with uncomplicated T1D, and which may independently

augment renal hemodynamic protective effects.

The present set of experiments demonstrates that modulation of TGF exerts greater renal

hemodynamic effects in patients with renal hyperfiltration compared to those with

normofiltration. However, the initiating factor leading to hyperfiltration in those with the

condition compared to those with normal GFR values remains unknown. Although speculative,

several possibilities exist. First, genetic polymorphisms in SLC5A2 encoding SGTL2 leading to

increased glucosuria have been described 46. This phenotype would be expected to increase distal

sodium delivery, causing afferent constriction thereby preventing hyperfiltration. While the

present study was not designed to examine between-group genetic differences, future work

comprising larger cohorts should consider evaluating the pharmacogenomic influence of SGLT2

polymorphisms. Second, hyperfiltration may be the result of interactions between intrarenal

activation of neurohormonal factors, such as the sympathetic nervous system or RAAS, with

TGF, which are both known to influence TGF sensitivity 31, 47, 48. As a consequence, underlying

differences in neurohormonal activation due to genetic variability or previous glycemic control

may contribute to a predisposition to hyperfiltration. Next, glycemia-related factors could also

contribute to the pathogenesis of hyperfiltration. For example, if the daily filtered glucose load

was higher in T1D-H, then the resulting increase in SGLT2 activity in this group could promote

afferent vasodilatation. While 24-hour glucose was not different in T1D-N vs. T1D-H at

baseline, daily carbohydrate intake did tend to be higher in T1D-H. Adequately powered future

studies should therefore examine the interaction between carbohydrate intake, GFR and SGLT2

inhibition. A final possible mechanism that may contribute to the initiation of hyperfiltration

relates to the fetal environment. Previous work in animals and in patients with essential

present study was not designed to examine between-group genetic differences, fufuutuurerr wwwororork k k

comprising larger cohorts should consider evaluating the pharmacogenomic influence of SGLT2

poolylylymomomorprphihihismsmms. SSSeececond, hyperfiltration may be e tththeee result of interararactioonsnsns between intrarenal

acctiivav tion of neneuuuroohohororrmomomonananal l l fafafactctorororss,s, ssucucch asss tthhe sssyymmpaaatththetetticic nneerrvrvouous s ssysyststememm oor r RARARAASASAS, wiwwiththth

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differences inin nnneueueurororohohohormrmmononnalalal aactctctivivi aaatitt ononon dddueueue ttto o o gegegenenenetitit c vavavariririabababilililititity yy ororor ppprerereviviv ououo ss glglglycycycemememic control



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hypertension have suggested that intrauterine abnormalities leading to low birth weight and

intrauterine growth restriction may have long term renal effects, including low nephron number,

leading to compensatory hyperfiltration 49, 50. Although speculative, a similar sequence of events

could occur in the context of T1D, when the effect on hyperfiltration may be more pronounced

due to the additional “second hit” physiological insults of neurohormonal activation and

enhanced proximal sodium-glucose cotransport.

One limitation of our investigational study is the relatively small sample size, potentially

leading to heterogeneity in relevant clinical parameters of the study population at baseline, as

well as in individual responses to empagliflozin. We attempted to minimize the effect of a small

sample size by using homogeneous study groups and by conducting a careful pre-study

preparation phase with a particular emphasis on factors that could potentially influence

neurohormonal activation, such as dietary sodium intake. We also decreased variability by using

a study design that allowed each subject to act as his/her own control. While this reduction in

baseline physiologic variability likely improved our ability to detect changes in renal function,

the use of homogenous groups of participants did not permit an in-depth analysis of other factors

that may inform the research community about the pathobiology of SGLT2 inhibitors such as

age, diabetes duration, degree of proteinuria or clinical nephropathy, which should be considered

in future work. Finally, study participants were not placed on a controlled nitrate diet, and this

may have limited the magnitude of the between-group differences in urine NO excretion.

