Sodium Glucose Cotransporter 2 Inhibitors in the Treatment ...circ.ahajournals.org/content/circulationaha/early/2016/07/28/... · glycosuric mechanism, SGLT2 inhibitors also reduce

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Sodium Glucose Cotransporter 2 Inhibitors in the Treatment of Diabetes:Cardiovascular and Kidney Effects, Potential Mechanisms and Clinical

Applications

Heerspink et al: Cardiovascular Risk and SGLT2 Inhibition

Hiddo J.L. Heerspink, PharmD, PhD1; Bruce A. Perkins, MD, MPH2; David H. Fitchett3, MD; Mansoor Husain, MD4; David Z. I. Cherney, MD, PhD5,6,7

1 Department of Clinical Pharmacy & Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands 2Department of Medicine, Division of Endocrinology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada 3Department of Medicine, Division of Cardiology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada 4Ted Rogers Centre for Heart Research and Department of Medicine, Division of Cardiology, Peter Munk Cardiac Centre, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada 5Department of Medicine, Division of Nephrology, Toronto General Hospital, Department of Physiology, Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada 6Department of Physiology, University of Toronto, Toronto, Ontario, Canada 7Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada

Correspondence to David Cherney, MD CM, PhD, FRCP(C) Toronto General Hospital 585 University Ave, 8N-845 Toronto, Ontario, M5G 2N2 Phone: 416.340.4151 Fax: 416.340.4999 Email: [email protected]

DOI: 10.1161/CIRCULATIONAHA.116.021887

p yCanada Department of Medicine, Division of Nephrology, Toronto General Hospital, DeDeDepapapartrtrtmemementntnt ooo

Physiology, Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, CanadaDepartment of Physiology, University of Toronto, Toronto, Ontario, Canada Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada

Coorrrrespondence too Davivivid dd Cherneyy,y, MMMDD CMCMCM, PhPhPhDD, FRCP((CCC) Torooontntnto oo Genenenerararal HoHHospppiiital 585858555 UUnUniviverersisiitytty AAAveve,, 8N8N8N 8-88454545 T t O t i

by guest on May 20, 2018

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AbstractSodium glucose co-transporter-2 (SGLT2) inhibitors, including empagliflozin, dapagliflozin and canagliflozin, are now widely approved anti-hyperglycemic therapies. Due to their unique glycosuric mechanism, SGLT2 inhibitors also reduce weight. Perhaps more importantly are osmotic diuretic and natriuretic effects contributing to plasma volume contraction, and decreases in systolic and diastolic blood pressures (BP) by 4-6/1-2 mmHg, respectively, which may underlie cardiovascular and kidney benefits. SGLT2 inhibition is also associated with an acute, dose-dependent reduction in eGFR by ~5 ml/min/1.73m2 and ~30-40% reduction in albuminuria. These effects mirror pre-clinical observations suggesting that proximal tubular natriuresis activates renal tubuloglomerular feedback through increased macula densa sodium and chloride delivery, leading to afferent vasoconstriction. On the basis of reduced glomerular filtration, glycosuric and weight loss effects are attenuated in patients with chronic kidney disease (CKD, eGFR <60 ml/min/1.73m2). In contrast, BP lowering, eGFR and albuminuric effects are preserved, and perhaps exaggerated in CKD. With regards to long term clinical outcomes, the EMPA-REG OUTCOME trial in patients with type 2 diabetes (T2D) and established cardiovascular disease randomized to empagliflozin versus placebo reported a 14% reduction in the primary composite outcome of CV death, nonfatal myocardial infarction, nonfatal stroke, and >30% reductions in cardiovascular mortality, overall mortality and heart failure hospitalizations associated with empagliflozin – even though by design, the HbA1c difference between the randomized groups was marginal. Aside from an increased risk of mycotic genital infections, empagliflozin-treated patients had fewer serious adverse events, including a lower risk of acute kidney injury. In light of the EMPA-REG OUTCOME results, some diabetes clinical practice guidelines now recommend that SGLT2 inhibitors with proven cardiovascular benefit be prioritized in patients with T2D who have not achieved glycemic targets and who have prevalent atherosclerotic cardiovascular disease. With additional cardiorenal protection trials underway, sodium-related physiological effects of SGLT2 inhibitors and clinical correlates of natriuresis - such as the impact on BP, heart failure, kidney protection and mortality – will be a major management focus. Key Words: SGLT2 inhibition, cardiovascular, heart failure

30% reductions in cardiovascular mortality, overall mortality and heart failure hosps italizationassociated with empagliflozin – even though by design, the HbA1c differennncecece bbbetetetweweweenenen thehandomized groups was marginal. Aside from an increased risk of mycotic genennitititalalal iiinfnfnfececectititionononsss

empagliflozin-treated patients had fewer serious adverse events, including a lower risk of acutekidney injury. In light of the EMPA-REG OUTCOME results, some diabetes clinical practiceguidelines now recommend that SGLT2 inhibitors with proven cardiovascular benefit beprioririritititizezezeddd ininin pppataa ieeenntn s with T2D who have not achihihieved glycemic cc tatt rgetetets and who have prevalenatheeerororosclerotic cc cacacardrdrdiooovavavascscsculuu ararar dddisisiseaeaease. With adaa ddittionnnalalal cccararardidd orororenenenalalal ppprorr tectctctioioion n n trtrtrialsss uuundndnderererwaww yodddiuuum-related phyhhysiolololooogiccalalal effecctststs ofoff SGLT2T22 inhnhibbbitititors ananand d cclinnicaaal corororrrrelatess ooof nnnaata riuresesesisiuchchch as the immmpapapactt ooonn n BPPP, heart failluuure, kidididneyyy ppprororotetetectctctioioionn n annndd d mooorrtaaaliiity – wwwillll bbbe a mamamajo

manaaagegegememm ntntnt fffooocusss.KeKeyy WoWordrds:s: SSGLGLT2T2 iinhnhibibititioionn, ccarardidiovovasascuculalarr, hheaeartrt ffaiailulurere



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Introduction

Anti-hyperglycemic interventions have traditionally focused on restoring -cell activity, insulin

sensitivity, or tissue glucose uptake to normalize plasma glucose levels in patients with diabetes.

An alternative strategy is to enhance urinary glucose excretion by targeting renal sodium-glucose

cotransporters (SGLTs). Studies in the 1930s documented the important role of the kidney in

glucose homeostasis, demonstrating that nearly all filtered glucose undergoes tubular

reabsorption, except when a threshold is exceeded and glycosuria thereafter increases linearly in

proportion to increasing plasma glucose1. Fifty years later, two distinct sodium-dependent

transport systems, SGLT1 and SGLT2, were characterized and cloned2. Low capacity, high

affinity SGLT1 transporters are located in cells isolated from the intestine, heart, skeletal

muscles, and kidney. By contrast, high-capacity, low affinity SGLT2 transporters are almost

exclusively located in the proximal tubule epithelium and are responsible for the reabsorption for

most filtered glucose3, 4 (Figure 1). Tubular glucose that "escapes" SGLT2 is subsequently

reabsorbed by SGLT1 in more distal tubular segments. Studies in SGLT1 and SGLT2 knock-out

mice have confirmed that 97% of filtered glucose is reabsorbed by SGLT2 and the remaining 3%

by SGLT1 under otherwise normal physiologic conditions3.

Studies have also shown that SGLT2 expression is increased in hyperglycemic

conditions, likely representing an ‘adaptive’ response to increased plasma glucose filtration, and

paradoxically increasing the threshold for urinary glucose excretion in patients with diabetes5.

Glucose kinetic studies have documented that SGLT2 inhibitors decrease the maximum capacity

of the renal tubules to reabsorb glucose and thereby increase urinary glucose excretion and

decrease plasma glucose concentrations6.

affinity SGLT1 transporters are located in cells isolated from the intestine,,, hhheaeaeartrtrt, , , skskskeleleleeeta

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Importantly for safety, the efficacy of SGLT2 inhibitors to increase urinary glucose

excretion attenuates at lower plasma glucose levels, thereby accounting for their reduced risk of

causing hypoglycemia. In contrast to the expected proportion predicted by animal studies, only

~50% of all filtered glucose reabsorption is blocked by SGLT2 inhibitors. This could suggest

incomplete inhibition by pharmacological SGLT2 blockade, insufficient target concentration or a

compensatory increase in SGLT1 expression/activity when SGLT2 is blocked3, 7.

Pharmacological agents that non-selectively inhibit SGLT1 and SGLT2 are being developed to

potentially achieve greater glycosuria and HbA1c lowering8.

At present, clinically available SGLT2 inhibitors include canagliflozin, dapagliflozin and

empagliflozin, which are approved as anti-hyperglycemic therapies for the treatment of patients

with T2D. In this review, we will summarize the glycemic-lowering effects of SGLT2 inhibitors

and their potential adverse effects. In addressing the remarkable results of the Empagliflozin,

Cardiovascular Outcomes, and Mortality in Type 2 Diabetes (EMPA-REG OUTCOME) trial, we

will also discuss the effects of SGLT2 inhibitors on non-glycemic parameters including

natriuresis and direct effects on glomerular hemodynamics, which may play an important role in

mediating the cardiovascular and kidney protection observed in this study.

