Systemic and Organ-Specific Anti-Inflammatory Effects of Sodium-Glucose Cotransporter-2 Inhibitors

Abstract

Inflammation plays an essential role and is a common feature in the pathogenesis of many chronic diseases. The exact mechanisms through which sodium-glucose cotransporter-2 (SGTL2) inhibitors achieve their much-acclaimed clinical benefits largely remain unknown. In this review, we detail the systemic and tissue- or organ-specific anti-inflammatory effects of SGLT2 inhibitors using evidence from animal and human studies. We discuss the potential pathways through which SGLT2 inhibitors exert their anti-inflammatory effects, including oxidative stress, mitochondrial and inflammasome pathways. Finally, we highlight the need for further investigation of the extent of the contribution of the anti-inflammatory effects of SGLT2 inhibition to improvements in cardiometabolic and renal outcomes in clinical studies.

SGLT2 Inhibitors: From Discovery to Clinical Use

Sodium-glucose cotransporter-2 (SGLT2, see Glossary) is a protein predominantly expressed in the kidneys, and is responsible for the reabsorption of 90–97% of filtered glucose in the proximal tubule. The glucosuric effects of SGLT2 inhibition were first discovered with the use of phlorizin and expanded to include multiple pharmacologic inhibitors now available for use in patients. Initially designed to lower blood glucose in individuals with diabetes, FDA-mandated large-scale cardiovascular outcomes safety trials revealed a surprising benefit in reducing major adverse cardiovascular events [1–3]. SGTL2 inhibitors have now also emerged as robust renal-protective drugs [4]. Nearly two decades after their introduction into clinical practice, the precise mechanisms of the beneficial effects of SGLT2 inhibitors above and beyond the glucosuric effect is still being actively investigated. Multiple preclinical and human studies have suggested that reductions in inflammation may play a role. In this review, we will evaluate existing data on the anti-inflammatory effects of SGLT2 inhibition, including pre-clinical and clinical data, and discuss potential mechanisms. Given the close interplay between oxidative stress, pro-fibrotic factors, and inflammatory pathways (i.e. inflammation can increase oxidative stress and reactive oxygen species (ROS) can modulate inflammatory signaling [5], and similarly pro-fibrotic factors can be mediated by and can mediate inflammatory signaling [6]), we broadly define “inflammation” in this review to encompass all these pathways, but list specific findings individually for clarity.

Systemic Anti-Inflammatory Effects of SGLT2 Inhibition

In rodent models, SGLT2 inhibition decreases pro-inflammatory mediators including interleukin (IL)-6, C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor (TNF)a, as reviewed elsewhere [7]. Several large-scale human studies have measured systemic markers of inflammation after treatment with SGLT2 inhibitors; overall, the data suggests a decrease in circulating inflammatory mediators, including measures such as CRP, IL-6, TNFα, plasminogen activator inhibitor-1 (PAI-1) and ferritin, although to varying degrees across studies [8]. In support of systemic anti-inflammatory effects of this class of medications, an observational study of individuals with type 2 diabetes demonstrated that use of an SGLT2 inhibitor decreased adverse outcomes of COVID-19 [9], which is known to be associated with a hyperinflammatory state. There are limitations to interpreting changes in systemic measures of inflammation, as these measures are not sensitive enough to pick up all changes in tissue-level inflammation. In addition, the low-grade inflammation in chronic inflammatory states, such as in obesity and diabetes, is harder to quantify than the inflammation associated with overt autoimmune diseases. The remainder of this review will focus on tissue-level inflammatory changes associated with SGLT2 inhibition (Figure 1).

Figure 1. Anti-Inflammatory Effects of SGLT2 Inhibitors.

Global and systemic anti-inflammatory effects of SGLT2 inhibition are illustrated in the top box and include decreases in pro-inflammatory cytokines, reactive oxygen species, and inflammasome activation. Anti-inflammatory effects of SGLT2 inhibition in tissues are illustrated in the bottom box, including effects on the heart, vasculature, kidney, adipose, and liver, respectively from left to right. ROS: reactive oxygen species; RAAS: renin-angiotensin-aldosterone system. Figure created using BioRender.

Cardiovascular System

SGLT2 Inhibitors Reduce Hospitalization for Heart Failure and Major Adverse Cardiovascular Events.

Multiple large-scale clinical trials have consistently demonstrated the ability of SGLT2 inhibitors to decrease the incidence of heart failure-related hospitalizations and cardiovascular death and, in many but not all of the studies, major adverse cardiovascular events [10]. An observational study in individuals with type 2 diabetes also demonstrated decreased in-stent restenosis with SGLT2 inhibitor therapy after acute myocardial infarction [11]. The proposed mechanisms underlying the cardiovascular benefits of SGTL2 inhibitors are complex and multifactorial, and those relating to anti-inflammatory benefits will be discussed below (Table 1).

Table 1.

