Copeptin, the C-terminal fragment of pre-provasopressin (CT-proAVP), represents a reliable biomarker of the activity of the antidiuretic system, being secreted by magnocellular hypothalamic neurons in equimolar amount with arginine-vasopressin (AVP) in response to osmotic, hemodynamic and stressful stimuli (Christ-Crain, 2019). The main task of AVP is to maintain osmotic homeostasis, promoting the passive reabsorption of water in the renal collecting ducts by activating the V2 receptors (V2R) located on the basal membrane of the principal cells.

It was shown that even in type 2 diabetes mellitus (T2DM) the antidiuretic system participates in the adaptation to osmotic diuresis, further increasing urinary osmolality by reducing the electrolyte-free water (EFW) clearance (Marton et al., 2021); in this context, sodium glucose co-transporter type 2 inhibitors (SGLT2i) appear to emphasize this mechanism, promoting persistent glycosuria and natriuresis.

In recent years, both empagliflozin (EMPA) and dapagliflozin (DAPA) have gained indication in the treatment not only of T2DM, but also of chronic heart failure (HF) with reduced ejection fraction and chronic kidney disease (CKD), thanks to their ability to reduce disease progression and exacerbations, together with overall cardiovascular mortality (Zinman et al., 2015; Wanner et al., 2016; McMurray et al., 2019; Wiviott et al., 2019).

The pathophysiological basis of these favorable effects is not yet fully understood, but one of the hypotheses is that SGLT2i could induce a greater reduction of interstitial fluids (ISF) than traditional diuretics, thus avoiding hypovolemia and acute kidney injury due to intravascular volume depletion (Lambers Heerspink et al., 2013; Hallow et al., 2018).

Indeed, significant losses of isotonic fluid able to reduce the effective circulating volume (ECV) cannot be immediately replaced by the water present in the interstitial space, since these two compartments share the same osmotic pressure. This process, called extracellular dehydration, leads to extensive neurohormonal activation, in turn associated with known harmful effects on the cardiovascular system when persisting for a long time (Cheuvront and Kenefick, 2014).

Conversely, hypotonic or electrolyte-free water losses are responsible for a mainly intracellular dehydration process, associated with a lower activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system, in favor of an isolated osmotic response of the antidiuretic system (Cheuvront and Kenefick, 2014).

The aim of this proof-of-concept study was to investigate the copeptin adaptive response to persistent osmotic diuresis due to SGLT2i administration, as well as the induced changes in body fluid distribution, in T2DM.

Our results confirm an isolated response of the antidiuretic system to the administration of SGLT2i in patients with T2DM. In particular, the increase in copeptin levels seems attributable to a prevalent intracellular dehydration phenomenon, in the absence of evident signs of hypovolemia or a wide neurohormonal activation. Moreover, the dehydration process due to SGLT2i treatment led to remarkable changes in body fluid distribution only in basally overhydrated patients.

SGLT2i represent new euglycemic drugs able to induce sustained glycosuria by the inhibition of glucose reabsorption in the renal proximal tubule. SGLT2i proved to be effective in reducing cardiovascular risk in patients with T2DM beyond glycemic control, even preventing HF exacerbation and CKD progression (Zinman et al., 2015; Wanner et al., 2016; Neal et al., 2017; Wiviott et al., 2019). Moreover, regardless of T2DM, SGLT2i treatment seems able to improve all hydro-retention states such as HF, liver cirrhosis, CKD and even the syndrome of inappropriate antidiuresis (Saffo and Taddei, 2018; McMurray et al., 2019; Refardt et al., 2020; Bioletto et al., 2023). So far, several hypotheses have been formulated to explain at least some of the unexpected pleiotropic effects associated with the administration of SGLT2i, some authors arguing that these drugs can allow effective drainage of interstitial fluid (ISF), thus avoiding hypovolemia and acute kidney injury (Lambers Heerspink et al., 2013; Hallow et al., 2018).