Role of SGLT2 inhibition as a therapeutic target for glycemic control

SGLT2 inhibitors have received extensive evaluation in Phase-2 and -3 clinical trials and

represents a well-defined class of anti-hyperglycemic agents with a detailed evidence-base of

knowledge of glycemic efficacy and adverse events. Some of these placebo-controlled studies

have included patients with renal impairment, and participants with eGFR as low as 30

ml/min/1.73m2.

empagliflozin, which are approved as anti-hyperglycemic therapies for the treatmtmtmenenent t t ofofof pppatatatieieient

with T2D. In this review, we will summarize the glycemic-lowering effects of SGLT2 inhibitor

and their popp tential adverse effects. In addressing the remark Empagliflozinable results of the

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Placebo-controlled studies have demonstrated adjusted mean treatment differences in

HbA1c of -0.7% vs. placebo (from average baselines of 7.5% to 9.2%) over study durations

ranging from 12 to 78 weeks, which are similar in magnitude across the different background

therapies and across the systematic evaluation of the 3 most widely-approved SGLT2

inhibitors.9-11 To this point, a network meta-analysis that compared efficacy of the individual

SGLT2 inhibitors implied a benefit of high-dose canagliflozin over dapagliflozin and

empagliflozin, but this was limited to the context of monotherapy and absolute differences in

HbA1c-lowering between agents were small in magnitude.12 Among those with renal

dysfunction, the efficacy on HbA1c reduction was attenuated for all 3 medications, though for 2

of the 3 agents, it remained statistically and clinically significant. For example, in participants

with stage-2 CKD (eGFR 60 to <90 ml/min/1.73m2) treated with empagliflozin, the adjusted

mean difference in HbA1c was -0.68%, while the mean estimate was -0.42% in those with eGFR

30 to <60 ml/min/1.73m2.13 Such magnitude of HbA1c-lowering was observed in patients with

similarly impaired kidney function evaluated with canagliflozin14, though not in a trial of

dapagliflozin.15

In terms of clinical efficacy, it is important to emphasize the metabolic benefit of the

SGLT2 inhibitor class when used in place of alternate anti-hyperglycemic agents such as

sulfonylureas. For example, a 104-week study of empagliflozin vs. glimepiride revealed that

while the latter was associated with greater short-term HbA1c reduction at 12 weeks, by the end

of the trial, there was a significant (0.11%) advantage in favor of empagliflozin.16 Beyond this

small magnitude difference in HbA1c, compared with the sulfonylurea, there was a substantial

mean 4.5 kg reduction in body weight and a 90% lower risk of hypoglycemia with the SGLT2

inhibitor.16 Similar results were observed for canagliflozin,17 while a longer 208-week study of

of the 3 agents, it remained statistically and clinically significant. For examplee,e, iiinnn papapartrtrticicicipipipaaant

with stage-2 CKD (eGFR 60 to <90 ml/min/1.73m2) treated with empagliflozin, the adjusted

mean difffefererence in HbA1c was -0.68%, while the mean estimate was -0.42% in those with eGFR

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dapagliflozin confirmed this metabolic benefit (HbA1c-lowering was 0.3% lower than the active

comparator glipizide), with substantial benefits in terms of BP-lowering and eGFR

preservation.18 Placebo-controlled studies of SGLT2 inhibition in patients on complex insulin

regimens similarly revealed further HbA1c reduction, less insulin dose requirement and greater

weight loss, each observed without additional hypoglycemia.19-21 This extensive clinical trial

record emphasizes the series of metabolic benefits observed when an SGLT2 inhibitor is used

particularly in place of an insulin-providing agent such as a sulfonylurea, or when used to

attenuate total daily insulin doses.

Summary of Adverse Effects of SGLT2 inhibition

There is a series of well-recognized and suspected adverse events associated with augmenting

the presence of glucose and sodium in urine. Though a mild-to-modest increase in the incidence

of urinary tract infections have been observed in some studies10, but not other large-scale

studies,22 the strong causal relationship with mycotic genital infections, usually with candida

species, is indisputably the most common adverse event associated with SGLT2 inhibition.10 The

magnitude of the absolute risk increment rages between ~3% in the placebo vs. ~9-18%% with

the active comparators in women,20 and about half those rates have been reported in men.22

The risk of volume depletion arising from the osmotic diuresis of glucose - and from

natriuresis - represents an infrequent but clear causal adverse event from SGLT2 inhibition, more

common in older adults, among those who use higher doses of the SGLT2 inhibitors, use loop

diuretics and have kidney dysfunction.20, 23 However, such an increase was not observed in

EMPA-REG OUTCOME, which was a large-scale controlled clinical trial involving patients

with established cardiovascular disease, as discussed below.22

Summary of Adverse Effects of SGLT2 inhibition Summary of Adverse Effects of SGLT2 inhibition

There is a series of well-recognized and suspected adverse events associated with augmenting

he presence of glucose and sodium in urine. Though a mild-to-modest increase in the incidence

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Earlier concerns for a numerically higher case-finding of bladder cancer, particularly in

the dapagliflozin development program, and of breast cancer, have not been consistent and the

totality of evidence does not suggest a causal relationship.10, 24 Rather, one potential speculation

is that of a differential surveillance bias induced by SGLT2 inhibition. For example, any increase

in urinary symptoms – such as urinary tract or genital infections – may increase the likelihood of

investigations that identify bladder cancer, and a loss in weight may increase the likelihood of

identifying breast lesions.

Although small mean changes in electrolytes have been described in a controlled trial of

canagliflozin,25 such changes have not been observed in large-scale study in which plasma levels

of sodium, potassium, calcium, magnesium and phosphate did not differ in over 2300

participants exposed to each of placebo and low- and high-dose empagliflozin.22 However,

hyperkalemia may be seen among those who develop kidney dysfunction and who are

concomitantly exposed to antihypertensives that may elevate potassium levels, independent of

SGLT2 inhibitor use.26 Early reports of minor elevations in serum phosphate26 raised concerns of

an exaggerated phosphate resorption induced by distal sodium delivery, which could explain an

increase in parathyroid hormone and its consequent effect on bone turnover, density, and fracture

risk. However, observations, made primarily in the canagliflozin trial program, have been

inconsistent and inconclusive regarding a risk of bone loss, and the small loss in bone mineral

density might relate to measurement bias associated with bone densitometry in the setting of

weight loss. Furthermore, the types of fractures described are primarily distal small bone

fractures that may represent a consequence of volume depletion and falls rather than a bone-

specific causal association.27 Importantly, there was no increase in bone fractures in the EMPA-

REG OUTCOME trial.

of sodium, potassium, calcium, magnesium and phosphate did not differ r r ininin ooovevever rr 23232300m

participants exposed to each of placebo and low- and high-dose empagliflozin.22 However

hyperkalemia may be seen among those who develop kidney dysfunction and who are

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Despite elevations in high-density lipoprotein cholesterol and reduction in triglyceride

concentrations, the finding of minor elevations in low-density lipoprotein cholesterol

concentrations raised concerns over cardiovascular disease risk. However, findings from the

large-scale EMPA-REG OUTCOME trial (discussed below) have refuted this concern, along

with recent data from an animal model demonstrating that an SGLT2 inhibitor switches

metabolism from carbohydrate toward lipid utilization, which moderately increases ketogenesis

and low-density lipoprotein concentrations despite net lipid metabolic utilization28

The elevation in ketones that may be observed with SGLT2 inhibition requires attention.

While it was first reported as potentially modifying the presentation of diabetic ketoacidosis

(DKA) in two individuals in a mechanistic study involving T1D patients29 - in whom use of the

SGLT2 inhibitor was not regarded as the cause of DKA but rather permitting an atypically less

marked elevation in plasma glucose - other case series30 and trials31 have emphasized that use of

SGLT2 inhibitors for patients with T1D is “off-label” and should be undertaken only in the

context of clinical research studies and trials that implement systematic ketone monitoring.32

After the first year of post-marketing surveillance in the United States, a warning was released

by the FDA that reported 20 cases of DKA or ketosis. Though some occurred in patients with

T1D receiving off-label therapy against indication, an unexpected finding was that most cases

occurred in patients with T2D. Supported by the lack of association in large-scale clinical trials,22

these observations may represent a theoretical surveillance bias, in which cases of DKA that

would have occurred regardless of therapy in certain patients with T2D are reported because of

concomitant SGLT2 inhibition. However, several potential mechanisms exist to explain

elevations in circulating ketones in patients with T2D: 1) the switch from carbohydrate to lipid

metabolism – compounded by frequently-observed reductions in insulin doses in human trials –

DKA) in two individuals in a mechanistic study involving T1D patients29 - in wwwhohohom m m usususe e e ofofof tthe

SGLT2 inhibitor was not regarded as the cause of DKA but rather permitting an atypically lesf

marked elevation in plasma glucose - other case series30 and trials31 have emphasized that use o

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promotes lipolysis and ketogenesis33; 2) inhibition of SGLT2 expressed on pancreatic -cell

promotes glucagon hypersecretion, which is a fundamental component to the pathophysiology of

ketoacidosis;34 and 3) ketone reabsorption from the urine is enhanced by the distal delivery of

sodium seen in SGLT2 inhibition by way of the tubular sodium-monocarboxylate transporter-1

(SLC5A8).35

Non-glycemic effects of SGLT2 inhibitors: physiological mechanisms

SGLT2 inhibitors exert a variety of additional metabolic effects, including improvements in

insulin sensitivity, reduced glucose toxicity and weight loss, as discussed elsewhere36. In

addition, SGLT2 inhibitors exert important non-glycemic effects (Figure 2) including 1) BP

lowering; 2) renal hemodynamic modulation and reduced albuminuria; and 3) uric acid-lowering.

Beyond interest around the potential mechanisms responsible for these effects, the impact of

these non-glycemic pathways deserves significant attention in light of the results of the EMPA-

REG OUTCOME trial, which demonstrated significant reductions in mortality, heart failure

hospitalization and nephropathy, even though HbA1c-lowering was, by design (to examine

glycemic equipoise), on average only 0.24% [95% CI, 0.40 to 0.08] and 0.36% [95% CI,

0.51 to 0.20] in the 10 and 25 mg treatment arms, respectively vs. placebo.

Blood pressure lowering:

Consistent systolic 5 mmHg and diastolic 2 mmHg BP lowering effects of SGLT2 inhibitors

have been reported in patients with T2D37. Notwithstanding the fact that very little head-to-head-

data exist, the magnitude of the BP-lowering effects is largely similar across members of the

class37. Additionally, empagliflozin reduces systolic BP by 2.7 mmHg on average in young

addition, SGLT2 inhibitors exert important non-glycemic effects (Figure 2) iiincncnclululudididingngng 111))) BPB

owering; 2) renal hemodynamic modulation and reduced albuminuria; and 3) uric acid-lowering

Beyond interest around the potential mechanisms responsible for these effects, the impact o

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patients with uncomplicated T1D38, and BP-lowering effects may extend to healthy individuals

without diabetes39, 40. In terms of circadian effects of SGLT2 inhibitors on BP, animal studies

involving obese salt-sensitive rats have suggested that SGLT2 inhibition may restore normal

nocturnal BP dipping41. In patients with T2D, 24 hour ambulatory BP monitoring (ABPM)

studies have suggested that empagliflozin successfully reduces systolic BP at night, including in

patients with nocturnal non-dipping42, 43.

Despite a paucity of data around effects on nocturnal dipping, ABPM studies in patients

with T2D have suggested that daytime BP lowering is attenuated at night42, 43. This observation

may reflect 1) a lack of glycosuric/natriuretic effects at night while patients are fasting; 2)

changes in renal perfusion and the reduction in GFR characteristic of being supine; and/or 3)

redistribution of sodium to the central circulation at night – mitigating the expected volume-

related BP lowering effects.