Studies Evaluating Inflammation in the Cardiovascular System

Pre-Clinical Studies
Reference	Model	Treatment	Primary Finding	Inflammatory Measures	Additional Findings
Zhang X et al. [12]	Sprague Dawley rats with HFpEF induced by a high-fat diet and nitro-L-arginine methyl ester (L-NAME)	Dapagliflozin for 6 weeks	↓Cardiac fibrosis ↑Cardiac function ↑Cardiac energetics	↓TGF-β1	↓ERK, NOX1/2 & P65
Zhang XL et al. [13]	Male C57BL/6 mice with HFpEF induced by unilateral nephrectomy, continuous infusion of d-aldosterone for 4 weeks, and maintenance on 1.0% sodium chloride drinking water	Empagliflozin in vitro for 24 hours	↓Cardiac fibrosis ↑Cardiac function ↓Lung congestion	↓Intracardiac neutrophil recruitment	↓HMGBl expression and neutrophil extracellular traps (NETs) formation
Byrne NJ et al. [14]	Dahl salt-sensitive rats with HFpEF induced by high salt diet	Empagliflozin for 2 weeks	↓Cardiac fibrosis ↑Cardiac function	↓NLRP3 inflammasome activation ↓Il6, Tnfa and Illb expression ↓IL-1β and TXNIP ↓Caspase-1 activity	Calcium ionophore blunted empagliflozin’s effect
Li C et al. [15]	Diabetic KK-Ay mice on a high-fat diet	Empagliflozin for 8 weeks	↓Cardiac fibrosis ↑Cardiac function	↓TGF-β ↓Oxidative stress	↑ Nrf2/ARE ↓ TGF-β/Smad signaling
Daud E et al. [16]	Sprague–Dawley rats with left anterior descending artery ligation-induced myocardial infarction	Empagliflozin for 4 weeks	↓Cardiac fibrosis ↑Cardiac function	↓TGF-β1	↓TGF-β1/Smad3 signaling
Ye Y et al. [17]	BTBR ob/ob or wild-type mice	Dapagliflozin, vehicle, or dapagliflozin + saxagliptin for 8 weeks	↓Cardiac fibrosis ↑Cardiac function	↓lllb, Il6, Tnfa expression ↓Expression and protein levels of NLRP3 inflammasome components	↑AMPK phosphorylation
Sabe SA et al. [18]	Yorkshire swine with chronic myocardial ischemia induced by a constrictor on the left circumflex artery	Canagliflozin for 5 weeks	↑Myocardial perfusion ↓Cardiac Fibrosis ↑Cardiac function	↓MCP-1 ↑Oxidative stress	↑AMPK phosphorylation ↓Jak/STAT ↓eNOS phosphorylation ↓ERK
Lee TM et al. [19]	Male Wistar rats with anterior descending artery ligation induced myocardial infarction	Dapagliflozin versus phlorizin for 4 weeks	↓Myofibroblasts ↓Cardiac Fibrosis	↑Myocardial IL-10 ↓Pro-inflammatory M1/homeostatic M2 macrophage ratio ↓ROS	↑STAT3 activity and translocation
Han JH et al. [21]	ApoE−/− mice on a Western diet for 20 weeks	Empagliflozin versus glimepiride for 8 weeks	↓Blood glucose ↓Insulin resistance ↓Atherosclerosis ↓Weight and fat mass	↓CRP ↓IL-6 ↓TNFα ↓MCP-1	↓Serum amyloid A ↓Urinary microalbumin ↑Adiponectin
Pennig J et al. [22]	Streptozotocin induced diabetic C57BL/6J mice	Empagliflozin for 3 weeks	↓Atherosclerosis ↑Plaque stability	↓Pro-inflammatory M1/ homeostatic M2 macrophage ratio	↓Leukocyte adhesion
Nakatsu et al. [23]	ApoE−/− mice with diabetes induced by nicotinamide/streptozotocin	Luseogliflozin for 7 days or 6 months	↓Atherosclerosis	↓TNFα ↓IL-1β ↓IL-6	↓ICAM-1, PECAM-1, MMP2, and MMP9 ↔ Serum lipid parameters
Mroueh A et al. [27]	Porcine coronary artery endothelial cells incubated with human plasma from patients with COVID- 19	Empagliflozin for 30 minutes or 24 hours in vitro	↓SGLT2 expression ↓Thrombogenici1y ↓Endothelial dysfunction ↓Senescence	↓IL-1β ↓IL-6 ↓TNFα	↓NF-κB activation
Abdollahi E et al. [28]	Human umbilical vein endothelial cells and macrophages under normoglycemic or hyperglycemic conditions for 24 hours treated with LPS	Dapagliflozin for 24 hours in vitro	↓NF-κB activation	↓TLR-4 expression ↓Pro-inflammatory M1/ homeostatic M2 macrophage ratio ↓IL-1β, IL-6, IL-8 and TNFa from macrophage cells ↓IL-6 and IL-8 from human umbilical vein endothelial cells ↑miR-146a	Effects similar in normoglycemic and hyperglycemic conditions
Li X et al. [29]	Human coronary artery endothelial cells with 10% stretch for 24 hours	Dapagliflozin, empagliflozin, or canagliflozin for 2 hours in vitro	↓Endothelial barrier dysfunction	↔ IL-6 and IL-8	Inhibition of NHE1 and NOXs
Juni RP et al. [30]	Human cardiac microvascular endothelial cells stimulated with either TNFα for 6 hours followed by coincubation with rat cardiomyocytes for 2 hours	Empagliflozin for 6 hours in vitro	Improve cardiomyocyte contraction and relaxation ↑NO	↓Cytoplasmic and mitochondrial ROS