In conclusion, we have demonstrated for the first time in humans that inhibition of

SGLT2 is associated with a significant attenuation of renal hyperfiltration. While several factors

may have contributed to the decrease in GFR in T1D subjects with hyperfiltration to near normal

levels, it may be hypothesized that activation of TGF by empagliflozin made a large

ample size by using homogeneous study groups and by conducting a careful prree-e-ststtudududy y y

preparation phase with a particular emphasis on factors that could potentially influence

neeurururohohohoorormomomonnnal acacacttitivvation, such as dietary sodiumumm iinntake. We alsooo deccrerereaaased variability by using

a sttuudy designn tthhah ttt alalllolol weweweddd eaeaeacchch sssubububjejectct to aacactt as hihiis/hheherrr owowwnnn cocoonttrool.l WWWhihilelee ttthihis s rerer dududuccctioioon n inini

baaseseselililinenen ppphyhyhysissiolologogogicic vvvarrriaiabibilililitytyty lllikikikelele y y imimimprprproovovededed oooururr aabibib lililitytyty to oo dededetetectctct cchhahanngngeseses iiin n n rereenananal ffufunncnctitioonon,,,rr

he use of homommogogogenenenououous s grg ouououpspp ooof f f papaartr iciccipipipananantststs dddididd nnnototo ppererermimim t t t ananan iiin-n-dededeptptpth h h anananalaltt ysysysisisis ooof f f otoo her factorsss



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contribution. Future work should focus on the long-term use of SGLT2 inhibitors as potential

renoprotective therapies that may reduce intraglomerular pressure thereby reducing the risk of

developing overt diabetic nephropathy.

Acknowledgments: The authors would also like to thank Dr. Paul Yip and Jenny Cheung-Hum

for their invaluable assistance with biochemical assays included in this work. The authors

acknowledge editorial assistance, supported financially by Boehringer Ingelheim, from Wendy

Morris, Fleishman-Hillard Group, Ltd. Finally, the authors are grateful to the study participants

whose time and effort are critical to the success of our research program. Contributions: D.Z.I.C,

B.A.P., H.P., M.M., V.L., A.L. researched data, wrote the manuscript. N.F., H.J.W., N.S., O.E.J.,

U.C.B., M.v.E. contributed to discussion, reviewed/edited manuscript. All authors have approved

the final version of this manuscript.

Funding Sources: This work was supported by Boehringer Ingelheim (to D.Z.I.C. and B.A.P.)

D.Z.I.C. was also supported by a Kidney Foundation of Canada Scholarship and a Canadian

Diabetes Association-KRESCENT Program Joint New Investigator Award and receives

operating support from the Heart and Stroke Foundation of Canada.

Conflict of Interest Disclosures: The results presented in this paper have not been published

previously in whole or in part. D.Z.I.C. has received speaker honoraria from Boehringer

Ingelheim and B.A.P received operational funding with D.Z.I.C. for this work. N.S., N.F.,

H.J.W. O.E.J., U.C.B., M.v E. are employees of Boehringer Ingelheim. These data were

presented at the American Diabetes Association Meeting on June 22, 2013

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4777. BlBlBlanana tztz RRRC,CC, PPPelllayayooo JCJCC.. A AA fufuncncnctititioononaalal rrrololole ee fofoor r r ththhee tutubububulolologgglomomomeererululularara fffeeeedbdbaacack kk mememecchchanannisssm.m. Kidndneyey IIntnt. 1998484;225:5:7739-747 6.6

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Table 1. Clinical Characteristics and Biochemistry before and after Empagliflozin in Patients with Type 1 Diabetes Mellitus with Normofiltration or Hyperfiltration (mean SD)

Normofiltration group (n=13) Hyperfiltration group (n=27) Baseline End of Treatment Baseline End of Treatment