In terms of interactions with antihypertensive agents, pre-clinical studies in animals have

thus far not demonstrated clear, additive BP-lowering effects with SGLT2 inhibitors when these

agents are combined with other diuretics (e.g. thiazides, furosemide)44. Studies in humans have

failed to demonstrate an additive BP-lowering effect of combining the SGLT2 inhibitor

canagliflozin with a thiazide, nor does the combination produce a greater natriuretic effect

compared with either drug alone45. Similar to this report using canagliflozin45, the addition of a

thiazide diuretic to dapagliflozin does not yield additional antihypertensive effect, whereas beta-

blocker and calcium channel blocker combinations with dapagliflozin did accentuate the degree

of BP lowering46. Similarly, the addition of dapagliflozin to a background of a RAAS inhibitor in

patients with T2D lowers systolic BP by an additional 3-4 mmHg47 – an effect that has been

supported by results from animal studies48. Whether this apparent synergy is on the basis of

changes in renal perfusion and the reduction in GFR characteristic of being suuupipipinenene;;; ananand/d/d/ororor 3

edistribution of sodium to the central circulation at night – mitigating the expected volume

elated BP lowering effects.

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plasma volume contraction leading to RAAS activation, which is then inhibited

pharmacologically by ACE inhibition or an ARB, resulting in enhanced BP-lowering is not

known. Alternatively, angiotensin II increases SGLT2 mRNA expression and proximal tubular

sodium uptake, perhaps promoting volume expansion and worsening hypertension49. It is

therefore conceivable that the greater BP-lowering effect observed with the combination of an

SGLT2 inhibitor with a RAAS blocking agent vs. either agent alone could represent suppressed

proximal tubular sodium reabsorption and volume contraction.

While the mechanisms responsible for BP lowering effects of the SGLT2 inhibitors are

still being elucidated, several factors are likely to be involved, including changes in plasma

volume and reduced arterial stiffness (discussed below). Thiazide diuretics cause a 5-8% initial

contraction of plasma volume, which then attenuates over time so that by 10-12 weeks, the BP-

lowering effect with these agents occurs on the basis of a reduction in systemic vascular

resistance50. In contrast, when compared with placebo or thiazide treatment, dapagliflozin lowers

BP over 12 weeks and causes a persistent 7% contraction in plasma volume and an increase in

hematocrit51. Although increases in hematocrit which are consistently observed in other

studies52, may represent hemoconcentration from plasma volume contraction, meta-regression

analyses of SGLT2 inhibitor–related BP lowering did not show an association between systolic

or diastolic BP and changes in body weight, where weight was used as a surrogate measure of

salt and water loss - perhaps suggesting that additional pathways are involved in mediating

systemic hemodynamic effects37. Furthermore, SGLT2 inhibition may initially stimulate

erythropoietin secretion thereby accounting for changes in hematocrit via plasma volume-

independent mechanisms51. Erythropoietin is, however, unlikely to account for sustained

increases in hematocrit, since changes dissipate over 6-8 weeks51. In contrast with these findings

volume and reduced arterial stiffness (discussed below). Thiazide diuretics caussseee a a a 5-5-5-8%8%8% iiinininitit a

contraction of plasma volume, which then attenuates over time so that by 10-12 weeks, the BP

oweringgg effect with these agents occurs on the basis of a reduction in systemic vascula

esiiistststaance50. Innn ccconnntrtt asasast,t,t, wwwheheh n n n cococompmpmparededed with plpp acceebo o o ororor ttthihihiazaa ididideee trtrtreaee tmtmtmennt,t,t, dadadapapapaglgg ifflololozizizin n n lololoweww r

BP ooovevv r 12 wwweeeeeeksss andndnd cauuuseees a persissteent 7%% % coonntraraactctctioioionnn ininin plaaassms a vvolululumem anddd aaannn iiincreasesese in

hehemamatotocrcritit51. AlAlththououghgh iincncrereasaseses iinn hehemamatotocrcritit wwhihicchh araree coconsnsisistetentntlyly obobseservrveded iinn otothehe



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comparing dapagliflozin vs. hydrochlorothiazide or placebo, Sha, et al. reported that the plasma

volume contraction associated with canagliflozin at 1 week did not persist at 12 weeks, although

this study lacked a placebo group and the wide variability of the plasma volume change at 12

weeks makes definitive conclusions difficult53.

Measures of increased arterial stiffness have been associated with hypertension,

hyperglycemia, obesity and echocardiographic features of heart failure54, 55. More importantly,

increased arterial stiffness is a predictor of cardiovascular events, heart failure and death –

especially in patients with diabetes and those at elevated cardiovascular risk54, 56-59, and is further

associated with microvascular complications60. In addition to this associative data, major

cardiovascular protective medications such RAAS inhibitors and statins reduce arterial stiffness

indices31, 61, 62. A second mechanism that may play some part in the BP-lowering effect observed

with SGLT2 inhibitors relates to a reduction in arterial stiffness, demonstrated by pulse wave

velocity and augmentation index in young patients with uncomplicated T1D63. In patients with

T2D, markers of arterial stiffness such as pulse pressure also improve with SGLT2 inhibition in

conjunction with improvements in markers of myocardial oxygen consumption (i.e. “rate

pressure product”)64. Whether these improvements in measures reflecting arterial stiffness are a

consequence or a result of BP lowering is not known, nor is their clinical relevance.

Nevertheless, given the relationship between higher arterial stiffness and increased risks of

cardiovascular events, mortality or death57, salutary effects of SGLT2 inhibition on measures of

arterial stiffness may in part explain some of the cardiovascular benefits observed in EMPA-

REG OUTCOME, as discussed below. The effect of SGLT2 inhibition on other determinants of

BP such as endothelial function, cardiac output or systemic vascular resistance are unknown and

the subject of ongoing clinical studies (NCT02501616, NCT02608905, NCT02235298).

cardiovascular protective medications such RAAS inhibitors and statins reduce aaartrtrterereriaiaial l l stststifififfnfnfnes

ndices31, 61, 62. A second mechanism that may play some part in the BP-lowering effect observed

with SGLT2 inhibitors relates to a reduction in arterial stiffness, demonsrr trated by pulse wave

velooocicicity and aaaugugugmemementttatata ioioionnn innndededex x x ininin yooounuu g paaatitit enntts wwwititithhh unununcococommpmplililicacc teteted d T1T1T1DDD636363. InII pppatatatieieientntnts s s wiww th

T2D,D,D, markers ooof f araarterrriaaal stiiiffffffness suchh aas pullsesese ppreessssururureee alalalsososo impmpmproveee wiwiiththh SGLT2T2T2 iiinnnhibitiooonn n in

coconjnjununctctioionn wiwithth iimpmprorovevemementntss inin mmararkekersrs ooff mmyoyocacardrdiaiall oxoxygygenen ccff ononsusumpmptitionon ((ii.ee. “raratete



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Based on available data, natriuresis and arterial stiffness are probably the most significant

mediators responsible for antihypertensive effects with SGLT2 inhibitors. Although other

factors, such as circulating neurohormones, have been identified as candidate antihypertensive

mediators, it is unlikely that these pathways play an important role. For example, based on

existing data, it seems unlikely that suppression of major neurohormonal pathways such as the

RAAS cascade are involved in a relevant way. Prior studies have demonstrated that plasma

levels of RAAS mediators including aldosterone and angiotensin II and urinary levels of

angiotensinogen and ACE actually increase in response to SGLT2 inhibition, with similar trends

for plasma renin38, 51, 65, possibly in response to plasma volume contraction. On the other hand,

urinary ACE2 protein and enzymatic activity also increase, which may be important for BP-

lowering, as the ACE2-Ang(1-7) pathway promotes vasodilatation66. Vasodilators such as

plasma NO decrease in response to empagliflozin, while urinary NO metabolites remain

unchanged, arguing against NO bioactivity being related to BP-lowering. Other neurohormonal

pathways such as the sympathetic nervous system (SNS) remain unchanged in response to

SGLT2 inhibition63, 67. Furthermore and in support of neutral effects on the SNS, previous

glycemic control studies have uniformly reported that SGLT2 inhibition does not increase heart

rate, which is perhaps surprising given the plasma volume reduction associated with the class.

The neutral effect on heart rate may therefore be interpreted as 1) a “normalization” of plasma

volume by SGLT2 inhibition that is insufficient to activate the SNS, or 2) “suppression” of the

SNS by an unidentified factor or pathway. In summary, reported changes in neurohormones do

not appear to account for BP-lowering effects associated with SGLT2 inhibition, with the

possible exception of the ACE2-(Ang1-7) pathways, which requires further study.

urinary ACE2 protein and enzymatic activity also increase, which may be impmpmpororortatatantntnt fffororor BBBP

owering, as the ACE2-Ang(1-7) pathway promotes vasodilatation66. Vasodilators such a

plasma NO decrease in response to empagliflozin, while urinary NO metabolites remain

unchchchaaanged, argrgrguuuingngng aaagagagainininststs NNNO O O bibibioaoo ctititiviv ty beieieingg relatatatededed tttooo BPPP-lowowowerrrinini g. OOOthththererer neuuurororohohohormrmrmonoo a

pathhhwwways such h h asss the symmmppap thetic nnererrvous sssystememm (((SNSNSNS)S)S) remmmaaia n unuunchchchana ged innn reeesponssseee to

SGSGLTLT22 ininhihibibititionon63,33 6667. FuFurtrthehermrmororee anandd inin ssupuppoportrt ooff neneututrarall efeffefectctss onon tthehe SSNSNS, prprevevioiouu



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As a final comment, weight loss induced by lifestyle modification – even when modest –

has been reported to reduce BP68. For the relationship between weight loss and BP reductions

with SGLT2 inhibitors, findings from previous analyses have been heterogeneous as to whether

weight loss is responsible for BP-lowering in response to this class of drugs. Some reports have

suggested no interaction between weight loss and BP reduction37, while others have observed

that weight loss may account for between 28-40% of the BP-lowering observed with SGLT2

inhibition26, 69. Based on these data, most of the BP-lowering effect of SGLT2 inhibition is

independent of longer-term weight loss, although there may be a contribution from the expected

initial plasma volume contraction and associated weight loss. For example, after canagliflozin

treatment for 1 week and systolic blood pressure lowering by 10.4 mmHg, body weight was

decreased modestly by 1.7 kg – a difference most likely to be accounted for by salt and water

loss53. There may be at least an initial modest contribution of weight loss to BP-lowering in

patients with normal renal function who ultimately achieve up to 3 kg of weight loss. However,

in patients with CKD, HbA1c and weight-lowering effects of SGLT2 inhibition are consistently

attenuated, probably because of the attenuation of glycosuria in the setting of CKD (Figure 3)70.