Clinical Studies
Reference	Patient Characteristics	Study design	N	Primary Finding	Inflammatory Measures	Other Measures
Requena-Ibáñez JA et al. [20]	Nondiabetic patients with HFrEF (sub-analysis of EMPA-TROPISM study)	Placebo-controlled RCT of empagliflozin versus placebo for 6 months Cardiac magnetic resonance imaging at baseline and after treatment	100	↓Epicardial adipose tissue ↓Subcutaneous adipose tissue ↓Cardiac fibrosis ↓Aortic stiffness ↑Cardiac function	↓Proteins involved in inflammatory pathways including CCL16, PAI-1, E-selectin, TNFRSF10C, IL-1RT2	↓Myocardial matrix volume ↓MMP-9, ICAM-2, PECAM-1
Striepe K et al. [33]	Type 2 diabetes	Placebo-controlled crossover RCT of empagliflozin versus placebo for 6 weeks Vascular and hemodynamic measures at baseline and after treatment	76	↓Blood pressure ↓Central systolic pressure, central pulse pressure ↓Arterial stiffness	ND
Bosch A et al. [34]	Type 2 diabetes	Placebo-controlled crossover RCT of empagliflozin versus placebo for 6 weeks (sub-analysis of above study)	58	↓Arterial stiffness	↓High-sensitivity CRP	Change in CRP significant determinant of change in arterial stiffness even after controlling for HgA1c or fasting blood glucose

SGLT2 Inhibitors Improve Outcomes in Heart Failure with Preserved Ejection Fraction.

Inflammation is a critical element of the pathophysiology of heart failure with preserved ejection fraction (HFpEF), and SGLT2 inhibitors are the only class of medications demonstrating consistent improvements in outcomes in individuals with HFpEF to date. Consequently, it is critical to understand whether the beneficial effects of SGLT2 inhibition in HFpEF are related to an anti-inflammatory effect on the myocardium. Indeed, current evidence from preclinical models suggests that this could be the case. In a Sprague Dawley rat model of HFpEF, the SGLT2 inhibitor dapagliflozin inhibited the infiltration of macrophages, reduced superoxide production, and decreased the pro-fibrotic cytokine transforming growth factor-β (TGF-β) in the myocardium [12]. In a mouse model of HFpEF, the SGLT2 inhibitor empagliflozin decreased neutrophil infiltration, formation of neutrophil extracellular traps and development of cardiac fibrosis, resulting in improvements in diastolic function [13]. Finally, in rats with HFpEF, the SGLT2 inhibitor empagliflozin decreased activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, a cellular sensor of infection or cellular damage that results in the production of IL-1β upon assembly/activation, and decreased cardiac dysfunction and IL-1β levels [14].

SGLT2 Inhibitors Decrease Myocardial Fibrosis.

Myocardial fibrosis, whether resulting from ischemic or non-ischemic injury, can be regulated and driven by tissue-resident immune cells including macrophages and monocytes. Several pre-clinical studies have evaluated the effect of SGLT2 inhibition on myocardial fibrosis. In diabetic or ischemic models, the SGLT2 inhibitor empagliflozin decreased excessive deposition of collagen, reduced myocardial oxidative stress injury and cardiac fibrosis [15], decreased pro-fibrotic cytokines [16], and reduced inflammasome activation [17]. Further, in a swine model of chronic myocardial ischemia, the SGLT2 inhibitor canagliflozin reduced JAK/STAT signaling, increased expression and activation of adenosine monophosphate-activated protein kinase (AMPK), and reduced oxidative stress and myocardial fibrosis [18]. Importantly, both JAK/STAT signaling pathways and oxidative stress are involved in pro-inflammatory processes. In addition, in a rat model of myocardial infarction, the SGLT2 inhibitor dapagliflozin decreased myocardial reactive oxygen and nitrogen species production, increased anti-inflammatory M2 macrophages and IL-10, decreased pro-inflammatory M1 macrophages, and decreased myofibroblast infiltration and cardiac fibrosis [19]. Finally, in a randomized placebo-controlled study of individuals with heart failure (EMPA-TROPISM study), empagliflozin decreased myocardial fibrosis as measured by cardiac MRI, and reduced inflammatory biomarkers [20]. Thus, SGLT2 inhibitor-mediated reduction in myocardial inflammation and fibrosis may be one mechanism of cardioprotection.

SGLT2 Inhibitors Decrease Atheroma Burden and Promote Plaque Stability.

Inflammation is a central component in the pathogenesis of atherosclerosis, being involved from the initial lesion formation to the progression of plaques and thrombotic events. SGLT2 inhibitors decreased atherosclerotic plaque areas in the aortic arch and valves in obese atherosclerosis-prone mice, which correlated with reduced inflammatory cells in both the plaques and in adipose tissue, and decreased circulating markers of inflammation, namely TNFa, IL-6, and MCP-1 [21]. Similar results were seen in diabetic atherosclerosis-prone mice [22,23]. In a study of individuals with type 2 diabetes and non-obstructive coronary artery disease, SGLT2 inhibitors reduced inflammatory markers such as NLRP3, caspase-1, and IL-1β, and increased coronary fibrous cap thickness, indicating enhanced plaque stability and a reduced risk of plaque rupture [24]. Thus, SGLT2 inhibitor-mediated reduction of inflammation with resulting improvement in atherosclerosis may be another mechanism of cardioprotection, and is further explored in the following section on endothelial function. Nonetheless, reduction of atherosclerotic outcomes by SGLT2 inhibitors has not been definitively demonstrated in humans and is debated [25].