Clinical characteristics: Male n (%) 8 (62) - 12 (44) - Age (years) 25.4 6.6 - 23.5 4.1 - Diabetes duration (years) 18.8 6.2 - 16.3 7.4 - Smoking status - never smoked (%) 92 - 85 - Weight (kg) 75.8 13.1 72.8 13.0† 71.1 12.5 68.6 11.9† Body mass index (kg/m2) 24.8 3.4 23.8 3.4 24.4 3.2 23.5 3.0 HbA1c (%) 7.8 1.0 7.3 0.8|| 8.2 0.9 7.8 1.0|| Daily insulin dose (units/day) Total 55.9 23.3 48.1 22.0** 54.1 19.3 44.6 17.4** Basal 26.6 9.2 19.2 8.0** 25.3 11.4 19.6 8.0** Bolus 29.3 19.7 29.0 17.8 28.8 14.0 26.0 12.3 Daily carbohydrate intake (grams/day) 140.6 40.1 184.4 82.5 194.3 142.4 250.8 184.9*** Biochemistry: Hematocrit (%) 0.381 0.032 0.395 0.038‡ 0.369 0.045 0.382 0.038‡ Blood urea (mmol/L) 4.4 1.2 5.4 1.2§ 4.6 1.8 5.3 1.6§ Urine albumin/creatinine ratio (mg/mmol) 0.88 0.48 1.05 0.42 1.31 0.96 1.86 2.05 24-hour urine volume (ml) 1900 848 2159 861 1463 920 2284 1081* 24-hour sodium excretion (mmol/day) 168 112 149 76 133 71 147 70 24-hour glucose excretion (gram/day) 18.2 19.4 106.7 39.5¶ 19.3 19.3 146.5 65.9¶# 24-hour protein intake (gram/kg/day) 0.91 0.18 0.95 0.24 0.99 0.35 1.14 0.48 24 hour protein intake estimated according to the formula [(24 hour urine urea X 0.18) +14] / body weight 27. Hematocrit and blood urea values are for V3 versus V12; Urine albumin/creatinine ratio is the average of values taken on V3/V4 compared to V12/V13. The calculation for mean diabetes duration was performed outside the clinical trials database, which only included categorical data as presented in the results section. *p<0.01 for change in urine volume in the T1D-H group; †p<0.01 for the within group changes in weight and body mass index; ‡p<0.02 for within group changes in hematocrit; §p<0.04 for within group changes in blood urea concentration; ||p<0.01 for T1D-H and p=0.0003 for T1D-N for within group changes in HbA1c; ¶p<0.01 for within group increase in urine glucose excretion; #p<0.05 for the change in urine glucose excretion in T1D-H vs. T1D-N; **p<0.05 for within group decreases in insulin doses; ***p<0.01 for within group change in carbohydrate intake in T1D-H

(y ) 18.8 6.2 16.3 7.4 king status - never smoked (%) 92 - 85 --ht (kg) 75.8 13.1 72.8 13.0† 71.1 12.55 6668.88.666 1111.99††mass index (kg/m2) 24.8 3.4 23.8 3.4 24.4 3.2 2 232323 5.55 333.00

1c (%) 7.8 1.0 7.3 0.8|| 8.2 0.9 7.8 1.0|| insulin dose (units/day)

al 55.9 23233 33.3 48.1 22.0** 54.1 19.3 44.6 17.4**alal 26.6 9.9.2 2 19.2 8.0**** 252 .3 11.4 19.6 8.0**ususu 292929.3.3. 919.7 2229.99 0 171717 8.8 28.88 141414 00.0 226.6.6.000 1212.3 carbrbr ohydrate intatakekeke (grgg amams/s dadaay)y)y) 4414 ..0 6 0040 1.1 118484.4.4 288 55.5 191 .4.333 14142.2.4 44 2522 0.0.0.88 1811 4.44 9*eheh imim stry:

aatototo rcrititi (%)% 0..3833 11 0.0 0300 2 2 0.0. 933 555 0..0300 8‡8‡8 000.3369696 0.0.04045 .0.383822 .0.030 8d d urureaeaea (((mmmmmmololol/L/L/L) ) 4.4..444 1.1.1.22 5.5.5 44 1.1 2§2§2§ 44 6.6.6 1.1.888 5.5.5 333 1.1.6§6§6§ e albubumim n/crreae tiniinen rattioio ((mgmg/mmom l)l) 0.888 0.0 48 11.0055 0.4242 11.3311 0.0 9696 11.8866 2.0505 our urine volumeme (((mlmlm )) 19191 000000 84848 888 212121595959 8686861 14141463636 9292920 00 2284 1081**ouourr sosodidiumum eexcxcreretitionon ((mmmmolol/d/dayay)) mm 161688 111122 141499 7676 113333 7171 114747 7070



