The first and most acceptable intrarenal mechanism is the control of glomerular hyperfiltration by SGLT2Is. Inhibition of sodium and glucose uptake by PCT increases sodium delivery to the distal segment. The macula densa within the distal convoluted tubules (DCT) interprets increased sodium delivery as circulating volume expansion and through tubuloglomerular feedback cause hemodynamic changes in the afferent arterioles. Administration of Empa to diabetic rats cause vasoconstriction of afferent arteriole within 30min together with decreased intraglomerular tuft pressure and reduction of single nephron GFR. Adenosine A1 receptors blocking abolished these effects.21 Adenosine increases as a consequence of excess ATP consumption during increased active sodium reabsorption by DCT (Fig. 1). The impact of SGLT2Is on GFR can occur even in euglycemic state. SGLT2Is reduced the renal glucose concentration threshold similarly in T2DM and non-diabetic individuals to less than 40mg/dL.22 Increased single nephron ultrafiltration of the surviving nephrons is one of the main causes of progressive decline in kidney function in CKD patients. By controlling single nephron hyperfiltration, SGLT2Is can mitigate CKD progression even in non-diabetic patients. In view of the observed decrease of glomerular hyperfiltration after use of Dapa in T2DM patients in spite of the unchanged renal vascular resistance, a recent study suggested that the decrease in glomerular tuft pressure is more likely caused by post-glomerular efferent arteriolar vasodilatation instead of preglomerular afferent arteriolar vasoconstriction. The authors hypothesized that the potential vasoconstrictive action of adenosine on the afferent arteriole was counteracted by a concomitant RAS blockade or an increase in prostaglandin synthesis. Subsequently, prostaglandins may have induced efferent arteriolar vasodilation, which could not be prevented by angiotensin II, since this was pharmacologically blocked.23

The kidney eliminates 70% of the daily UA production in humans.24 The offending effect of high serum uric acid (SUA) on the kidney was clearly demonstrated in experimental animals after raising their SUA. Animals have low SUA due to the uricase enzyme that breaks down UA. Oxonic acid inhibits the uricase enzyme and is used in experimental animals to raise their SUA. By increasing SUA, experimental animals develop systemic hypertension, glomerular hypertension, glomerulosclerosis, and interstitial fibrosis.25–29 SUA is a strong predictor of increased urine albumin excretion. On follow-up of T1DM patients having normal urine albumin for 6 years, every 1mg/dL SUA above normal level increases the risk of development of albuminuria by 80%.30 The risk for albuminuria and accelerated decline of GFR in T2DM patients suffering hyperuricemia is further illustrated by 2 prospective studies.31,32 In patients suffering T2DM for fifteen years or more, SUA >7mg/dL in males and >6mg/dL in females increases the risk of DKD progression, and overall mortality.33 Treatment of patients suffering T2DM and DKD and high SUA with allopurinol decreased albuminuria and serum creatinine significantly over three years of follow-up.34 A prospective observation study of 900 blood donors followed for 5 years showed that the basal SUA is a significant predictor of eGFR decline in healthy normotensive subjects lacking signs of CKD at entry to the study.35 In contrast, recent studies failed to support the therapeutic benefit of urate lowering treatment. Allopurinol failed to improve kidney outcomes among patients with T1DM and early-to-moderate DKD.36 Treatment of hyperuricemia using allopurinol also failed to slow the rate of decline in eGFR as compared with placebo in patients with CKD and a high risk of progression.37 Similarly, febuxostat failed to mitigate the progression of CKD among patients with asymptomatic hyperuricemia and stage 3 CKD.38 Based on clinical and experimental evidence, it seems that intracellular UA behaves differently in comparison to extracellular UA. While extracellular UA has anti-oxidant properties, intracellular UA imposes a detrimental pro-oxidant effect.39