SGLT2 Inhibitors Improve Endothelial Cell Function.

Vascular endothelial cells significantly influence cardiovascular health by regulating vascular tone and integrity, hemostasis and thrombosis, redox balance, and atherosclerosis. Immune and endothelial cells within the vasculature are vitally linked at the molecular level, and pro-inflammatory cytokines and ROS can decrease endothelial production of nitric oxide which is crucial for vascular health. Pre-clinical studies in animal models have demonstrated that SGLT2 inhibition prevents endothelial cell dysfunction and enhances endothelial-dependent vasodilation, and studies in human endothelial cells in vitro have demonstrated that SGLT2 inhibition has direct anti-inflammatory effects (as reviewed elsewhere [26]). In addition, inflammatory mediators caused upregulation of SGLT2 expression within porcine endothelial cells which promoted endothelial injury, which was blocked by the SGLT2 inhibitor empagliflozin in vitro [27]. Dapagliflozin treatment in vitro decreased human umbilical vein endothelial cell production of the pro-inflammatory cytokines IL-6 and IL-8 in response to stimulation by bacterial lipopolysaccharides (LPS) [28]. A similar benefit of SGLT2 inhibition has been demonstrated in stretch-induced endothelial cell dysfunction [29]. Finally, in studies using human cardiac microvascular endothelial cells co-cultured with rat cardiomyocytes, empagliflozin blocked TNFa-mediated ROS generation and restored endothelial cell nitric oxide production and subsequent cardiomyocyte contractility [30], a finding that was later expanded to uremic serum-induced endothelial dysfunction from patients with chronic kidney disease [31]. This study demonstrated that inflammation-induced endothelial cell dysfunction can be reversed by SGLT2 inhibition, leading to improved cardiomyocyte function.

Clinical studies have demonstrated some conflicting effects of SGLT2 inhibition on endothelial function depending on the population studied and duration of treatment, and interpretation has been limited by small sample sizes and lack of controls in some studies. In a meta-analysis of six randomized controlled trials, SGLT2 inhibitors significantly improved flow-mediated dilation [32], a nitric oxide-dependent measure of endothelial function that independently predicts cardiovascular events and all-cause mortality. Further, empagliflozin significantly improved arterial stiffness, a measure of vascular function and pressures, in a randomized controlled trial of individuals with type 2 diabetes [33]. Importantly, the SGLT2 inhibitor-induced improvement in vascular function was mediated by a reduction in the pro-inflammatory factor high-sensitivity CRP, but not by changes in glucose metabolism [34].

Renal System

SGLT2 Inhibitors Slow the Progression of Renal Disease.

Large clinical trials and meta-analyses have confirmed renal protective effects of SGLT2 inhibitors in patients with diabetes as well as other chronic medical conditions irrespective of diabetes status [35]. Investigations into potential causative mechanisms outside of glycemic control are ongoing and include: osmotic diuresis, regulation of the renin-angiotensin-aldosterone system (RAAS), reduction of oxidative stress, nutrient deprivation signaling, restoration of tubulo-glomerular feedback, increased renal uric acid elimination, decreased renal fibrosis, and multiple inflammatory pathways [36]. Below we discuss findings related to SGLT2 inhibitor inflammatory regulation and the renal system (Table 2).

Table 2.