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Table 2. Hemodynamic Responses to Empagliflozin During Clamped Euglycemia in Patients with Type 1 Diabetes with Normofiltration or Hyperfiltration (mean SD) Normofiltration group Hyperfiltration group

Baseline EMPA p-value Baseline EMPA p-value Renal hemodynamic function Effective renal plasma flow 642 90 637 88 0.89 1042 287 724 148 <0.01 Filtration fraction 0.185 0.026 0.200 0.025 0.19 0.176 0.047 0.197 0.042 0.02 Renal blood flow 1030 160 1042 138 0.82 1641 458 1156 219 <0.01 Renal vascular resistance 0.081 0.013 0.077 0.011 0.35 0.052 0.016 0.071 0.014 <0.01 Blood pressure Heart rate (bpm) 71 11 69 14 0.50 76 14 73 14 0.27 Systolic blood pressure (mmHg) 111 7 109 9 0.21 111 10 108 9 <0.05 Diastolic blood pressure (mmHg) 63 9 64 10 0.83 64 9 63 7 0.43 Plasma biochemistry Plasma angiotensin II (pmol/L) 3.1 3.9 10.4 20.2 0.14 3.6 3.9 6.4 6.8 0.04 Plasma aldosterone (pmol/L) 49 31 81 41 <0.01 43 26 66 44 0.02 Plasma renin activity (ng/ml/hr) 0.539 0.528 0.502 0.432 0.81 0.570 0.417 0.883 1.07 0.16 Plasma nitric oxide ( mol/L) 46 21 44 24 0.76 48 21 29 23 <0.01 Urine biochemistry Prostaglandin D2 (pmol/mg creatinine) 0.487 0.349 0.463 0.241 0.81 0.342 0.301 0.422 0.371 0.23 Prostaglandin E2 (pmol/mg creatinine) 0.087 0.041 0.086 0.071 0.97 0.119 0.083 0.103 0.062 0.26 Prostaglandin F1 (pmol/mg creatinine) 0.185 0.070 0.204 0.060 0.43 0.196 0.059 0.241 0.145 0.08 Thromboxane B2 (pmol/mg creatinine) 0.188 0.094 0.160 0.035 0.27 0.216 0.156 0.212 0.110 0.89 Urine nitric oxide ( mol/mmol creatinine) 132 96 123 96 0.80 153 148 126 91 0.25

EMPA = Empagliflozin Effective renal plasma flow in ml/min/1.73 m2; renal blood flow in ml/minute/1.73 m2; renal vascular resistance in mmHg/L/min

al blood flow 1030 160 1042 138 0.82 1641 458 1111156565 21219 9 <0al vascular resistance 0.081 0.013 0.077 0.011 0.35 0.052 0.016 0.0 07070 11 0.0.0 0101014 44 <0d pressure t rate (bpm) 71 11 69 14 0.50 76 14 73 14 0.2olic blood pressure (mmHg) 111 7 109 9 0.21 111 10 108 9 <0toliicc blblooooodd d prprp esessusus re (((mmmmm Hg) 63 9 6466 10 0.83 64646 9 63 7 0.4mamaa bbbiooiochchemmisistrtrt y y mamam aaangn iotensin IIII II (p(ppmomomol/l/L)L) 33.3.111 3.3..999 010.4. 20200.2.2.2 0.0..14141 33.3 66 3.9 9 6.6.44 66.6.8 8 0.0mma alala dod sterone (ppmomol/L)LL 4949 31 881 41414 <0<0 00.01 334 26626 6666 4444 0.0mamm rrenene in activitty y (n /g/g lmm /h )r) 0.0 3535 99 0. 22528 ..0 005022 0.0.0.434343222 0. 188 0.575 00 0.00 11417 0.00 8888 33 .1 07 0.