In such patients with CKD, the magnitude of BP lowering is preserved and possibly

augmented13-15. This discordance between glycosuria-related effects (HbA1c and weight

reduction) and BP-lowering in patients with CKD is striking, and argues against a prominent role

for longer-term weight loss, which mainly consists of a reduction in adipose tissue, as an

important mediator of the BP response to SGLT2 inhibition. While the mechanisms responsible

for BP-lowering in the setting of CKD in the absence of HbA1c or weight effects are not fully

understood, it is perhaps relevant that patients with CKD tend to be more salt-sensitive71. As a

consequence, even if natriuresis and plasma volume contraction are attenuated with SGLT2

reatment for 1 week and systolic blood pressure lowering by 10.4 mmHg, booodydydy wwweieieighghght t t wwwa

decreased modestly by 1.7 kg – a difference most likely to be accountet d for by salt and wate

oss53. There may be at least an initial modest contribution of weight loss to BP-lowering in

patiiienenents with nononormmmalaa rrrenenenalalal funununctctctioioion n n whhho oo ultimamamatelyly achchchieieieveveve uuup pp tototo 333 kggg of wwweieieighghghttt lossss. HoHoHowewewevev r

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atattetenunuatateded, prprobobabablyly bbececauausese ooff tthehe aattttenenuauatitionon ooff glglycycososururff iaia iinn ththee sesettttiningg ofof CCKDKD (F(Figigururee 3)3)7070



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inhibition in CKD, these patients may display a heightened sensitivity to even small changes in

plasma volume, leading to persistent BP-lowering effects. Alternatively, CKD patients tend to

have higher BPs, and SGLT2 inhibition may lead to greater anti-hypertensive effects in those

with higher BP at baseline64.

Renal hemodynamic function and albuminuria:

In addition to systemic hemodynamic effects, SGLT2 inhibitors impact renal function. In

patients with T2D, eGFR changes are characterized by acute, dose-dependent reductions of 5

ml/min/1.73m2 over several weeks. After this initial “dip”, eGFR subsequently tends to return

toward baseline and remains stable over time (Figure 4). The impact of SGLT2 inhibition on

eGFR is consistent in patients with and without CKD, in those with established cardiovascular

disease and is observed after treatment for 3-4 weeks (Figure 4)13-15, 17, 72, 73. In addition, the

eGFR dip is reversible within two weeks of drug discontinuation13, an effect also observed in the

EMPA-REG OUTCOME trial73.

Effects of the SGLT2 inhibitor class on renal function are both important mechanistically

and clinically, since the underlying changes in intraglomerular pressure contribute to

improvements in renal endpoints in EMPA-REG OUTCOME73. In considering the pattern of

renal function change with SGLT2 inhibitors and possible benefits, previous work has compared

and contrasted these effects with what is expected in response to agents that block the RAAS.

Inhibitors of the RAAS reduce intraglomerular pressure through efferent arteriolar

vasodilatation, leading to reductions in intraglomerular hypertension and renal hyperfiltration

and resulting in nephroprotection74. Renal hyperfiltration – generally defined as a GFR 135

ml/min/1.73m2 - is a marker of intraglomerular hypertension and is a risk factor for the initiation

oward baseline and remains stable over time (Figure 4). The impact of SGLT2T2T2 iiinhnhnhibibibitititioioionnn on

eGFR is consistent in patients with and without CKD, in those with established cardiovascula

disease anand d is observed after treatment for 3-4 weeks (Figure 4)13-15, 17, 72, 73. In addition, the

eGFRFRFR dip is reeevvverrsibii leee wwwititithihih n n n twtwtwoo o weww ekekeks of drruuug discocoontntntinininuauauatit ononnr 1333,, ananan eeeffececect t t alalalsososo ooobssserrrveveved dd ininin the

EMMMPAPAPA-REG OOOUTUTUTCOCOOMMEM triririaala 73.

EfEffefectctss ofof tthehe SSGLGLT2T2 iinhnhibibititoror cclalassss oonn rerenanall fufuncnctitionon aarere bbotothh imimpoportrtanantt memechchananisistiticacallllyy



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and progression of diabetic nephropathy75, 76. Importantly, hyperfiltration at the single nephron

level is a risk factor for renal disease progression in animals, and likely important in humans as

well. When analyzed according to eGFR change over time after treatment with RAAS blockade,

patients with T2D who have the greatest tertile of eGFR change within 3 months have the best

preservation of renal function77. The impact of RAAS blockade on eGFR and the positive

association between initial eGFR decline and preservation of renal function has been interpreted

to reflect a beneficial decline in intraglomerular pressure though efferent vasodilatation. Whether

the magnitude of change in eGFR with an SGLT2 inhibitor similarly correlates with the degree

of renal protection is not known.

In contrast to the effects of RAAS blockers, the initial decline in eGFR observed with

SGLT2 inhibitors is likely related to afferent arteriolar vasoconstriction, through a

tubuloglomerular feedback mechanism. In non-diabetic conditions, SGLT2 is responsible for

about 5% of total renal NaCl reabsorption. However, in the context of hyperglycemia, SGLT2

and SGLT1 mRNA expression increase by 36% and 20%, respectively78-80. As a consequence,

SGLT1/2 activity accounts for as much as 14% of total renal NaCl reabsorption in the setting of

hyperglycemia of diabetes78-80, thereby leading to a marked reduction in distal NaCl delivery to

the macula densa (Figure 5)81. According to the tubular hypothesis for glomerular

hyperfiltration, the decline in macula densa NaCl delivery is sensed incorrectly as a reduction in

effective circulating plasma volume by the juxtaglomerular apparatus. This process is called

tubuloglomerular feedback, and leads to maladaptive glomerular afferent arterial vasodilatation

and increased intraglomerular pressure82. In animals, non-specific SGLT1/SGLT2 inhibitors83

and selective SGLT2 inhibition increase distal renal NaCl delivery, causing increased afferent

tone, thereby suppressing hyperfiltration84.

In contrast to the effects of RAAS blockers, the initial def cline in eGFR R R obobobseseservrvrvededed wwwith

SGLT2 inhibitors is likely related to afferent arteriolar vasoconstriction, through a

n non-diabetic conditions, SGLT2 is responsible foubuloglomerular feedback mechanism. I

abouououtt t 5% of totototttal l l rerr nananall NaNaNaClClCl rerereabababsoss rppptitt on. HoHoHowwevver,r,r, iiin n n thththeee cccononontetetextxx ooof hyhyhypepepergrgrglylylycemimimia,a,a, SSSGLGLGLT2

andd d SSGS LT1 mRmRmRNANNA eeexxpx ressssiioi n increaaseee by 363636% aand d d 202020%%%, rrrespppectc iveleely78-78-78-80. As aaa conononsequenenence

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In animal models of diabetes, the non-selective SGLT1/SGLT2 inhibitor phlorizin

decreases hyperfiltration and suppresses proteinuria78, 82, 83. Selective SGLT2 inhibitors have

similar effects on markers of diabetic nephropathy, including reductions in hyperfiltration,

proteinuria, glomerular hypertrophy and mesangial expansion, and SGLT2 knock out models

also exhibit a reduction in hyperfiltration, as reviewed elsewhere85. Since increases in

intraglomerular pressure and resultant increases in glomerular wall tension are associated with

glomerular fibrosis and inflammation, it would perhaps be expected that SGLT2 inhibitor-

mediated reductions in hyperfiltration would suppress markers of inflammation and fibrosis. In

animals, SGLT2 inhibition suppresses blood and tissue markers of inflammation, oxidative

stress, and fibrosis, although it is unknown if these effects are based on improved glycemia or

instead due to hemodynamic-mediated effects84, 86-90. The difficulty in distinguishing

hemodynamic from non-hemodynamic, anti-proteinuric pathways is even more complex due to a

lack of data around anti-fibrotic/anti-inflammatory markers with SGLT2 inhibition in humans,

except for the observation that dapagliflozin reduces C-reactive protein91. Historically, very little

has been known about the effect of SGLT1/2 inhibition with phlorizin on renal function in

humans92. We have reported the effect of empagliflozin on renal hemodynamic function in

patients with T1D, demonstrating reductions in renal hyperfiltration of similar magnitude to what

has been observed with ACE inhibition93, suggesting a protective decline in intraglomerular

pressure.

While the mediators responsible for reducing hyperfiltration in animals and/or humans in

response to SGLT2 inhibition are not yet known, renal adenosine production may play a key

role. In response to an increase in NaCl delivery to the macula densa after blockade of proximal

tubular NaCl reabsorption, increased uptake of NaCl by macula densa cells occurs largely via the

tress, and fibrosis, although it is unknown if these effects are based on improveveved d d glglglycycycemememiaiaia o

nstead due to hemodynamic-mediated effects84, 86-90. The difficulty in distinguishing

hemodyynamic from non-hemodynamic, anti-proteinuric pathways is even more complex due to a

ackkk ooof data aaarororounnnd dd ananantititi f-f-fibibi rororotititic/c/c/anananti-iiinfnn lammmmataa ooryy mmmarararkekekersrsrs wwwititith h SGSGSGLTLL 2 inininhihihibibibititit on iiin n n huhuhumamam ns

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Na/K/2Cl co-transporter, which is an energy-requiring process leading to breakdown of

adenosine triphosphate to adenosine (Figure 5). Adenosine then acts in a paracrine fashion via

adenosine type-1 receptors on afferent arteriolar vascular smooth muscle cells causing

vasoconstriction94. In the normotensive, modestly hyperglycemic streptozotocin-induced diabetic

mouse model of T1D, knock out of the adenosine type 1 receptor attenuates renal hyperfiltration,

suggesting a physiological link between adenosine action and hyperfiltration95, at least under

these experimental conditions. In humans, consistent with the hypothesis that SGLT2 inhibition-

mediated increases in distal NaCl augment adenosine release, we recently reported that SGLT2

inhibition for 8 weeks increases the urinary adenosine/creatinine ratio as measured by LC-

MS/MS under clamped hyperglycemic conditions96. Of note, another proximal tubular diuretic –

acetazolamide – reduces hyperfiltration in animals97 and in patients with T1D98, and in obese,

patients without diabetes with hyperfiltration at baseline – likely reflecting similar changes in

tubuloglomerular feedback99. Other agents that increase NaCl delivery to macula densa may

therefore activate some of the same tubuloglomerular feedback pathways and attenuate

hyperfiltration. The obvious exception to this is the use of loop diuretics such as furosemide,

which block macula densa NaCl entry by inhibiting the Na/K/2Cl channel, and in doing so

attenuate tubuloglomerular feedback99.