Indeed, consistent losses of body water and electrolytes, as observed during secretory diarrhea or loop diuretics administration, may be responsible for large ECF volume contraction, primarily involving the intravascular volume. In similar conditions of marked extracellular dehydration, the activation of baroreceptors determines a reduction in the tonic inhibition exerted on the release of AVP (Cheuvront and Kenefick, 2014). Furthermore, the baroreceptor reflex itself, together with the strong stimulation of AVP 1a receptors (V1aR) by the hypothalamic nonapeptide, induces the activation of the sympathetic nervous system and the RAAS, aiming to restore the ECV both through arterial vasoconstriction and through the reabsorption of sodium (Na⁺) and water from the kidney. Unfortunately, a similar persistent neurohormonal activation also leads to harmful cardiovascular effects and cardiac remodeling, at least in part attributable to chronic water retention and the combined profibrotic action exerted by AVP, angiotensin II and aldosterone (Szczepanska-Sadowska et al., 2018).

Conversely, intracellular dehydration occurs during prevailing losses of electrolyte-free water. This condition produces a consequent flow of water from the ICF to the ECF and is detected by both the central and peripheral osmoreceptors, in turn responsible for an isolated stimulus to the release of AVP from the hypothalamus (Cheuvront and Kenefick, 2014).

Copeptin is co-secreted by the neurohypophysis in an equimolar proportion with AVP and in response to the same stimuli, being mainly osmoregulated. Due to low pre-analytical variability and reliable automated testing, copeptin levels are strongly correlated with p-Osm in healthy subjects, even better than AVP itself (Balanescu et al., 2011).

Copeptin levels were found slightly higher in patients with both type 1 and T2DM than in healthy individuals (Roussel et al., 2017; Jensen et al., 2019) and, in this regard, an acceleration in the turnover of body fluids due to glycosuria, as well as a reset of the osmostat have been hypothesized (Marton et al., 2021). Nevertheless, a definitive justification for this endocrine adaptation has not yet been provided (Bankir et al., 2001).

Furthermore, a modest increase in copeptin was previously observed even after a few weeks of treatment with SGLT2i (Heerspink et al., 2019; Lytvyn et al., 2020); thus, further supporting the theory of a compensatory mechanism to limit volume depletion in response to osmotic diuresis, but also of a possible adaptive mechanism alteration of the AVP-renal axis.

In this context, our results show for the first time that copeptin adaptation to SGLT2i in diabetic patients persists for over 3 months and that its levels remain strongly associated with p-Osm even during long-term treatment. Most importantly, BIVA recorded a proportional dehydration process between ECF and ICF; this phenomenon was associated with a condition of basal hyperhydration, in the absence of significant RAAS activation.

These data support the hypothesis of a progressive and coupled reduction of both ECF and ICF, as observed in the process of intracellular dehydration, able to counteract ECF overload without inducing hypovolemia.

Indeed, our results confirm that treatment with SGLT2i in well-controlled diabetic subjects increases u-Osm inducing glycosuria, with only a transient increase in total daily diuresis and natriuresis.

A possible explanation for these findings is the unique mechanism of action of SGLT2i. As known, the natriuretic effect of SGLT2i is exerted by the selective inhibition of Na⁺ reabsorption in the proximal tubule, with consequent increase of the luminal Na⁺ content in the distal convoluted tubule and in the collecting duct. Increased luminal Na⁺ and flow rate result in increased Na⁺ reabsorption in the aldosterone-sensitive distal nephron via epithelial Na⁺ channels (ENaC), thereby reducing effective Na⁺ losses (Szczepanska-Sadowska et al., 2018). Finally, it should be considered that an increase in AVP release would further improve both ENaC and Na⁺/K⁺-ATPase activity in the kidney (Nicco et al., 2001; Mordasini et al., 2005).

On the other hand, in physiological conditions (i.e., euglycemic state), serum glucose substantially represents an ineffective osmole, due to its presence both in the extracellular and intracellular compartment (Verbalis, 2003); conversely, in severe hyperglycemic state, glucose significantly increases effective p-Osm by attracting water from the ICF.

The result of the sum of these combined effects is that, in a population of diabetic subjects not severely hyperglycemic, but affected by ECF overload, SGLT2i would favor a progressive loss of substantially hypotonic fluids compared to plasma; thus, determining a prevalent phenomenon of intracellular dehydration (Figure 5).

Noteworthy, our results also confirm an evident increase in HCT a few months after treatment with SGLT2i. Although HTC levels are reliable indicators of a prevalent extracellular dehydration phenomenon, several evidence gathered in recent years supports the hypothesis that this new class of drugs directly and indirectly improves erythropoietin production (Marathias et al., 2020; Kanbay et al., 2022).