Studies Evaluating Inflammation in the Renal System

Pre-Clinical Studies
Reference	Model	Treatment	Primary Finding	Inflammatory Measures	Additional Findings
Hasan R et al. [37]	Long Evans rats exposed to isoprenaline to induce renal oxidative damage	Canagliflozin for 2 weeks	↑Antioxidant/anti-inflammatory signaling pathways	↓Inflammatory cell infiltration ↑AMPK ↑Akt ↑eNOS ↓iNOS ↓NOX4 ↓Caspase 3	↓Apoptosis ↓Renal fibrosis
Vallon V et al. [38]	Type 1 diabetic Akita mice	Empagliflozin for 15 weeks	↓Renal hyperfiltration	↓CCL2 ↓CD14 ↓IL-6 ↓TIMP2	↓NF-κB ↓Albuminuria ↓Kidney growth makers ↔NOX2, NOX4, CCL5, TGFβ, renal collagen
Terami N et al. [39]	Diabetic db/db mice and cultured tubular epithelial cells (mProx24)	Dapagliflozin for 12 weeks in vivo and incubation with dapagliflozin in vitro	↓Progression of diabetic nephropathy	↓Macrophage infiltration (CD14, CD11c, CD206) ↓Mcp1, Tgfb expression ↓ROS	↓β-cell damage ↓Albuminuria ↓Renal fibrosis
Birnbaum Y et al. [40]	BTBR ob/ob or wild-type mice	Dapagliflozin, vehicle, or dapagliflozin + saxagliptin for 8 weeks	↓BUN	↓NLRP3 inflammasome activation ↓Tnfa, Il1b, Il6 expression	↓AMPK phosphorylation
Zaibi N et al. [41]	Human proximal tubular cells (HK-2)	Dapagliflozin for 24 hours in vitro	↓Oxidative stress	↓ROS	↓Apoptosis Modified calcium dynamics
Das NA et al. [42]	Human proximal tubular cells (HK-2)	Empagliflozin for 24 hours in vitro	↓Epithelial to mesenchymal transition ↓Epithelial cell migration	↓ROS ↓NF-κB activation	↓p38 MAPK ↓miR-21 ↓MMP2
Ishibashi Y et al. [43]	Primary cultured human proximal tubular cells from normal kidney	Tofogliflozin for 24 hours in vitro	↓Oxidative stress ↓Profibrotic factors	↓ROS ↓MCP-1	↓Apoptosis
Yao D et al. [44]	Human proximal tubular cells (HK-2)	Dapagliflozin for 48 hours in vitro	↓HMGB1-RAGE-NF-κB signaling pathway	↓ROS ↓MCP-1 ↓NF-κB activation	↓Fibrosis makers (FN and Col 1) ↓ICAM-1
Satou R et al. [46]	Mouse proximal tubular cells cultured in high glucose conditions	Canagliflozin in vitro	↓Angiotensinogen	↓ROS
Woods TC et al. [47]	New Zealand obese mice on high fat diet to induce diabetes	Canagliflozin for 6 weeks	↓Angiotensinogen ↓Tubular fibrosis	↓ROS ↓Monocyte/macrophage infiltration	Normalization of systolic BP
Ke Q et al. [48]	Mouse C57BL/6 with kidney fibrosis induced by ischemic/reperfusion injury and primary proximal tubular epithelial cells	Dapagliflozin for 1 week in vivo and incubation with dapagliflozin in vitro	↓Renal fibrosis ↑Itaconate (metabolic from TCA cycle)	↓NLRP3 inflammasome activation ↓Caspase 1 ↓IL-1β ↓IL-18	↓mTOR signaling ↓HIF-1α signaling
Pirklbauer M et al. [49]	Human proximal tubular cells (HK-2 and RPTEC/TERT1) treated with TGF-β	Empagliflozin or canagliflozin in vitro	↓Mediators of renal fibrosis	ND	↓Tenascin C (TNC) ↓Thrombospondin 1(THBS1) ↓Platelet derived growth factor subunit B (PDGF-B)

SGLT2 Inhibitors Decrease Oxidative Injury and Inflammation in the Kidneys.

Multiple studies have demonstrated an antioxidant and anti-inflammatory effect of SGLT2 inhibition in the renal system. First, canagliflozin reduced renal oxidative damage and stimulated anti-inflammatory signaling pathways in a rat model of sympathetic hyperactivity [37]. empagliflozin reduced expression of genes associated with inflammation in the kidneys of diabetic Akita mice, including the transcription factor NF-κB, pro-inflammatory mediators IL-6 and MCP-1, and the macrophage marker CD14 [38]. Similar results were seen in diabetic db/db mice, where dapagliflozin was also found to reduce macrophage infiltration into the kidneys [39]. Further, in diabetic ob/ob mice, dapagliflozin decreased activation of the NLRP3 inflammasome and development of diabetic nephropathy [40]. Finally, SGLT2 inhibition of human proximal tubular cells in vitro decreases oxidative stress [41] and hyperglycemia [42–44] induced cell injury, ROS production, inflammatory gene expression and cytokine production. Thus, SGLT2 inhibitor-mediated reduction of oxidative stress and inflammation contributes to the renal benefits of these drugs.

SGLT2 Inhibitors Modulate RAAS.

RAAS overactivation leads to systemic hypertension and subsequent multiorgan damage, inflammation and fibrosis, and agents that reduce RAAS activity mitigate these effects. SGLT2 inhibitors have been shown to modulate RAAS activation in several studies [45]. Notably, canagliflozin reduced hyperglycemia-induced angiotensinogen production and subsequent ROS generation in murine proximal tubular cells in vitro [46], and additionally reduced angiotensinogen production and prevented renal inflammation in diabetic mice in vivo [47]. Thus, part of the renal protective effects of SGLT2 inhibitors may be via modulation of RAAS-induced inflammation.

SGLT2 Inhibitors Reduce Renal Fibrosis.

Renal inflammation plays a central role in the initiation and progression of chronic kidney disease and renal fibrosis. Renal inflammation is characterized by increased pro-fibrotic molecules such as TGF-β and decreased anti-fibrotic cytokines including IL-10. In a mouse model of renal fibrosis, dapagliflozin significantly protected against fibrosis development through modulation of metabolites and inflammation [48]. Further, in diabetic db/db mice, dapagliflozin similarly protected against renal fibrosis and decreased macrophage infiltration and expression of genes involved in promoting inflammation and oxidative stress [39]. SGLT2 inhibition in human proximal tubular epithelial cells in vitro decreased expression of markers of inflammation and oxidative stress in both hyperglycemic [44] and normoglycemic [49] conditions, and additionally decreased mediators of fibrosis. Thus, SGLT2 inhibitors decrease pro-inflammatory and profibrotic factors that reduce renal fibrosis.