Aside from the intrarenal effects described above, it has been suggested that blood

pressure or weight loss in response to SGLT2 inhibition mediate changes in kidney function. It

is, however, unlikely that BP-lowering effects in response to SGLT2 inhibition significantly

impact kidney function over the relatively short duration of trials reported to date. In support of

BP-independent kidney effects, in patients with T1D, modest BP-lowering effects were similar in

patients with (GFR 135 ml/min/1.73m2) and without glomerular hyperfiltration. In contrast,

MS/MS under clamped hyperglycemic conditions96. Of note, another proximal tuuubububulalalar r r dididiururureteteticii –

acetazolamide – reduces hyperfiltration in animals97 and in patients with T1D98, and in obese

patients without diabetes with hyperfiltration at baseline – likely reflecting similar changes in

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only the hyperfiltration group exhibited a fall in eGFR (19.2%) – an effect that was greatly out of

proportion to the 2.7 mmHg change in systolic BP. Furthermore, SGLT2 inhibitors reduce

albuminuria by 30-40%, possibly on the basis of reductions in intraglomerular hypertension100,

since anti-albuminuric effects are largely independent of concomitant changes in BP, weight or

HbA1c100, 101. It seems likely that similar to the impact of agents that block the RAAS, intrarenal

hemodynamic effects observed with SGLT2 inhibitors are mediated by BP-independent

pathways via afferent arteriolar vasoconstriction.

It is similarly unlikely that acute reductions in eGFR simply reflect improvements in

glycemic control. Although improved glycemic control can lower eGFR102, the observation that

CKD patients exhibit blunted or complete loss of HbA1c-lowering and still have an acute eGFR

dip and significant lowering of albuminuria argues instead the notion that intrarenal

hemodynamic mechanisms are principally responsible for short-term changes in renal function

with SGLT2 inhibitors. Moreover, in our cohort of patients with T1D, those with hyperfiltration

and normofiltration had similar modest changes in HbA1c, whereas only the hyperfiltration

group had a change in inulin-derived eGFR, renal blood flow and renal vascular resistance –

highlighting the concept of intrarenal effects of SGLT2 inhibition.

Finally, in animal models, increased atrial natriuretic peptide (ANP) levels have been

associated with the renal hyperfiltration and volume expansion that is characteristic of

diabetes103. To test whether blockade of ANP-related pathways would modify hyperfiltration,

studies in the 1990s reported that ANP antiserum and ANP receptor antagonists reduce

hyperfiltration in animals, although results were not uniform and may have differed based on the

model, varied BP levels and the degree of hyperglycemia104-106. In patients with T1D, most

previous work has failed to report differences in ANP levels in those with and without

CKD patients exhibit blunted or complete loss of HbA1c-lowering and still haveee aaan n n acacacututute e e eGeGeGFRf

dip and significant lowering of albuminuria argues instead f the notion that intrarena

hemodyynamic mechanisms are principally responsible for short-term changes in renal function

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hyperfiltration, and investigative studies in patients with T2D have even reported that ANP

levels increase with improved glycemic control, even while GFR decreases, which is discordant

from conclusions drawn from animal studies107-109.

Due to the interaction between ANP, SGLT2 overactivity and volume expansion in

diabetes, is it possible that by blocking SGLT2, volume expansion can be normalized, leading to

reduced ANP levels and attenuated hyperfiltration? To test this hypothesis, we measured plasma

ANP levels in a well characterized cohort of patients with T1D with or without hyperfiltration at

baseline, and then in response to SGLT2 inhibition for 8 weeks38, 63, 110. At baseline, ANP levels

did not differ between normofiltering and hyperfiltering patients (Figure 6). In response to

empagliflozin, ANP levels remained unchanged in both groups (Figure 6). In contrast, in

patients with newly diagnosed T2D, SGLT2 inhibition for 24 weeks reduced ANP levels111.

Although no information about renal hemodynamic function or hyperfiltration were included in

this report, hyperfiltration is less common in this setting compared with young patients with

T1D. While SGLT2 inhibition may therefore reduce volume expansion in older patients with

T2D and thereby suppress ANP, data from younger patients with T1D suggest that SGLT2

inhibition-mediated effects on renal hemodynamic function are independent of ANP.

Uric acid levels:

As reviewed elsewhere, plasma uric acid levels have been associated with hypertension,

cardiovascular disease and renal disease112. As a consequence, uric acid-lowering therapies have

been the subject of ongoing renal and cardiovascular protection studies112. The SGLT2 inhibitor

class of drugs has been associated with a 10-15% reduction in plasma uric acid levels as a result

of increased glycosuria, leading to secretion of uric acid in exchange for glucose reabsorption via

empagliflozin, ANP levels remained unchanged in both groups (Figure 6). InInIn cccononontrtrtrasasasttt, in

patients with newly diagnosed T2D, SGLT2 inhibition for 24 weeks reduced ANP levels111

Althoughg no information about renal hemodynamic function or hyperfiltration were included in

hisss rrreport, hypypyperrrfififiltraraatititiononon is s s lelelessssss cccommmmomm n innn thhiss setetettititingngng cccommmpapaparerered d d wiww thhh yyyouououngngng ppatatatieieientntnts s s wiww th

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the GLUT9 transporter110. In light of the possible relationship between plasma uric acid levels

and cardiorenal disease, SGLT2 inhibitor-associated uricosuria and uric acid-lowering may be

beneficial, although the clinical relevance of this effect remains to be elucidated.

EMPA-REG OUTCOME trial

Background and brief study description:

The EMPA-REG OUTCOME trial was a long term, non-inferiority cardiovascular safety trial

carried out in 7,020 patients with T2D at high cardiovascular risk assigned in a 1:1:1 ratio to

placebo vs. empagliflozin 10 mg daily vs. empagliflozin 25 mg daily22. After the first 12 weeks

of the study when baseline glycemic agents remained unchanged, glucose-lowering medications

were adjusted to achieve HbA1c levels to local standards resulting in a <0.4% difference in

HbA1c between treatment and placebo groups.

By hierarchical testing, non-inferiority was shown in subjects treated with empagliflozin

compared to placebo. In subsequent order, superiority was analyzed. The primary 3-point major

adverse cardiovascular event (MACE) endpoint (cardiovascular death, non-fatal myocardial

infarction, non-fatal stroke) was reduced by 14%, driven by a 38% reduction in cardiovascular

death, without significant reductions in either non-fatal myocardial infarction or stroke. Death

from any cause was reduced by 32%, and hospitalization for heart failure, which was a pre-

specified secondary outcome, was reduced by 35% in patients randomized to receive

empagliflozin. Reduction of cardiovascular mortality and heart failure hospitalization was

observed within 3 months of starting treatment, and was observed in a wide range of subgroups,

including those with CKD, and whether or not patients were taking antihypertensive agents,

of the study when baseline glycemic agents remained unchanged, glucose-loweririringngng mmmedededicicicatatatioioion

were adjusted to achieve HbA1c levels to local standards resulting in a <0.4% difference in

HbA1c between treatment and placebo groups.

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compmpmpared to plplplaaacebeebo. IIIn suuubbsbsequent ordrdder, suuupperiiioorittty y y wawawas s anananalyzyzyzed. TTheee prpp imaryyy 3-3--pppoint mamamajo

adadveversrsee cacardrdioiovavascsculularar eeveventnt ((MAMACECE)) enendpdpoiointnt ((cacardrdioiovavascsculularar ddeaeathth, nonon-n-fafatatall mymyococarardidiaa



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diuretic, insulin or lipid-lowering drugs. Similar improvements in outcomes were observed with

both doses of empagliflozin.

Empagliflozin was well tolerated with no increase in serious adverse outcomes. Mycotic

genital infections occurred in 4.5% more patients on empagliflozin than placebo, yet less than

0.6% had to discontinue treatment. Rates of serious adverse events leading to drug

discontinuation (19.4% vs. 17.3%), acute renal failure (6.6% vs. 5.2%), acute kidney injury

(1.6% vs. 1.0%) were lower in empagliflozin-treated patients, while rates of DKA, volume-

depletion and “thromboembolic events” were not increased by empagliflozin.

The secondary, pre-specified renal endpoints from EMPA-REG OUTCOME have also

been reported73. Empagliflozin reduced the endpoint of “new onset or worsening nephropathy”

(new onset macroalbuminuria, doubling of creatinine and eGFR<45 ml/min/1.73m2, initiation of

renal replacement therapy, death due to renal disease, HR 0.61, 95% CI 0.53-0.70, p<0.0001)

and also reduced the composite of doubling of creatinine initiation of renal replacement therapy,

and death due to renal disease (HR 0.54, 95% CI 0.40-0.75, p=0.0002). The kidney protective

effects therefore appear to be complementary to the cardiovascular benefits.

Mechanistic analysis:

With the wide range of systemic and renal physiological effects, it is currently not possible to

ascertain which mechanisms are most responsible for the remarkable cardiovascular and/or renal

protection observed with empagliflozin in EMPA-REG OUTCOME39. Nevertheless, an analysis

of potentially relevant mediators is important to better understand these effects, which have not

been observed with any other anti-hyperglycemic therapy with the possible exceptions of

liraglutide and semaglutide in the LEADER and SUSTAIN-6 trials, respectively. The LEADER

been reported73. Empagliflozin reduced the endpoint of “new onset or worseningngng nnnepepephrhrhropopopatatathyhh ”f

new onset macroalbuminuria, doubling of creatinine and eGFR<45 ml/min/1.73mf 2, initiation o

enal replpp acement therapy, death due to renal disease, HR 0.61, 95% CI 0.53-0.70, p<0.0001

and d d alalalso reducccededed ttthehh cccomomompopop sisisitetete ooofff dodd ubububling oooff f creaeatinnnininineee inininitititiaaatititiononon ooof f rerer nal l l rerereplplplacacacemmmenenenttt thththerererapaa y

andd d dded ath dueee tototo reenaaal diseaeaease (HR 0.5.554, 95%%% CICI 000.4.4.40-0-0-000.75775, p===00.0 0002002).).. TThe kidddnnneyyy protecccttit ve

efeffefectctss ththererefefororee apappepearar ttoo bebe ccomomplpleemementntararyy toto tthehe ccarardidiovovasascuculalarr bebenenefifitsts.