Increased intracellular production of uric acid (UA) occurs in epithelial cells of PCT in diabetic state. Excess glucose absorption by PCT in diabetic patients increases intracellular glucose availability. A consequent increase in the activity of polyol pathway leads to increased fructose synthesis. Fructose metabolism leads to increased intracellular adenosine and UA synthesis.40 Increased intracellular UA stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme causing increased intracellular oxidative stress, mitochondrial injury, adenosine triphosphate (ATP) depletion,41,42 endothelial injury, RAS activation and increased epithelial- mesenchyme transition (EMT). Increased EMT leads to overproduction of fibroblasts that infiltrate the interstitium with consequent progressive interstitial fibrosis.43 Increased NADPH oxidase activity can also stimulate production of macrophage chemo-attractant factor (MCP1) with consequent increased macrophage infiltration of the kidney44 (Fig. 2). By inhibiting glucose absorption, SGLT2Is decrease intracellular glucose concentration, decrease activity of polyol pathway, decrease fructose and thus decrease intracellular UA synthesis.45 Moreover increased availability of luminal glucose as a consequence of SGLT2Is administration triggers apical GLUT9 isoforms in PCT epithelium trying to absorb glucose in exchange with UA. GLUT9 activation thus depletes intracellular UA within PCT. In contrast, GLUT9 in the collecting ducts increase absorption of uric acid from the lumen. Excess glucose within the tubular lumen inhibits UA absorption by GLUT9 in the collecting ducts46 (Fig. 3). Contrary to the debatable impact of serum UA reduction, it seems that reduction of intracellular UA within PCT have a crucial role in the renal protection of SGLT2Is. Among the different angiotensin receptor blockers, only losartan has the ability to lower serum UA through its uricosuric effect.47 Decreased uptake of UA by PCT cells result in decreased intracellular UA within these cells. Losartan is the only member among its family that showed a reduction for the need for dialysis in patients suffering DKD.7

Fig. 2.

Different pathogenic mechanisms of kidney injury possibly induced by uric acid. UA: uric acid; ROS: reactive oxygen species; NF-κB: nuclear factor kappa B; MCP1: macrophage chemo-attractant protein-1; RAS: renin angiotensin system; EMT: epithelium mesenchyme transition; VSMC: vascular smooth muscle cells.

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Fig. 3.

Mechanism of increased uric acid excretion induced by SGLT2 inhibitors. SGLT2I: sodium glucose cotransporters inhibitor; PCT: proximal convoluted tubules; DCT: distal convoluted tubules; UA: uric acid.

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Sodium hydrogen exchangers (NHE) exist in nine isoforms.48,49 NHE3 isoform is encountered on renal tubular and intestinal epithelium. When NHE3 in PCT and ascending loop of Henle are activated, excess sodium retention occurs and contributes to systemic hypertension in diabetic patients.50,51 Diabetes and excess insulin stimulate while SGLT2Is inhibit renal NHE351 (Fig. 4). The natriuretic effect of SGLT2Is is mediated mainly through NHE3. Empa failed to induce natriuresis in normo-glycemic NHE3 knock out mice in contrast to wild mice.52 The natriuretic effect of SGLT2Is is responsible for the drop of blood pressure observed after the use of these agents. In salt treated obese and metabolic syndrome rats that develop hypertension, treatment with SGLT2Is significantly decreased the blood pressure associated with an increase in urinary sodium excretion. This was associated with normalized circadian rhythms of both blood pressure and sympathetic nerve activity and might explain why the observed hemoconcentration induced by SGLT2Is is not associated with change in heart rate.53

Fig. 4.

Increased activity of NHE3 isomer within the proximal convoluted tubules increases sodium absorption from the lumen of these tubules in exchange with the secreted hydrogen. Decreased sodium delivery to the distal nephron segments results in glomerular hyperfiltration. Diabetic state and insulin administration increase NHE3 activity while SGLT2Is and GLP1RAs inhibit it. NHE: sodium hydrogen exchanger; SGLT2Is: sodium glucose transporter-2 inhibitors; GLP1Ras: glucagon like peptide receptor agonists.