Metabolic Organs

SGLT2 Inhibitors Decrease Adipose Tissue Volume and Inflammation in Obesity.

Several adipose depots exist throughout the body, including visceral, subcutaneous, epicardial, and perivascular depots, and in obesity and diabetes ectopic adipose tissue can be deposited in the liver and skeletal muscle. Obesity is associated with a chronic inflammatory state due, in part, to inflammation arising from adipose tissue, and obesity-associated inflammation contributes to insulin resistance and cardiovascular disease. In animal models of diet-induced obesity, treatment with SGLT2 inhibitors decreases adipose tissue inflammation by decreasing pro-inflammatory macrophage populations and increasing homeostatic/anti-inflammatory macrophage populations [50]. Multiple studies have also demonstrated that SGLT2 inhibition decreases visceral, subcutaneous, and ectopic liver adiposity in humans (as recently reviewed elsewhere [51]).

Epicardial adipose tissue (EAT), which surrounds the heart and can influence myocardial function by paracrine signaling, has also been hypothesized to mediate some of the cardiovascular benefits of SGLT2 inhibition [52]. SGLT2 expression has previously been demonstrated in human EAT, and treatment with dapagliflozin in EAT explants decreased secreted chemokines and improved wound healing in endothelial cells [53]. In vitro, empagliflozin decreased IL-6, MCP-1, and ROS in human primary epicardial adipocytes [54]. EAT thickness or volume has additionally been correlated with systemic inflammation, insulin resistance and coronary artery disease. Several studies have demonstrated decreased EAT volume after treatment with SGLT2 inhibitors [55], which correlated with systemic measures of inflammation [20,56–58].

SGLT2 Inhibitors Improve Fatty Liver Disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) can range from simple steatosis to steatohepatitis, an inflammatory condition characterized by elevation of liver enzymes and the development of hepatic fibrosis. Obesity and insulin resistance are major risk factors for development of MASLD, and those with MASLD have increased prevalence of comorbid cardiovascular disease. SGLT2 inhibition in rodent models of fatty liver disease reduces pro-inflammatory cytokines such as IL-6, TNFα, IL-1β, and IL-17 [59,60], reduces the pro-fibrotic cytokine TGFβ [61], enhances macrophage autophagy [62], and results in improvement in liver steatosis and fibrosis. Further, in a diabetic-steatohepatitis model, the SGLT2 inhibitor dapagliflozin decreased hepatic ROS and reduced inflammasome activation [63], which was also seen in other models of obesity and insulin resistance [64,65]. Finally, given that the inflammatory pathways in steatohepatitis lead to the development of hepatocellular carcinoma, some have postulated that the anti-inflammatory effects of SGLT2 inhibitors in the liver may drive their anti-neoplastic benefit which has been demonstrated in animals [66].

SGLT2 inhibitors are associated with improvement in liver function and steatosis by imaging in individuals with diabetes [67,68]. Improvement in hepatic steatosis has been shown to be independent of weight loss in animals [69], suggesting possible alternative mechanisms of this benefit including reduction of inflammation [70]. Importantly, few studies have evaluated the effects of SGLT2 inhibition on liver tissue by pathology in humans. However, small case series have demonstrated improvement in steatosis by biopsy after treatment with an SGLT2 inhibitor [71–74], as well as fibrosis in a few studies [75,76]. In one study, pathology revealed improvement in inflammation scores, steatosis and fibrosis compared to pre-treatment despite worsening obesity [76], again suggesting that this benefit is not due to weight loss.

Other Tissues

Overall, there is a paucity of data on the anti-inflammatory effects of SGLT2 inhibitors in tissues other than those described in the previous sections. The available data is summarized below.

In the skeletal muscle, canagliflozin decreased macrophage accumulation, pro-inflammatory cytokines, and expression of the atrophic factor atrogin-1 in high-fat diet (HFD)-fed obese mice [77]. Further, canagliflozin decreased expression of the macrophage marker F4/80 and the pro-inflammatory cytokine Tnfa, both of which were significantly increased with obesity in HFD mice [77]. Finally, SGLT2 inhibitors may exert beneficial anticatabolic effects in the muscle via generation of ketone bodies [78], which can suppress oxidative stress and activation of the NLRP3 inflammasome, as discussed below.

In the nervous system of HFD mice, canagliflozin decreased expression of the macrophage/microglia marker Iba1 and the inflammatory cytokines Il6 and Tnfa, as well as the number of macrophages/microglia in the nodose ganglion and hypothalamus [77]. Empagliflozin also significantly reduced expression of the pro-inflammatory mediators Il6, Tnf and Il1b in LPS-activated primary microglia from rats [79].

In the lungs, canagliflozin has been shown to reduce acute inflammation in response to LPS. First, canagliflozin decreased pro-inflammatory cytokines IL-1b, IL-6, and TNFα after LPS stimulation in mouse and human immune cell lines in vitro [80]. In addition, canagliflozin reduced pro-inflammatory M1 and promoted homeostatic M2 macrophage phenotypes in bone marrow-derived macrophages in vitro [81]. Finally, in in vivo LPS-induced acute lung injury models, canagliflozin decreased circulating IL-6, TNFα, and IL-1b, decreased bronchoalveolar lavage fluid pro-inflammatory cytokines and macrophages, and reduced lung injury [80,81].