amama nn tititririr c c oxo idide e e ((( momol/l L)L)L 46464 21211 44444 2424 0.00 6767 884 12121 229 3323 <0<e biiocochhehe imimiststryry ttata lglglanandididinn D2D2D2 (((ppmomol/l/l/mgmg ccrere tatatiininiinin )e)e) 000.484848777 000.343434999 000.464646333 000.242424111 000.818181 000.343434222 000.303030111 000.424242222 000.373737111 0.0.0 2taglandin E2 (ppmomomol/l//mgmgmg ccrerereatatininninine)e)e) 0.0 08088777 00.04040 111 0.0 08080 666 0.0707711 0.00 979 0.00 11111999 0.0 08083 33 00.0 10101 333 0.062 ..0 2



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Table 3. Hemodynamic Responses to Empagliflozin During Clamped Hyperglycemia in Type 1 Diabetes Patients with Normofiltration or Hyperfiltration (mean SD)

Normofiltration group Hyperfiltration group Baseline EMPA p-value Baseline EMPA p-value

Renal hemodynamic function Effective renal plasma flow 706 157 688 101 0.74 1051 251 748 144 <0.01 Filtration fraction 0.195 0.024 0.196 0.019 0.95 0.185 0.046 0.192 0.032 0.40 Renal blood flow 1117 240 1140 154 0.79 1633 430 1193 227 <0.01 Renal vascular resistance 0.078 0.015 0.073 0.012 0.37 0.054 0.015 0.072 0.015 <0.01 Blood pressure Heart rate (bpm) 68 10 63 11 0.06 74 11 75 12 0.79 Systolic blood pressure (mmHg) 115 8 113 8 0.41 111 10 110 10 0.47 Diastolic blood pressure (mmHg) 65 9 63 9 0.54 66 8 64 6 0.34 Plasma biochemistry Plasma angiotensin II (pmol/L) 2.6 2.4 4.2 4.0 0.17 2.3 2.5 3.6 3.0 0.03 Plasma aldosterone (pmol/L) 27 4 47 32 0.04 29 8 50 43 0.03 Plasma renin activity (ng/ml/hr) 0.317 0.281 0.368 0.389 0.36 0.328 0.273 0.356 0.241 0.61 Plasma nitric oxide ( mol/L) 40 23 42 20 0.73 46 20 28 23 <0.01 Urine biochemistry Prostaglandin D2 (pmol/mg creatinine) 0.610 0.356 0.633 0.295 0.85 0.509 0.344 0.631 0.404 0.15 Prostaglandin E2 (pmol/mg creatinine) 0.096 0.092 0.084 0.070 0.69 0.090 0.054 0.099 0.060 0.52 Prostaglandin F1 (pmol/mg creatinine) 0.225 0.099 0.265 0.100 0.20 0.223 0.095 0.298 0.162 0.03 Thromboxane B2 (pmol/mg creatinine) 0.175 0.056 0.191 0.061 0.18 0.206 0.075 0.207 0.066 0.98 Urine nitric oxide ( mol/mmol creatinine) 98 58 112 58 0.43 138 108 162 124 0.37

EMPA = Empagliflozin Effective renal plasma flow in ml/min/1.73 m2; renal blood flow in ml/min/1.73 m2; renal vascular resistance in mmHg/L/min

al blood flow 1117 240 1140 154 0.79 1633 430 11111939393 2222227 7 <0< .al vascular resistance 0.078 0.015 0.073 0.012 0.37 0.054 0.015 0.00 07772 0.0 1010155 5 <0< .d pressure t rate (bpm) 68 10 63 11 0.06 74 11 75 12 0.7olic blood pressure (mmHg) 115 8 113 8 0.41 111 10 110 10 0.4toliicc blbllooooooddd prprpresessusus re (((mmmmm Hg) 65 9 636363 9 0.54 666666 8 64 6 0.3maama bbioioiochch memisistrtrtry yy mamm aaangn iotens nin IIII II (p(ppmomom l/l/L)L)) 2.2.666 2.22.4 4 4.44 22 4.0 0 0..171717 22.2 333 2.55 3.3.66 333.00 0 0.0mma alala dod sterone (ppmomol/L)LL 2727 4 4 4747 232 0.0. 40404 992 8 8 0505 4344 0.0mamam renene in activitty y y (n /g/g lmm /h )r) 0.313177 .0.281 0. 663 88 0.0.38383899 9 0.0 6636 0. 233 888 0.0.272733 0.353566 0.00 4242 1 0.6mama nnn titririric cc oxoxididde e e ((( momol/l L)L)L) 4040 32323 4422 0020 00.0.7373 646 2020 2228 2322 <0<0.e biiocochehe imiststryry ta lglandidin D2D2 ((ppmomol/l/mggg creatiiniine)e) 0.0.61610 0.0.0 35356 6 0.0.636333 00. 9295 5 00.8585 0.0.50509 .0.34344 4 00.636311 0.404 00.11taglg andin E2 ((ppmomol/l//mgmg ccrereatatinininine)e)) 00.0 09096 00.090922 00.0 080844 0.00 707000 00.6969 00.09090 00 00.050544 0.00 0909999 0.060 0.55