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NHE1 isoform present on the surface of cardiomyocytes, endothelial cells, and platelets are also stimulated in diabetic patient and are inhibited by SGLT2Is. The favorable impact of SGLT2Is on the heart and endothelium are mediated, in part, through NHE1 inhibition.50,51

PCT epithelial cells incubated in high glucose medium increase the expression of Toll-like receptor-4, increase nuclear DNA binding for NF-κB and activator protein 1, and increase collagen IV expression as well as interleukin-6 secretion. All these processes are inhibited on addition of Empa to the culture medium.54 SGLT2Is inhibit inflammation indirectly through suppression of oxidative injury. In diabetic kidney, generation of free radicals is a result of a pro-oxidant enzyme action, polyol pathways, lipid peroxidation, mitochondrial dysfunction, hemodynamic changes, and protein kinase C activation. Empa inhibits free oxygen radical production through inhibition of NADPH oxidase enzyme.55 Other pro-oxidant enzymes like endothelial nitric oxide synthase, and xanthine oxidase, were also modified by different SGLT2Is through alteration of their activity or their expression.56,57 Suppression of free oxygen radicals consequently suppresses MCP1 and NFκB. High mobility group box protein 1 (HMGB1) is a nuclear DNA-binding protein normally indulged in a variety of processes including repair, differentiation, and development of DNA. Under pathological circumstances, HMGB1 is released from the necrotic or activated cells and acts as a proinflammatory cytokine. HMGB1 exerts its functions via several receptors including RAGE and toll-like receptors (TLRs).58 Macrophages and activated dendritic cells transport HMGB1 from the nucleus to the cytoplasm and then release it via secretory inflammasomes.59 Through the interaction with TLR2/4 receptors, HMGB1 augments the translocation of NF-κB p65 subunit into the nucleus to promote synthesis of different inflammatory mediators.60 HMGB1 plays a crucial role in the pathogenesis of inflammation in diabetic patients.61 When Empa was administered for 4 weeks to male Wistar rats 8 weeks after induction of diabetes, renal cortical NF-κB was down regulated together with reduction of renal levels of HMGB1, RAGE, and TLR-4.62

Autophagy is an intracellular lysosome-dependent process that sweeps dysfunctional organelles out of the cytoplasm. Increased accumulation of dysfunctional organelles is a common feature of diabetic kidney as a sequence of increased oxidative stress and endoplasmic reticulum stress. These accumulating organelles pose changes in ion channels and trigger cellular inflammation and thus endanger cell survival. Autophagy is usually mitigated in diabetic patients and in cases receiving insulin. Impaired autophagy is due to deficiency of sirtuin-1 (SIRT1), and AMP-activated protein kinase (AMPK) in diabetic patients and manifests as decrease of autophagy of the podocytes and the renal tubular cells.63 By mimicking fasting state, SGLT2Is augment AMPK/SIRT1 signaling and stimulate autophagy, thereby decrease the accelerated damage of podocytes and renal tubular cells within the kidney.64

Chronic renal hypoxia is recently suggested as an important cause of renal damage encountered in DKD patients. This hypoxia is the consequence of a mismatch between oxygen delivery and oxygen demand. Decreased oxygen delivery is the result of microangiopathy affecting small intra-renal arteries together with the glomerular mesangial expansion that compromises the down stream tubular blood flow. Increased oxygen demand evolves as the result of increased activity of the sodium pumps in parallel with sodium glucose cotransporters up regulation and to face glomerular hyperfiltration. Once the imbalance between demand and delivery exists, subsequent tissue hypoxia causes capillary damage, extracellular matrix expansion, inflammation, tubular damage, and subsequent fibrosis. Nephron loss increases the workload of surviving units. This exaggerate the delivery/demand of the surviving nephrons and thus ensuing a vicious cycle that exaggerates subsequent damage.65 Tissue hypoxia stimulates hypoxia inducible factor (HIF) expression in renal tissue. HIF plays an important role in hypoxia-induced tubulointerstitial fibrosis. SGLT2Is inhibit HIF gene expression, and thus inhibit hypoxia induced renal fibrosis.66 This effect of SGLT2Is is likely related to improved tissue hypoxia thanks to the significant down regulation of oxygen consumption by PCT cells after SGLT2Is administration. Further renoprotection offered by SGLT2Is is related to their anti-inflammatory effect of these agents discussed above.54 As we discussed above, free oxygen radical injury causes mitochondrial damage and depletion. A recent study has demonstrated that the SGLT2I Dapa improves mitochondrial function. Dapa increases a panel of urinary metabolites linked to mitochondrial metabolism.67 Decreased sodium and glucose uptake by the PCT can thus reduce cortical oxidative stress and enhance recovery from tubulointerstitial damage that possibly restores renal erythropoietin (EPO) production. Increased erythropoiesis significantly improves hematocrit that improves tissue oxygen delivery68 (Fig. 5).