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trial recently reported a 13% reduction in the 3-point MACE primary endpoint, and a 22%

reduction in cardiovascular death113. SUSTAIN-6 topline results have been summarized in a

press release reporting superiority for MACE outcomes, but these data are yet to be published. A

mechanistic analysis of EMPA-REG OUTCOME is also important to determine how to best use

these agents in patients with T2D, and, as we have suggested elsewhere, potentially in other

disease states outside of T2D40.

The first mechanism that has perhaps been most widely credited with the positive

cardiovascular and renal outcomes of EMPA-REG OUTCOME relates to effects on diuresis-

both natriuresis and osmotic diuresis39, 40. Although data related to diuresis are surprisingly

sparse, previous studies with canagliflozin and empagliflozin have reported that 24 hour urine

volume increases by ~300 ml/day after even day-1 of treatment, but that daily urine volume

returns back to baseline after several weeks53, 114. In patients with T1D, 24 hour urine sodium

excretion is similarly unchanged after 8 weeks of treatment – likely reflecting the establishment

of a new steady state38. In conjunction with plasma volume data described above, SGLT2

inhibition induces a rapid but modest contraction of plasma volume over several days, which

stabilizes over time. As discussed above, long-term changes in hematocrit/red cell mass are

unlikely to be based on changes in erythropoietin-derived bone marrow production since initial

increases in erythropoietin are transient. Insight into whether or not volume overload improved

in patients enrolled in EMPA-REG OUTCOME may also be gleaned from a secondary analysis

of heart failure risk in this study. In this analysis, Fitchett et al., reported that the use of

furosemide was reduced in empagliflozin treated patients vs. those taking placebo115 –

suggesting restoration of relative euvolemia. Decrease in loop diuretic use may also be relevant

in light of the reductions in acute kidney injury, acute renal failure and CKD progression

parse, previous studies with canagliflozin and empagliflozin have reported thatatat 2224 44 hohohoururur uuurirr ne

~volume increases by ~300 ml/day after even day-1 of treatment, but that daily urine volume

eturns back to baseline after several weeks53, 114. In patients with T1D, 24 hour urine sodium

excrcrcreetetion is simimimilarararly uuuncncnchahahangngngededed afafaftet r 888 weekkksss off ttreatatatmemementntnt – lllikikikelelely yy rerereflf eccctititingngng ttthehehe estststababablililishshshmemm n

of aaa new steadadadyy sstattteee38. InInIn conjuncttiooon wittthhh pplaasmamama vvvolololumumu e ddad ta ddeese cccribed aaabobobovvve, SGGGLLTL 2

nnhihibibititionon iindnducuceses aa rrapapidid bbutut mmododesestt cocontntraractctioionn ofof pplalasmsmaa vovolulumeme ooveverr seseveverarall dadaysys, whwhicichh



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endpoints, since the volume depletion associated with loop diuretic use can lead to pre-renal

acute kidney injury. As a final point, the early separation of the survival curves for empagliflozin

in EMPA-REG OUTCOME are analogous to those observed with spironolactone in the

Randomized Aldactone Evaluation Study (RALES) – another diuretic that may mediate positive

effects via natriuresis and plasma volume contraction116. Nevertheless, mineralocorticoid

antagonists suppress fibrosis and remodeling – effects that are less clearly understood with

SGLT2 inhibitors, underscoring potential differences between these medication classes.

Beyond playing a possible role in mediating some of the anti-hypertensive effects of

SGLT2 inhibitors natriuresis-related changes in plasma volume my also be relevant in light of

the cardiovascular protective effects reported in EMPA-REG OUTCOME. The acute changes in

natriuresis leading to plasma volume contraction could induce relative euvolemia, thereby

reducing cardiac preload and heart failure hospitalization risk, which decreased rapidly over

several months in EMPA-REG OUTCOME. Moreover, plasma volume contraction-mediated

decreases in myocardial stretch may have reduced cardiac arrhythmogenesis – another

mechanism plausibly responsible for the reduced mortality observed in the empagliflozin-treated

subjects in EMPA-REG OUTCOME39, 40. Of note, previous individual studies and meta-

analyses of anti-hypertensive drugs have reported that other, non-SGLT2 inhibitor-based

diuretics reduce the risk of heart failure – an observation not uniformly observed with other

antihypertensive agents117, perhaps suggesting unique salutary effects with diuretic agents

overall. It is, however, difficult to compare heart failure effects of SGLT2 inhibitors with those

of thiazides because of differential effects on longer-term plasma volume contraction. For loop

diuretics, previous studies have reported a reduction in the risk of being hospitalized for heart

failure with the use of these agents118. Despite suggestions of an effect on mortality with loop

he cardiovascular protective effects reported in EMPA-REG OUTCOME. The aaacucucutetete ccchahahangngngeseses in

natriuresis leading to plasma volume contraction could induce relative euvolemia, thereby

educinggg cardiac preload and heart failure hospitalization risk, which decreased rapidly ove

eveveverraral monthshshs in n n EMMMPAPAPA-R-R-REGEGEG OOOUTUTUTCOCC MEEE. MoMoreovovovererer,, , plplplasssmamama vvvolllumuu e e cococontntntrararactiooon-n-n-mememedididiataa ed

decrcrcreeeases in mmmyoyyocardrdrdial sststretch maayyy havvev rreduduucececeddd cacaarrddiaccc arrhrhhyytthmhmhmogenesesesiis – anoootththe

memechchananisismm plplauausisiblblyy rerespspononsisiblblee foforr ththee rereduducecedd momortrtalalitityy obobseservrveded iinn ththee emempapaglglififlolozizin-n-trtreaeatetedd



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diuretics118, it is not known whether these agents reduce mortality because randomized

controlled trials with current standards have not been performed.

BP-lowering effects of empagliflozin have also been identified as a possible contributor

to the beneficial effects observed in EMPA-REG OUTCOME. BP-lowering may have reduced

cardiac afterload, improving coronary blood flow and contractility, thereby contributing to

improved cardiac performance and reduced heart failure risk. Afterload reducing effects may

have been augmented by improved arterial compliance with empagliflozin, an effect that also

increases myocardial blood flow63, 64. While this mechanism is plausible and may have acted in

concert with preload effects, it seems unlikely that a 4 mmHg reduction in systolic BP can fully

account for such substantial clinical benefits, given the relatively modest impact of lowering

systolic BP on heart failure or morality, even in high risk patients119. Furthermore, if reductions

in BP did account for improved outcomes, one might also have expected similar improvements

in “plaque-rupture” atherothrombotic events such as non-fatal myocardial infarction and stroke –

which were not seen in EMPA-REG OUTCOME.

Aside from effects on plasma volume and BP, weight loss has been identified as a

possible factor responsible for the protective effect of empagliflozin. The influence of weight

reduction strategies on clinical outcomes has been examined in the LOOK-AHEAD study

involving 5145 overweight or obese patients with T2D. In this trial, even though lifestyle

interventions led to an 8.7% decrease in body weight – much more than the 2-3% anticipated

with an SGLT2 inhibitor – there were no mortality or heart failure benefits120. In addition to

weight loss, other metabolic factors that may have contributed to mortality and heart failure

benefits include improvements in glycemic control. However, it seems unlikely that the modest

anti-hyperglycemic effect of empagliflozin alone accounted for the substantial reductions in

account for such substantial clinical benefits, given the relatively modest impapapactctct ooof f f lololoweweweririr ng

ystolic BP on heart failure or morality, even in high risk patients119. Furthermore, if reduction

n BP did account for improved outcomes, one might also have expected similar improvement

n “““plplplaque-ruppptututureee” atttheheherororoththt rororombmbmbotototicii eeevevv nts sususuchh aas nononon-n-n-fafafatatatal mymymyocococararardidid al iiinfnfnfarararctctctioioion ananand d d stststrororokekk –

whicicich hh were nooot sseeeenn inn EMPMPPA-AA REG OUOUUTCOMMME..

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mortality, heart failure and renal risks observed in the trial, since previous glycemic control

studies have overall failed to show this kind of benefit with much more robust lowering of

HbA1c121.

Whether the emerging renal protective effects of empagliflozin were on the basis of these

hemodynamic effects or rather due to reduced tissue inflammation/fibrosis is not known.

Whatever underlies the large clinical renal benefit in EMPA-REG OUTCOME, it is important to

recognize that the mortality, heart failure and renal risk reductions may not have been “cardio-

renal” (preservation of cardiac function leading to renal protection), but rather “reno-cardiac”. In

other words, preservation of renal function (Figure 4), including maintenance of total body salt

and water homeostasis, and avoidance of sympathetic nervous system activation and

inflammation associated with diabetic nephropathy, may have been responsible for reducing the

risk of heart failure (Figure 7). Ultimately, the impact of SGLT2 inhibition on primary renal

endpoints will be confirmed in the ongoing “CREDENCE” study (NCT02065791).

As described above, SGLT2 inhibitors have been associated with suppression of pro-

inflammatory and pro-fibrotic pathways in several animal models. Most of these studies have

either been in vitro, or have looked at effects in kidney tissue. Less is known about these effects

in the heart, and nothing to date has been reported in humans. In the heart, epicardial adipose

tissue has been linked with cardiac fibrosis, reduced contractility, arrhythmias and heart

failure122. If SGLT2 inhibitors reduce epicardial fat in addition to decreasing adipose tissue in

other areas of the body, this could provide a mechanistic link between empagliflozin and

cardiovascular effects observed in EMPA-REG OUTCOME (Figure 2). This hypothesis is being

tested in an ongoing clinical study (NCT02235298).

and water homeostasis, and avoidance of sympathetic nervous system aaactctctivivivatatatioioion n n aaand

nflammation associated with diabetic nephropathy, may have been responsible for reducing the

isk of heart failure (Figure 7). Ultimately, the impact of SGLT2 inhibition on primary rena

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Other mechanisms conceivably contributing to the reductions in cardiovascular death and

heart failure hospitalization observed in EMPA-REG OUTCOME may be worth noting. First, a

unique property of SGLT2 inhibitors is their ability to cause ongoing calorie loss through

glycosuria. While this effect is maintained over the long term, the body weights of subjects

treated with empagliflozin did not continue to fall, suggesting that the latter is either more

attributable to salt and water loss that stabilizes over time, or counteracted by increased caloric

intake or and/fundamental changes to the metabolic rates of individual taking these agents, which

although recently modelled has not been carefully studied123. The inherently catabolic state of

patients treated with an SGLT2 inhibitor is manifest by heightened glucagon levels124, and a

potential increased risk of (diabetic) ketoacidosis discussed above. Although caloric ‘loss’ has

not been as well studied as calorie restriction, there is a robust body of data on the

cardioprotective actions of the latter125. Indeed, in an animal model, as few as 7 days of calorie

restriction activates a robust cardiac gene program and reduces injury from experimental

myocardial infarction125.