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Table 4. Adverse events in 42 participants with T1D who took empagliflozin

Condition Number (%) Infections Nasopharyngitis 11 (26) Genitourinary tract infection 6 (14) Gastroenteritis (viral) 1 (2) Influenza 4 (10) Gastroenteritis 2 (5) Vaginal infection 2 (5) Ear infection 1 (2) Eye infection 1 (2) Lung infection 1 (2) Tooth infection 1 (2) Metabolism and nutrition Hypoglycemia – all episodes 40 (95) Hypoglycemia – severe requiring assistance 1 (2) Decreased appetite 2 (5) Diabetic ketoacidosis 2 (5) Increased appetite 1 (2) Psychiatric Anxiety 2 (5) Insomnia 2 (5) Stress 1 (2) Nervous system disorders Headache 13 (31) Dizziness 10 (24) Tinnitus 6 (14) Vertigo 1 (2) Respiratory conditions Nasal sinus congestion or 2 (5) Sinus congestion 1 (2) Throat irritation 1 (2) Gastrointestinal disorders Dry mouth 7 (17) Nausea 7 (17) Vomiting 6 (14) Abdominal pain 5 (12) Abdominal discomfort 1 (2) Abdominal distension 1 (2) Abdominal pain upper 1 (2) Haemorrhoids 1 (2) Mallory-Weiss tear 1 (2) Skin and musculoskeletal disorders Pruritus 1 (2) Skin irritation 1 (2)

Decreased appetite 2 (5) Diabetic ketoacidosis 2 (5) Increased appetite 1 (2)Psychiatric AAnxietyy 2 (5) IIInnsnsommnininiaaa 2 (5(5( ) SStress 111 (2(22)))NeNeervr ous systtememm dddissoorrdedeersrs HHeadachhe e 1333 ((331) DDDizizizzizinenenesssss 101010 ((242424)) TTinin initutus 6 (1(14)4) Vertigoo 111 (222)))ReRespspiriratatororyy cocondndititioionsns



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Back pain 4 (10) Myalgia 1 (2) Reproductive and breast disorders Dysmenorrhea 1 (2) Menstrual disorder 1 (2) Menstruation delayed 1 (2) Menstruation irregular 1 (2) Polymenorrhea 1 (2) Vaginal haemorrhage 1 (2) Vulvovaginal dryness 1 (2) General disorders Thirst 33 (74) Fatigue 2 (5) Pyrexia 2 (5) Chest discomfort 1 (2) Influenza like illness 1 (2) Pollakiuria 33 (79) Nocturia 1 (2) Urine output increased 1 (2) Dysuria 1 (2)

Figure Legends:

Figure 1. Postulated tubuloglomerular feedback (TGF) mechanisms in normal physiology, early

stages of diabetic nephropathy and following SGLT2 inhibition. A: Under physiological

conditions, TGF signalling maintains stable GFR by modulation of pre-glomerular arteriole tone.