Fig. 5.

Mechanism of renal hypoxia induced in diabetic kidney disease and role of SGLT2 inhibitors to improve oxygen status. SGLT2Is: sodium glucose co-transporter inhibitors; ROS: reactive oxygen species; HIF: hypoxia inducible factor.

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Decreased pancreatic insulin secretion and/or decreased insulin requirement after SGLT2Is administration render circulating insulin not enough to suppress lipolysis and ketogenesis. Increased pancreatic secretion of glucagon induced by SGLT2Is further increases ketogenesis.69 SGLT2Is mediated cardiac protection is related, in part, to enhanced ketogenesis.70 ATP production within epithelial cells of PCT is largely dependent on fatty acid oxidation that is suppressed in diabetic patients. Impaired ATP production is responsible for progression of CKD and DKD.71 Ketone bodies are an energy source substitute that is capable to ameliorate defective energy metabolism in the epithelial cells of PCT and decrease the progressive loss of PCT and fibrosis after administration of SGLT2Is. In addition to acting as direct energy source for damaged PCT instead of deficient fatty acids, ketone bodies prevent PCT epithelial and podocyte damage by inhibiting the mammalian target of rapamycin complex 1 (mTORC1) signaling. The last mechanism might explain the renoprotective value of SGLT2Is in normoproteinuric as well as proteinuric CKD72 (Fig. 6).

Fig. 6.

Ketone bodies as inhibitors of the renal mammalian target of rapamycin C1. Through this mechanism, SGLT2 inhibitors protect the kidney against the offending action of mammalian target of rapamycin that is induced by diabetic state. SGLT2Is: sodium glucose co-transporter inhibitors; PCT: proximal convoluted tubules; DKD: diabetic kidney disease.

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Up-regulation of SGLT2 by hyperglycemia results in accumulation of glucose within the cytoplasm of PCT cells with consequent aberrant glycolysis. Accumulating glycolytic products are thus diverted into four noxious pathways, namely, the polyol pathway to produce sorbitol and fructose, the hexosamine pathway to form hexosamine that is responsible for formation of transcription factor SP1 and transforming growth factor-β (TGF-β1) induction,73 the lipid synthesis pathway to form diacyl glycerol that induces protein kinase c (PKC) activity, and the formation of advanced glycation end-products. These paths eventually lead to overproduction of TGF-β1 and IL-8, as well as epithelial mesenchymal transition (EMT) of PCT epithelial cells and endothelial mesenchymal transition (EndMT) in the adjacent capillaries. These events eventually lead to interstitial inflammation and fibrosis. By inhibiting excess cellular glucose uptake, SGLT2Is abort all this cascade of events as proved by in- vitro and experimental animal studies.74,75

p21Cip1/Waf1 is a cyclin-dependent kinase inhibitor that increases in epithelial cells of PCT in patients suffering diabetes. This effect is aborted by knocking down SGLT2.76,77 These findings suggest that hyperglycemia causes PCT cell senescence via a SGLT2- and p21-dependent pathway in the diabetic kidney. SGLT2Is reduce the increased expression P21 and thus decrease senescence of renal tubular cells76,77 (Fig. 7).

Fig. 7.

Activation of SGLT2 in diabetic patients leads to overactivity of P21, the natural inhibitor of cyclin-dependent kinase 2. This kinase enzyme inhibits cell senescence. By inducing P21, diabetic patients suffer increased proximal tubular epithelium senescence. Through inhibition of SGLT2, SGLT2Is protect proximal tubular epithelial cells against increased senescence. SGLT: sodium glucose transporter; PCT: proximal convoluted tubule.

