Mechanisms of heart failure and chronic kidney disease protection by SGLT2 inhibitors in nondiabetic conditions.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), initially developed for type 2 diabetes (T2D) treatment, have demonstrated significant cardiovascular and renal benefits in heart failure (HF) and chronic kidney disease (CKD), irrespective of T2D. This review provides an analysis of the multifaceted mechanisms underlying the cardiorenal benefits of SGLT2i in HF and CKD outside of the T2D context. Eight major aspects of the protective effects of SGLT2i beyond glycemic control are explored: (i) the impact on renal hemodynamics and tubuloglomerular feedback; (ii) the natriuretic effects via proximal tubule Na+/H+ exchanger NHE3 inhibition; (iii) the modulation of neurohumoral pathways with evidence of attenuated sympathetic activity; (iv) the impact on erythropoiesis, not only in the context of local hypoxia, but also systemic inflammation and iron regulation; (v) the uricosuria and mitigation of the hyperuricemic environment in cardiorenal syndromes; (vi) the multiorgan metabolic reprogramming including the potential induction of a fasting-like state, improvement in glucose and insulin tolerance and stimulation of lipolysis and ketogenesis; (vii) the vascular endothelial growth factor A (VEGF-A) upregulation and angiogenesis, and (viii) the direct cardiac effects. The intricate interplay between renal, neurohumoral, metabolic, and cardiac effects underscore the complexity of SGLT2i actions and provides valuable insights into their therapeutic implications for HF and CKD. Furthermore, this review sets the stage for future research to evaluate the individual contributions of these mechanisms in diverse clinical settings.

Paracrine and endocrine regulation of renal K+ secretion.

The seminal studies conducted by Giebisch and coworkers in the 1960s paved the way for understanding the renal mechanisms involved in K+ homeostasis. It was demonstrated that differential handling of K+ in the distal segments of the nephron is crucial for proper K+ balance. Although aldosterone had been classically ascribed as the major ion transport regulator in the distal nephron, thereby contributing to K+ homeostasis, it became clear that aldosterone per se could not explain the ability of the kidney to modulate kaliuresis in both acute and chronic settings. The existence of alternative kaliuretic and antikaliuretic mechanisms was suggested by physiological studies in the 1980s but only gained form and shape with the advent of molecular biology. It is now established that the kidneys recruit several endocrine and paracrine mechanisms for adequate kaliuretic response. These mechanisms include the direct effects of peritubular K+, a gut-kidney regulatory axis sensing dietary K+ levels, the kidney secretion of kallikrein during postprandial periods, the upregulation of angiotensin II receptors in the distal nephron during chronic changes in K+ diet, and the local increase of prostaglandins by low-K+ diet. This review discusses recent advances in the understanding of endocrine and paracrine mechanisms underlying the modulation of K+ secretion and how these mechanisms impact kaliuresis and K+ balance. We also highlight important unknowns about the regulation of renal K+ excretion under physiological circumstances.

Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats.

This study aimed to investigate the antiproteinuric and hyperkalemic mechanisms activated by dual renin-angiotensin system (RAS) blockade in renovascular hypertensive rats (2-kidney 1-clip model [2K-1C]). Six weeks after clipping the left renal artery or sham operation (2K), rats were treated with losartan, enalapril, or both drugs for two weeks. We found that 2K-1C rats displayed higher tail-cuff blood pressure (BP), increased non-clipped kidney Ang II concentration, and more pronounced urinary albumin excretion than 2K. BP was decreased by the treatment with either enalapril or losartan, and the combination of both drugs promoted an additional antihypertensive effect in 2K-1C rats. Renal Ang II content and albuminuria were reduced by either enalapril or losartan in monotherapy and restored to control levels by dual RAS blockade. Albuminuria in 2K-1C rats was accompanied by downregulation of the glomerular slit protein podocin, reduction of the endocytic receptors megalin and cubilin, and a marked decrease in the expression of the ClC-5 chloride channel, compared to 2K animals. Treatment with losartan and enalapril in monotherapy or combination increased the expression of podocin, cubilin, and ClC-5. However, only the combined therapy normalized podocin, cubilin, and ClC-5 protein abundance in the non-clipped kidney of 2K-1C rats. Renovascular hypertensive 2K-1C rats had a lower concentration of plasma potassium compared to 2K rats. Single RAS blockade normalized potassium plasma concentration, whereas 2K-1C rats treated with dual RAS blockade exhibited hyperkalemia. Hypokalemia in 2K-1C rats was accompanied by an increase in the cleaved activated forms of α-ENaC and γ-ENaC and the expression of ß-ENaC. Combined RAS blockade but not monotherapy significantly reduced the expression of these ENaC subunits in 2K-1C rats. Indeed, double RAS blockade reduced the abundance of cleaved-α-ENaC to levels lower than those of 2K rats. Collectively, these results demonstrate that the antiproteinuric effect of dual RAS blockade in 2K-1C rats is associated with the restored abundance of podocin and cubilin, and ClC-5. Moreover, double RAS blockade-induced hyperkalemia may be due, at least partially, to an exaggerated downregulation of cleaved α-ENaC in the non-clipped kidney of renovascular hypertensive rats.