In cases of conditional increases in GFR, the macula densa within the juxta-glomerular apparatus

senses an increase in distal tubular sodium delivery and adjusts GFR via TGF accordingly. B:

Under chronic hyperglycemic conditions (diabetes mellitus), increased proximal SGLT2-

mediated reabsorption of sodium (Na+) and glucose impairs this feedback mechanism. Thus,

despite increased GFR the macula densa is exposed to lowered sodium concentrations. This

impairment of TGF signalling likely leads to inadequate arteriole tone and increased renal

perfusion. C: SGLT2 inhibition with empagliflozin treatment blocks proximal tubule glucose and

Nocturia 1 (2) Urine output increased 1 (2) Dysuria 1 (2)

FiFiiguuure Leggenenndsds:::

Figugurere 1. PoP sttulatteded ttubululogoglolomemerular fefeededbabackck ((TGFGF)) memechchana issmsms iin n noormrmalal pphyhysiiolo ogogy,y, eearrlyly

ttagageses ooff didiababeteticic nnepephrhropopatathyhy aandnd ffolollolowiwingng SSGLGLT2T2 iinhnhibibititioionn AA:: UnUndederr phphysysioiolologigicacall



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32

sodium reabsorption, which leads to increased sodium delivery to the macula densa. This

condition restores TGF via appropriate modulation of arteriolar tone (e.g. afferent

vasoconstriction), which in turn reduces renal plasma flow and hyperfiltration.

Figure 2. Flow diagram for study participants.

Figure 3. Study outline for renal hemodynamic function tests.

Figure 4. GFR responses to empagliflozin during clamped euglycemia (A) and hyperglycemia

(B) (mean SD). *p<0.01 for baseline GFR in T1D-N versus T1D-H. †p<0.01 for the within-

group change in GFR in T1D-H. ‡p<0.01 for the between-group effect of empagliflozin on

change in GFR.

B) (mean SD). *p<0.01 for baseline GFR in T1D-N versus T1D-H. †p<0.01 fororr theheh wwwittithihihin-n-n

group change in GFR in T1D-H. ‡p<0.01 for the between-group effect of empagliflozin on

chhananangegege iiin GFGFGFR.



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Figure 1



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Assessed for eligibility (n=52)

Excluded (n=8)•Not meeting inclusion criteria

Entered in placebo run in phase (n=44)


Entered in empagliflozin treatment phase (n=42)

Discontinued interventiondue to adverse events (n=2)

Analyzed (n=40)

Completed treatment and follow up phases (n=40)

Figure 2


EEnntteerreed iinn eemmppaagglliifflloozziinn ttrreeaaatmmenntt ppphhaase (((nnn===4422))

DDDiiscccooonntttiinuuueeddd iinnttteeerrrvvveeennttiiioonnnduuee ttoo aadveerse evveentss (((n=22)))



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Empagliflozin 25 mg OD for 8 weeks

1 week controlled Na and protein diet

Screening1 week

Clamped euglycemia

1 week controlled Na and protein diet

Placebo run-infor 2 weeks

V4V3 V12 V13

Clamped hyperglycemia

24-hour urine collection

V1 V2 V8V5-V7 phone visits V9-10 phone visits

V14 V15 V11

1 week 1 week Follow-up

Figure 3

Empapaglglififloloziinn 25 mmg g ODOD fforor 88 wweeeksks

1 1 weekek cconoo trololleled d NaN and rprprotoo eiein n dididietet

nnininggekek

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PlPlPlacacacebebebo oo rurun-n-inininfoforr 22 weweekekss

4V4V4V3VV V1VV 22 V1VV 33

hyperglycemia

urine collection

V2V2V V8VV55V5-V77 phhhonono e e vivv si sts V9V9V9-1-100 phpp nnone viv si ssts

V1VV 4 44V1V1111

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DM-N DM-H

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50

100

150

200

250BaselineEmpagliflozin* †‡

GFR

(ml/m

in/1

.73m

2 )

DM-N DM-H

0

50

100

150

200

250BaselineEmpagliflozin

*†‡

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(ml/m

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B.

Figure 4

DM-N DM-H

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Nora M. Fagan, Hans J. Woerle, Odd Erik Johansen, Uli C. Broedl and Maximilian von EynattenDavid Z. I. Cherney, Bruce A. Perkins, Nima Soleymanlou, Maria Maione, Vesta Lai, Alana Lee,

The Renal Hemodynamic Effect of SGLT2 Inhibition in Patients with Type 1 Diabetes

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