In the gastrointestinal system, empagliflozin alleviated acute dextran sulfate sodium-induced colitis in mice, decreasing the expression of pro-inflammatory cytokines TNFα, IL-1β and IL-6 [82]. In multiple models of rat colitis, SGLT2 inhibition increased AMPK phosphorylation, decreased mTOR, NF-κB and NLRP3 expression, and reduced pro-inflammatory cytokines including IL-1β, IL-6, TNFα and IL-18 [83–85].

Hypothesized Anti-Inflammatory Mechanisms of SGLT2 Inhibition

SGLT2 Inhibitors Reduce Inflammasome Activation.

The NLRP3 inflammasome is a macromolecular structure formed in response to pathogens or danger signals that results in the production of IL-1β, which is a key cytokine that is linked with the pathogenesis of cardiovascular and metabolic diseases. SGLT2 inhibition decreases NLRP3 inflammasome activation in rodent models of heart failure [14], diabetes [17,40], fatty liver [63,65], kidney fibrosis [48], and obesity [64]. SGLT2 inhibition for 30 days in individuals with type 2 diabetes and cardiovascular risk decreased NLRP3 inflammasome activation and production of IL-1β in blood-derived human macrophages [86]. Importantly, SGLT2 inhibitors increase the serum ketone beta-hydroxybutyrate, a known inhibitor of the NLRP3 inflammasome [87], and prior studies have demonstrated that blocking inflammasome activation through increased ketones via a ketogenic diet can reduce inflammation including relieving gout flares [88] and increasing metabolically protective T cell subsets in adipose tissue [89]. Thus, SGLT2 inhibitors may block inflammasome activation by increasing ketones [90].

SGLT2 Inhibitors Block NHE1 Activity and Reduce Intracellular Sodium and Calcium.

SGLT2 inhibitors, in addition to inhibiting the protein SGLT2, are also known to inhibit the protein sodium-hydrogen exchanger-1 (NHE1). NHE1 catalyzes the exchange of extracellular sodium and intracellular hydrogen, causes increased intracytoplasmic calcium accumulation and calcium-mediated signaling, activates protein kinase C and NFAT, all of which lead to immune activation. NHE1 is expressed more broadly than SGLT2, and off-target effects of SGLT2 inhibitors on NHE1 are hypothesized to contribute to cardiometabolic and renal benefits [91], and may contribute to anti-inflammatory effects of these drugs. SGLT2 inhibitors block NHE1 activity and reduce intracytoplasmic sodium and calcium in rodent cardiomyocytes in vitro [92,93]. In addition, direct binding of dapagliflozin with NHE1 was demonstrated in rat cardiomyocytes, and further supported by loss- and gain-of-function in vitro experiments [94]. Importantly, SGLT2 inhibition decreases pro-inflammatory mouse macrophage polarization in vitro by downregulating NHE1 expression and activity, and reducing intracellular calcium [95].

In studies using human tissues, atrial cardiomyocytes were found to express NHE1, which was increased in those with heart failure and atrial fibrillation, and in vitro treatment with empagliflozin inhibited NHE1 activity similarly to the NHE inhibitor cariporide [96]. Further, empagliflozin reduced TNFα-mediated NHE activity, intracellular sodium accumulation, and ROS generation in human endothelial cells [97]. Interestingly, an artificial intelligence model predicted that the effects of empagliflozin in heart failure were predominantly mediated by inhibition of NHE1, and less so by SGLT2 [98]. Future studies are needed to investigate what effects of SGLT2 inhibitors may be mediated via NHE1 in vivo. Of note, a study of the NHE inhibitor cariporide in individuals with coronary artery disease was negative for the primary outcome of death or myocardial infarction, but showed reduction of risk at the highest dose of 120mg in individuals undergoing bypass surgery [99].

SGLT2 Inhibitors Reduce Uric Acid.

Increased levels of uric acid, the final product of purine metabolism, is associated with chronic kidney disease, diabetes, and cardiovascular disease. Uric acid has been shown to increase production of ROS and contribute to inflammation, which is at least one mechanism of association with cardiometabolic diseases. In addition, in in vitro models of low-grade inflammation, uric acid further promotes production of IL-6 and IL-1b from monocytes and endothelial cells [100]. SGLT2 inhibitors increase urinary excretion of uric acid and lead to decreased serum uric acid levels, as seen in a meta-analysis of randomized trials [101]. Thus, a potential anti-inflammatory mechanism of SGLT2 inhibition may be via reduction of serum uric acid levels.

SGLT2 Inhibitors Modulate Mitochondrial Function and Immunometabolism.

Mitochondria are the energy centers of any mammalian cell and are involved in many cellular metabolic functions, such as ATP production, fatty acid synthesis and oxidation, and triglyceride balance. In addition, mitochondria participate in various cellular signaling events, such as the regulation of calcium homeostasis, orchestration of apoptosis, and production of ROS. Mitochondrial functions are tightly associated with inflammatory responses. For example, ROS generated from mitochondria play a crucial role in NLRP3 inflammasome activation, leading to the secretion of pro-inflammatory cytokines IL-1β and IL-18 [102].