Second, the ‘glucagonergic’ effects of SGLT2 inhibitors deserve further exploration. As

mentioned above, SGLT2 inhibitors increase glucagon secretion in humans with resultant

enhanced hepatic glucose production. Rodent and human pancreatic alpha cells express a

functional SGLT2 protein, which is downregulated in rodents with experimental hyperglycemia.

Genetic and pharmacological reduction of SGLT2 expression and activity, respectively enhance

glucagon gene expression in human islets by 4-fold and increased plasma glucagon levels in

rodents by 3-fold126. Although many pre-clinical and clinical studies have demonstrated

pleiotropic beneficial cardiovascular effects of glucagon-like peptide-1 (GLP-1) and drugs

targeting the GLP-1 pathway127, fewer data exist on this front for glucagon. Enhanced glucagon

potential increased risk of (diabetic) ketoacidosis discussed above. Although cacaalololoririric c c ‘l‘l‘losososs’s’s’ hhha

not been as well studied as calorie restriction, there is a robust body of data on the

cardioprotective actions of the latter125. Indeed, in an animal model, as few as 7 days of calorie

estttririricction actititivvvatetetes a a a rororobubub ststst cccararardidd accc gene prpp ogogrammm aaandndnd redededucucucesee iiinjnn urrry y y frfrfromomom eexpxpxperererimimimenee ta

myyocococardial infffararrctttioon125122 .

SeSecocondnd, ththee ‘gglulucacagogonenergrgicic’ efefffecectsts ooff SGSGLTLT22 ininhihibibitotorsrs ddeseseeffff rvrvee fufurtrtheherr exexplplororatatioionn. AA



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secretion has been demonstrated in several inflammatory conditions including severe sepsis, and

aside from its effects on hepatic gluconeogenesis, other important actions of glucagon (of direct

relevance to the physiology of SGLT2 inhibitors) include weight loss, increased energy

expenditure, and decreased adipose tissue expansion128. Pharmacological effects of glucagon in

humans include increased heart rate, blood pressure and cardiac output129, which have not been

observed in subjects treated with SGLT2 inhibitors. Although a study in mice suggested that

cardiac glucagon receptor activation is harmful in a model of myocardial infarction130,

historically, intravenous administration of glucagon has been studied as a treatment for patients

with heart failure, cardiogenic shock and bradycardia131, with animal studies also showing its

capacity to vasodilate splanchnic vessels, increase renal blood flow, and enhance glomerular

filtration and electrolyte excretion132. At the very least, these data suggest that the renal, cardiac,

and vascular effects of SGLT2 inhibitors described above may be influenced in part by the

cardiovascular effects of increased glucagon levels in subjects treated with SGLT2 inhibitors.

Finally, the potential role of increased lipolysis and enhanced bioavailability of free fatty

acids and ketone bodies in subjects treated with SGLT2 inhibitors may also be relevant33.

Patients with heart failure exhibit increased levels of circulating free fatty acids and ketone

bodies compared to control subjects in proportion to the severity of cardiac dysfunction and

neurohormonal activation133. Indeed, findings such as these have been thought to represent

metabolic evidence of the syndrome of ‘cardiac cachexia’ in patients with severe heart failure134.

In this context, it is hard to imagine that the increased catabolism and potential predisposition

towards ketoacidosis in subjects treated with SGLT2 inhibitors can be seen as a mechanism

mediating reduced cardiovascular mortality and heart failure hospitalizations observed in EMPA-

REG OUTCOME.

capacity to vasodilate splanchnic vessels, increase renal blood flow, and enhananancecece ggglololomememerururula

filtration and electrolyte excretion132. At the very least, these data suggest that the renal, cardiac

and vascular effects of SGLT2 inhibitors described above may be influenced in part by the

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Rather than simplistically attributing the clinical impact of empagliflozin to one pathway

vs. another, perhaps the results observed in EMPA-REG OUTCOME were instead due to an

overall beneficial profile – reduced BP and plasma volume, weight loss, and modest anti-

hyperglycemic and uric acid lowering effects. Moreover, due to the mechanism of action of

SGLT2 inhibitors, there was no increase in major hypoglycemic events to attenuate these

benefits, nor was there evidence of sympathetic activation-related reflex tachycardia. In terms of

activation of RAAS neurohormones, SGLT2 inhibition induces a modest rise in plasma RAAS

hormones such as aldosterone and angiotensin II, but within the suppressed range that is typical

of diabetes (“the RAAS paradox”)135, and similarly increases levels of urinary RAAS markers

(angiotensinogen, ACE, ACE2), perhaps as a consequence of plasma volume contraction38, 51.

From a mechanistic perspective, the RAAS activation in patients taking ACE inhibitors or

angiotensin II blockers may shunt the RAAS activation cascade toward vasodilatory/natriuretic

ACE2-Ang(1-7) pathways, as suggested elsewhere65, 66 (Figure 2). The natriuresis and

hemodynamic benefits with empagliflozin do not appear to have been counterbalanced by an

increase in the risk of volume depletion, acute kidney injury, or hyperkalemia that have been

seen in renal/cardiovascular protection studies such that used a dual RAAS blockade strategy136,

137. It is therefore possible that benefits in EMPA-REG OUTCOME were analogous to the

benefits seen in previous multi-faceted, clinical intervention studies such as STENO-2 that

simultaneously targeted global cardiovascular risk factor management in patients with type 2

diabetes138, 139.

angiotensinogen, ACE, ACE2), perhaps as a consequence of plasma volume cccononontrtrtracacactititiononon38,38,38, 51

From a mechanistic perspective, the RAAS activation in patients taking ACE inhibitors o

angiotensin II blockers may shunt the RAAS activation cascade toward vasodilatory/natriuretic

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nncrcreaeasese iinn ththee ririsksk ooff vovolulumeme ddepepleletitionon, acacututee kikidndneyey iinjnjururyy, oorr hyhypeperkrkalalememiaia tthahatt hahaveve bbeeeenn



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Potential Clinical Applications:

EMPA-REG OUTCOME is the first trial evaluating an anti-hyperglycemic therapy to

demonstrate benefits on important clinical outcomes that are commonly targeted by other

specialists, including cardiologists (MACE outcomes, heart failure hospitalization, hypertension)

and nephrologists (eGFR decline, proteinuria, hypertension, dialysis). It is therefore important to

consider how the results of EMPA-REG OUTCOME will impact clinical decision-making, as

well as greater familiarity with and use of SGLT2 inhibitors by cardiologists and nephrologists.

Based on EMPA-REG OUTCOME, clinical practice guidelines have already started to reflect

the shift toward the use of SGLT2 inhibitors in patients with established cardiovascular disease

who have not yet reached glycemic targets140. In addition, the European Society of Cardiology

clinical practice guidelines have included empagliflozin as a therapeutic option in patients with

diabetes to reduce hospitalization for heart failure141.

Approximately 40-50% of patients with coronary heart disease have T2D142. These

patients are more likely to be seen by cardiologists than either endocrinologist or general

practitioners. Consequently, cardiologists are likely to take an interest in the choice of anti-

hyperglycemic agents to 1) recommend agents with proven cardiovascular benefit such as

empagliflozin or liraglutide, 2) prefer an agent with proven cardiovascular safety without adverse

CV effects (e.g. metformin, sitagliptin, and lixisenatide) and 3) avoid agents with either potential

cardiovascular harm or no proven benefit (e.g. sulfonylureas, thiazolidinediones, saxagliptin).

Cardiologists are becoming more comfortable prescribing glucose lowering agents, especially

with the low incidence of adverse events with current agents. Now that agents that reduce

cardiovascular events are available, the cardiologist has a responsibility to at least consider the

who have not yet reached glycemic targets140. In addition, the European Societytyty ooof ff CaCaCardrdrdioioiololology

included empagclinical practice guidelines have liflozin as a therapeutic option in patients with

diabetes to reduce hospitalization for heart failure141.

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patiiienenents are mmmoooreee likekekely tttooo be seen bbby carrrdddiolloogisisistststs ttthahahann eiththther eeendnddooccrc inologggiiist t oor gennneere a

prpracactitititiononererss. CCononseseququenentltlyy, ccarardidioolologigiststss araree lilikekelyly ttoo tatakeke aann inintetererestst iinn ththee chchoioicece ooff anantiti



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use of the agents with cardiovascular risk reduction, in preference to agents with either no proven

benefit or potential harm.

Given the primacy of the cardiovascular benefits with empagliflozin, the use of agents in

this class with proven cardiovascular benefit in patients with T2D closely parallels the use of

statins in this population, although the impact of statins is mediated via a reduction in

atherosclerotic events rather than heart failure and renal disease risk with empagliflozin. A

single, 10 mg dose of empagliflozin is as efficacious as the higher 25 mg dose, and does not

require titration or monitoring in most patients, except for perhaps elderly patients with CKD

taking loop diuretics and RAAS inhibitors. Currently available data suggest that the mortality

benefit observed in EMPA-REG OUTCOME is most likely to be due to natriuresis-related

reductions in heart failure and renal disease. Although other mechanisms such as reduced

arrhythmogenesis are plausible, they will require further study. Regardless of the responsible

mechanism, the results of EMPA-REG OUTCOME are likely to have substantial impact on the

changing landscape of emerging anti-hyperglycemic, renal and cardiovascular protective

therapies.

benefit observed in EMPA-REG OUTCOME is most likely to be due to natatatriririurururesesesisisis-r-rrelelelaaatedt

eductions in heart failure and renal disease. Although other mechanisms such as reduced

arrhythmogggenesis are plausible, they will require further study. Regardless of the responsible

mechchchanism, thehehe resesesultststs ooofff EMEMEMPAPAPA-R-R-REGGG OUTCOCOC MEM aaarerere lllikikikelele y yy tototo hahahaveee subbbstststananantititialala impmpmpacacact tt ononon the

chananangigg ng landsdsdscacaappe ooof ememmerginggg antntti-hypeeperrrglyyceceemimimiccc, rrenenenal aaand ccarrardddiovascuuulllarrr protecccttit ve

hhererapapieiess.



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CONTRIBUTIONS

HLH, BAP, MH, DHF and DZIC wrote, edited and approved the final version of the manuscript.