We also found a significant increase in HRV in our population, accompanied by a reduction in mean nocturnal HR (Table 2), apparently not associated with resolution of fluid overload, as well as copeptin response.

Consistently, a recent Japanese study identified during SGLT2i administration a significant reduction in HR at rest, another well-known indicator of the autonomic nervous system activity (Sano, 2018). Taken together, these observations could suggest a beneficial reduction in sympathetic tone and an improvement in the function of the autonomic nervous system, notoriously impaired in diabetic subjects; although this constitutes a research area yet to be explored (Spallone et al., 2011). Conversely, ABPM did not show significant blood pressure variation during SGLT2i treatment, except for small reduction in diurnal values, of the order of 2–3 mmHg, in agreement with the results of other groups (Baker et al., 2017).

Finally, recent evidence, collected from large observational studies conducted on diabetic patients, has shown a clear association between the values of copeptin and albuminuria (Velho et al., 2013; Roussel et al., 2017). In our population, administration of SGLT2i led to a subclinical increase in both albuminuria and ACR, regardless of any sign of lower urinary tract infection (Table 1). Although there is evidence of a possible adverse effect of elevated circulating AVP levels on the degree of albuminuria, possibly mediated by V2R activation, a clear pathophysiological explanation for this phenomenon has not yet been provided (Bankir et al., 2001; Roussel et al., 2017). Most of all our patients were no longer receiving RAAS inhibitors at least 3 weeks prior to the initiation of SGLT2i and this could represent an explanation for a partially increased glomerular filtration pressure.

No variables among copeptin, HVR, TBW, ECF, and ICF were significantly associated with the arms of treatment in our population. Although the number of subjects required for our study was calculated to find a significant difference in copeptin levels before and after SGLT2i treatment, these data support the hypothesis of a class effect; furthermore, a low dose of EMPA may be sufficient to achieve the same beneficial draining effect of ECF, as hypothesized based on other previous studies (Zinman et al., 2015).

The main strengths of our study are the prospective design, the outpatient population enrolled in real life and the extensive analysis conducted on the endocrine regulation of water and electrolyte balance. On the other hand, the absence of a control group and the small sample size certainly represent the main limitations of this research. Furthermore, there is a complex relationship between AVP and glucose metabolism, as V1aR activation induces both glycogenolysis and gluconeogenesis in the liver (Whitton et al., 1978; Keppens and de Wulf, 1979) and diabetic patients also exhibit an apparent enhanced AVP response to various stimuli (Bankir et al., 2001). In addition, previous studies have shown that AVP increases the release of insulin and glucagon by acting directly on pancreatic beta and alpha cells, in a glucose-dependent manner (Abu-Basha et al., 2002); whereas insulin may be able to reduce urinary Na⁺ excretion, due to its ability to promote the adenylate cyclase system in the thick ascending limb of the nephron (Mandon et al., 1993).

Also for this reason we enrolled patients in good glycemic control to evaluate the copeptin response to SGLT2i; on the other hand this implies that our results are not applicable to a poorly controlled diabetic patient population.

In patients with T2DM, SGLT2i induces the release of AVP, thus compensating for persistent osmotic diuresis. This mainly occurs because of a proportional dehydration process between ECF and ICF (i.e., intracellular dehydration rather than extracellular dehydration). The extent of fluid reduction, but not the copeptin response, is affected by the patient’s baseline volume conditions. In view of the known association between T2DM, essential arterial hypertension and volume overload, even in the absence of HF, our results offer some new insight into the pleiotropic benefits derived from SGLT2i treatment in patients with T2DM.

The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Local Ethics Committees of the Turin and Novara University Hospitals (Turin: protocol n. D026280; Novara: protocol n. CE76/19). The patients/participants provided their written informed consent to participate in this study.

AMB and MP-C conceived and designed the study. FR, FP, and FS performed the biochemical analysis and collected the results. CL and CB performed the instrumental evaluation and edit the cases report form. AMB, MP-C, NP, MC, and FB performed the data analysis, the figures designing, and the manuscript writing. VG, GM were in charge for overall direction and planning. SG, MM, and ASB gave the needed encouragement and support to investigate and supervised the findings of this work. All authors revised the manuscript for important intellectual content and approved the definitive version.