Several studies suggest that SGLT2 inhibitors can regulate mitochondrial functions through distinct mechanisms. First, SGLT2 inhibitors significantly reduced mitochondrial ROS production under oxidative stress conditions in human proximal tubular cells (HK-2) and human endothelial cells [41,103]. Second, the SGLT2 inhibitor luseogliflozin was shown to reduce mitochondrial oxygen consumption and the expression of hypoxia-inducible factor-1α (HIF-1α), a key molecule in hypoxia-induced tubulointerstitial fibrosis, in human renal proximal tubular epithelial cells [104]. Moreover, these effects were mediated via activating the cellular energy sensor protein AMPK. However, it is interesting to point out that a pharmacological activator of AMPK, without disrupting the cellular energy balance, did not mimic the effect of luseoglfozin, while mitochondrial complex I inhibitors, leading to mild cellular energy deficiency, also suppressed HIF-1α expression similar to luseogliflozin, suggesting that the effects of luseogliflozin may be associated with cellular energy depletion independent of AMPK activation [104]. Finally, in HEK-293 cells, canagliflozin inhibited mitochondrial respiration by blocking Complex I, leading to an increase in AMPK activity [105].

The immune metabolic profile, or how immune cells use fuel to generate energy from pathways such as glycolysis and mitochondrial respiration, is intimately linked to the activation status of immune cells [106] and may be another target of SGLT2 inhibition. In one study, canagliflozin decreased T cell glycolysis, which is associated with T cell activation, and reduced pro-inflammatory cytokines after LPS stimulation in immune cell lines in vitro and in mice in vivo [80]. In another study, canagliflozin treatment in vitro decreased human T cell activation through metabolic reprogramming, and decreased effector functions of T cells derived from patients with systemic lupus erythematosus and rheumatoid arthritis [107]. Thus, SGLT2 inhibitor-mediated modulation of mitochondrial function and immunometabolism may contribute to the anti-inflammatory benefit of these drugs. However, further mechanistic studies are necessary.

Concluding Remarks and Future Perspectives

SGLT2 inhibitors have been shown to mitigate inflammation systemically and within several tissues/organ systems, including the cardiovascular system, the kidney, adipose tissue, and the liver. Whether the anti-inflammatory properties of SGLT2 inhibitors mediate some of the clinically observed cardiometabolic or renal benefits of this class of medications remains to be definitively demonstrated (See Outstanding Questions). Future studies will need to extend the anti-inflammatory mechanisms demonstrated in pre-clinical and in vitro studies to clinical outcomes. One such mechanistic study (NCT05972564) is an ongoing randomized controlled trial of SGLT2 inhibition versus placebo with simultaneous assessment of adipose tissue and systemic inflammatory measures combined with surrogate measures of cardiovascular health. Additional considerations for future studies include measurements of functional or structural surrogates of inflammation-related organ dysfunction or imaging-based measures of fibrosis. It is possible that with additional data on the anti-inflammatory potential of SGLT2 inhibition in humans, we may repurpose these drugs for use in autoimmune diseases [108] or other hyperinflammatory conditions such as COVID-19 infection [109] (see Outstanding Questions).

Outstanding questions

• What are the key molecular pathways by which SGLT2 inhibitors exert their anti-inflammatory effects?

• To what extent are the clinical improvements in cardiometabolic and renal outcomes driven by systemic and tissue-specific anti-inflammatory effects of SGLT2 inhibitors?

• Can SGLT2 inhibitors be repurposed for the management of autoimmune diseases and hyperinflammatory conditions?

Highlights

• Evidence from animal models and human studies clearly demonstrate that sodium-glucose cotransporter-2 (SGLT2)-inhibitors have systemic and tissue-specific anti-inflammatory effects that may be independent of glycemic control and weight loss.

• SGTL2 inhibitors may achieve their anti-inflammatory effects through various mechanisms, including reducing reactive oxygen species, inflammasome activation, intracellular sodium and calcium levels, circulating uric acid and mitochondrial dysfunction.

• The direct and indirect mitigation of inflammatory pathways by SGLT2 inhibitors may partially explain their cardiac and reno-protective effects. However, this still needs to be formally demonstrated in human studies.

Glossary

Glucosuric

This indicates the ability to produce glycosuria, which is the excretion of glucose into the urine. Sodium-glucose cotransporter-2 (SGLT2) inhibitors produce glycosuria as their primary mechanism of action.

Interleukins (IL)

Group of cytokines, which are secreted proteins and signal molecules, expressed and secreted by leukocytes and other cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties.

Reactive oxygen species (ROS)

Highly reactive chemicals containing at least one oxygen atom and one or more unpaired electrons, produced results of normal aerobic respiration. Their increased production leads to molecular damage, denoted as oxidative stress. These are also key signaling molecules that play an important role in the progression of inflammatory disorders.

Sodium-glucose cotransporter-2 (SGLT2)

A member of the sodium-glucose cotransporter family, which are sodium-dependent glucose transport proteins. SGLT2 is the main cotransporter involved in glucose reabsorption in the kidney.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors

Drugs, otherwise called gliflozins or flozins, that mainly act by inhibiting sodium-glucose cotransporter-2 (SGLT2).