ACKNOWLEDGEMENTS

MH and DZIC are each supported by funding from the Canadian Institutes of Health Research.

M.H. is a Career Investigator of the Heart and Stroke Foundation (Ontario). Finally, the authors

are grateful to the study participants whose time and effort are critical to the success of our

research program. DZIC is the guarantor of this work and, as such, had full access to all the data

in the study and takes responsibility for the integrity of the data and the accuracy of the data

analysis.

SOURCES OF FUNDING

Support for this work was in part provided by funding from the Canadian Institutes of Health

Research. The authors thank Boehinger Ingelheim for providing funding for the atrial natriuretic

peptide analyses described in this manuscript.

DISCLOSURES

DZIC has received speaker/consultant fees from Boehringer-Ingelheim, Eli Lilly, AstraZeneca,

Merck and Janssen and has received operational funding from Boehringer Ingelheim, Merck and

AstraZeneca. BAP has received speaker honoraria from Medtronic Inc., Johnson and Johnson,

Roche, Glaxo Smith Kline Canada, Novo Nordisk and Sanofi; has received research grant

support from Medtronic and Boehringer Ingelheim; and serves as a consultant for Neurometrix.

DHF has received speaker/consultant fees from Boehringer-Ingelheim, Eli Lilli, Astra-Zeneca,

Merck, and Sanofi. He is a member of the EMPA-REG OUTCOME trial steering committee and

analysis.

SOURCES OF FUNDING

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participates in Data and Safety Monitoring Boards for Novo Nordisk. HJLH is a consultant to

Abbvie, Astellas, AstraZeneca, Boehringer Ingelheim, Janssen, Merck and ZS-Pharma

(honoraria were paid to his employer). MH has been a consultant to AstraZeneca, Boehringer

Ingelheim, Janssen and has received speaker/consultant fees from Merck and Novo Nordisk.

MH has also received investigator-initiated research funding from Astra-Zeneca, Merck and

Novo-Nordisk.

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139. GGGaeaeaededede PPP. IInttensive glucose control and cardididiovascular diseasasase innn ttype 2 diabetes--should wechangee ttthehehe recccomomommememendddededed tttaraa get tt for glllycyy atted hhemememogogoglololobiiin?n?n? CCComomommemm nttarararyyy tototo ACCCCCCORORORD D D anddADVANCEEE ttriaaalsss.. PPPol l l ArArArch MMMeeed WWWewn. 2020008;111888:666119-666212121.

14000. Harper W,WW CClememm nt MMM, Goldenbeerrrg R, HaHaannnaa A,A,A, MMMaiaiainn n AA,A RRReettnakkkarrannn R, Sherrriffafaliii D, Woooooo VYale JJJF.F.F. Phahharmmmaccologggiccc managagagemememeeent off tttyyype 22 dddiaaabetess.. CCaC nn JJ J Diabaabettteseses.. 201666;3;3;377 7 SuSuSuppl 1:::SS6S 1686868.

141. Ponikikow kskii P, Voors AA, A knker SD, Bueno H, lCl lelandd JG, Coats AJ, Falklk V, Gonz lalez-Juanatey



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FIGURE LEGENDS

Figure 1: The sodium glucose cotransporter-2 (SGLT2) mechanism in the proximal tubule Modified from Bakris et al4 with permission of the publisher. ©2009. Elsevier.

Figure 2: Physiological mechanisms implicated in the cardiovascular and renal protection with SGLT2 inhibition

Figure 3: The relationship between urinary glucose excretion and eGFR. With worsening renal function impairment (eGFR), the change in urinary glucose excretion over 24 hours ( UGE0-24h) diminishes.http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/EndocrinologicandMetabolicDrugsAdvisoryCommittee/UCM334550.pdf70

Figure 4: Effects of SGLT2 inhibitors on GFR. The effects of canagliflozin (100 mg daily, square symbols; 300 mg daily, circle symbols) vs. glimepiride (triangle symbols) in patients with preserved renal function (PANEL A)17 (Reproduced with permission of the publisher. ©2013, Elsevier), dapagliflozin (DAPA; 5 mg daily, square symbols; 10 mg daily, triangle symbols) vs. placebo (PBO) in patients with CKD (PANEL B)15 (Reproduced with permission of the publisher. ©2014. Elsevier), and empagliflozin vs. placebo in patients with enrolled in EMPA-REG OUTCOME (PANEL C)73 (Reproduced with permission of the publisher. ©2016, Massachusetts Medical Society).

Figure 5: Putative mechanism for sodium-mediated changes in adenosine bioactivity at the afferent arteriole.

LEGEND: During normal conditions (A), sodium-glucose cotransport leads to minimal glycosuria. If, under these non-diabetic conditions, NaCl delivery to the macula densa was reduced in the context of a physiological stress such as hypotension, renal perfusion would decrease, leading to a reduction in NaCl transit across macula densa cells, thereby causing less adenosine triphosphate (ATP) release and breakdown to adenosine, which is a vasoconstrictor. Consequently, less vasoconstrictive adenosine would act via the adenosine type-1 receptor on vascular smooth muscle cells (VSMCs) to cause less afferent arteriolar vasoconstriction. The resulting afferent vasodilation would serve to preserve renal blood flow and GFR, thereby avoid acute kidney injury – the inverse relationship between changes in the NaCl transit across macula densa cells and GFR is a process called tubuloglomerular feedback. Adenosine is generated by both intracellular and extracellular sources, and extracellular generation involves ecto-5’-nucleotidase (5’-NT). Under conditions of ambient hyperglycemia (B), sodium glucose cotransport-2 activity (SGLT2) is increased, thereby reducing macula densa NaCl delivery. This affects the same tubuloglomerular feedback mechanisms, leading to afferent vasodilatation. Since renal blood flow and GFR start off within a ‘normal’ range, afferent vasodilatation under these circumstances resulting in renal hyperperfusion and glomerular hyperfiltration. The goal of using an SGLT2 under these conditions (C) would therefore be to restore distal tubular flow and NaCl delivery to the macula densa, thereby increasing local adenosine generation and afferent vasoconstriction to attenuate the hyperfiltration state81.

placebo (PBO) in patients with CKD (PANEL B) (Reproduced with permmisisission of thepublisher. ©2014. Elsevier), and empagliflozin vs. placebo in patients with enrrrolololleleled d d ininin EEEMPMPMPAREG OUTCOME (PANEL C)73 (Reproduced with permission of the publblblisisisheheher.r.r ©©©202020161616Massachusetts Medical Society).

Figure 55: PPutative mechanism for sodium-mediated changes in adenosine bioactivity at theafferererentntnt a ttrterererioioiolell .

LEEEGGGEND: Durinnngg nononormalalal condiddittitiononns (A)), sooddiumumum-gggluuucococose cccotraaannspopoporrt leaadsdsds tooo minnnimmmaglycyccoososuria. Ifff,,, ununundder ttht eseee non n-diiiababbeetiiic connnddditionons,s,s, NNN CCaCl dded liiivvev ry to thhhe macccuululaa dddensa wwaweducececeddd ininin ttthehehe conoontextxtxt of a aa phphphysysysioioiolooogigigicacacalll stststrrresss sucucuchhh asasas hhhypyy oootenenensionon, rererenananalll pepeperfuususion wowowoulu d

dedecrcreaeasese, leleadadiningg toto aa rrededucuctitionon inin NNaCaCll trtranansisitt acacrorossss mmacacululaa dedensnsaa cecellllss, tthehererebyby ccauausisingng lleses



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43

Figure 6: ANP levels in subjects with type 1 diabetes before and after treatment with an SGLT2 inhibitor. Baseline levels of atrial natriuretic peptide levels (ANP, pg/ml) in 40 patients with type 1 diabetes and ether normofiltration (T1D-N: GFR<135 ml/min/1.73m2) or hyperfiltration (T1D-H: GFR 135 ml/min/1.73m2) at baseline (A); effects of empagliflozin 25 mg daily treatment for 8 weeks on ANP levels (B); effects of empagliflozin treatment 25 mg daily for 8 weeks on ANP levels in normofilterers (C) and in hyperfilterers (D) LEGEND: Pre-SGLT2i-Eu Pre Dose (baseline) SGLT2i Euglycemia; Pre-SGLT2i-Hyper Pre Dose (baseline) SGLT2i Hyperglycemia; Post-SGLT2i-Eu Post Dose SGLT2i Euglycemia; Post-SGLT2i-Hyper Post Dose SGLT2i Hyperglycemia; T1D-N – T1D with Normofiltration; T1D-H – T1D with Hyperfiltration. In this study we quantified Atrial Natriuretic Peptide (ANP) by using Sigma-Aldrich's Atrial Natriuretic Peptide EIA kit (St. Louis, MO, USA). The EIA assay was performed by Eve Technologies Corp (Calgary, AB, Canada) according to Sigma-Aldrich protocol. The assay sensitivity for ANP begins at 1.02 pg/mL. Figure 7: The “renal-cardio” hypothesis for cardiovascular protection with SGLT2 inhibition: a nephrocentric perspective. nephrocentric perspective.



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0

20

40

60

80

Day

1, Δ

UGE

0-24

h (g)

GFR (mL/min/1.73m2)

eGFR Normal Renal Function (n=3) ≥90 mL/min/1.73m2

eGFR Mild Renal Impairment (n=10) 60 to 89 mL/min/1.73m2

eGFR Moderate Renal Impairment (n=9) 30 to 59 mL/min/1.73m2

eGFR Severe Renal Impairment (n=10) 15 to 29 mL/min/1.73m2

y = -12.2 + 0.697 (r2adj: 0.783)

95% Confidence Band

20 40 60 80 100

20

40

Day

1, ΔΔ

UG

UE 0

-24h

(g)

eGFR Normal Renal Function (n=3) ≥90 mL/min/1.73m2

eGFR Mild ReR nal Impairment (n=10) 600 tto 8989 mL/L/mmin/1.1.7373mm2

eGFR Modeeraate Reenal Impairmmennt (n=9) 30 to 59 mLL/mminn/11.73m2

eGFR Severe Renal Impairment (n=10)15 to 29 mL/min/1 73m2



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Hiddo J.L. Heerspink, Bruce A. Perkins, David H. Fitchett, Mansoor Husain and David Z.I. CherneyKidney Effects, Potential Mechanisms and Clinical Applications

Sodium Glucose Cotransporter 2 Inhibitors in the Treatment of Diabetes: Cardiovascular and

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2016 American Heart Association, Inc. All rights reserved.

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