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Despite the widespread use of different therapies for DKD and non- diabetic CKD, including statins and RAS blockers, over the past decades, there is still a substantial residual burden of CVD. The significant reduction in cardiovascular adverse events and progression to ESRD associated with the use of SGLT2Is is a distinguished advantage of this novel group of hypoglycemic agents. The most outstanding cardiovascular effect of this group is the highly significant reduction in the incidence and hospital admission for heart failure among the wide spectrum of urine protein excretion and GFR. The cardioprotective benefits of SGLT2Is are multifactorial, including blood sugar control, body weight reduction, systolic blood pressure reduction, diuretic effect, hypouricemic effect, improved erythropoiesis, reduced sympathetic nerve activity,53 and inhibition of sodium hydrogen exchangers. Microalbuminuria was found associated with the future development of heart failure with reduced ejection fraction (HFrEF) and with systolic dysfunction in a community-based sample. On the other hand, CKD was modestly associated with heart failure whether having HFrEF or heart failure with preserved ejection fraction (HFpEF).78 Conversely, heart failure increases the risk of renal function decline and of adverse renal outcomes.79

SGLT2Is have also a significant favorable effect on arterial stiffness.9 Increased pulse wave velocity, as an index of arterial wall stiffness, is associated with a higher risk of incident CKD. The greater the carotid artery stiffness, the higher is the chance of incident CKD and the steeper is the rate of annual GFR decline.80

These cardiovascular benefits offered by SGLT2Is in diabetic and non-diabetic CKD patients might cast their impact on the improved renal outcome in these patients.

Proteinuria is recognized as a predictor of the decline in glomerular filtration rate. The higher the protein excretion rate the more likely the expected severity of renal disease as well as its rate of progression. Independent of the initial insult, different causes of CKD common pathogenic mechanisms that lead to single nephron hyperfiltration, proteinuria, progressive tubular injury, and renal scarring. Protein leakage into the glomerular ultrafiltrate stimulates excess proximal tubular protein reabsorption. Excess protein load within PCT epithelial cells triggers tubular chemokine expression and complement activation. As a consequence, the interstitium of the kidney become a site for inflammatory cell infiltration and subsequent fibrosis. For this reason, proteinuria is considered a valuable surrogate end point for clinical trials in CKD patients and a target for renal protection studies.81 To look for the anti-proteinuric effect of Dapa, a multicenter randomized, double-blind, placebo-controlled crossover trial was done in adult non-diabetic CKD patients having 24h protein excretion between 0.5 and 3.5g/day and eGFR ≥25ml/min/m2 (DIAMOND study). In this trial, six weeks treatment of 52 patients with Dapa 10mg daily failed to achieve a significant decrease in urine protein excretion.82 The short duration of treatment might underlie this failure. In DAPA-CKD study, administration of 10mg Dapa for a mean period of 2.4 years was associated with a highly significant decline of urine protein excretion rate.13 The anti-proteinuric effect of Dapa was also demonstrated in a pooled analysis of 11 phase 3 randomized controlled clinical trials. These studies included 136 diabetic patients having urine albumin/creatinine ratio (UACR) ≥30mg/g that received 5 or 10mg Dapa in comparison to 69 patients that were kept on placebo. eGFR of these cases ranged between 11 and 45ml/min/1.73m2. Over 102 weeks, Dapa 5mg and Dapa 10mg reduced UACR by 47% and 35% respectively compared to placebo. There was no appreciable difference in glycemic control between the Dapa groups and the control cases.83 This is the first report of renoprotective effect of SGLT2Is in CKD patients having eGFR below 25ml/min/1.73m2.

A novel animal study has demonstrated that SGLT2 is expressed in mice podocytes. Induction of proteinuria in these mice was associated with upregulation of SGLT2 in podocytes. Dapa provided glomerular protection in these mice and limited proteinuria, podocyte dysfunction and loss induced by SGLT2 upregulation.84