Empagliflozin Inhibits Proximal Tubule NHE3 Activity, Preserves GFR, and Restores Euvolemia in Nondiabetic Rats with Induced Heart Failure.

BACKGROUND: SGLT2 inhibitors reduce the risk of heart failure (HF) mortality and morbidity, regardless of the presence or absence of diabetes, but the mechanisms underlying this benefit remain unclear. Experiments with nondiabetic HF rats tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) inhibits proximal tubule (PT) NHE3 activity and improves renal salt and water handling. METHODS: Male Wistar rats were subjected to myocardial infarction or sham operation. After 4 weeks, rats that developed HF and sham rats were treated with EMPA or untreated for an additional 4 weeks. Immunoblotting and quantitative RT-PCR evaluated SGLT2 and NHE3 expression. Stationary in vivo microperfusion measured PT NHE3 activity. RESULTS: EMPA-treated HF rats displayed lower serum B-type natriuretic peptide levels and lower right ventricle and lung weight to tibia length than untreated HF rats. Upon saline challenge, the diuretic and natriuretic responses of EMPA-treated HF rats were similar to those of sham rats and were higher than those of untreated HF rats. Additionally, EMPA treatment prevented GFR decline and renal atrophy in HF rats. PT NHE3 activity was higher in HF rats than in sham rats, whereas treatment with EMPA markedly reduced NHE3 activity. Unexpectedly, SGLT2 protein and mRNA abundance were upregulated in the PT of HF rats. CONCLUSIONS: Prevention of HF progression by EMPA is associated with reduced PT NHE3 activity, restoration of euvolemia, and preservation of renal mass. Moreover, dysregulation of PT SGLT2 may be involved in the pathophysiology of nondiabetic HF.

Cardioprotection conferred by sodium-glucose cotransporter 2 inhibitors: a renal proximal tubule perspective.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, also known as gliflozins, improve glycemia by suppressing glucose reuptake in the renal proximal tubule. Currently, SGLT2 inhibitors are primarily indicated as antidiabetic agents; however, their benefits extend far beyond glucose control. Cardiovascular outcome trials indicated that all studied SGLT2 inhibitors remarkably and consistently reduce cardiovascular mortality and hospitalization for heart failure (HF) in type 2 diabetes (T2D) patients. Nevertheless, the mechanisms underlying the unprecedented cardiovascular benefits of gliflozins remain elusive. Multiple processes that directly or indirectly improve myocardial performance may be involved, including the amelioration of proximal tubular dysfunction. Therefore, this paper provides a perspective on the potential cellular and molecular mechanisms of the proximal tubule that may, at least in part, mediate the cardioprotection conferred by SGLT2 inhibitors. Specifically, we focus on the effects of SGLT2 on extracellular volume homeostasis, including its plausible functional and physical association with the apical Na+/H+ exchanger isoform 3 as well as its complex and its possible bidirectional interactions with the intrarenal angiotensin system and renal sympathetic nervous system. We also discuss evidence supporting a potential benefit of gliflozins in reducing cardiovascular risk, attributable to their effect on proximal tubule handling of uric acid and albumin as well as in erythropoietin production. Unraveling the mechanisms behind the beneficial actions of SGLT2 inhibitors may not only contribute to a better understanding of the pathophysiology of cardiovascular diseases but also enable repurposing of gliflozins to improve the routine management of HF patients with or without T2D.

The potential role of myosin motor proteins in mediating the subcellular distribution of NHE3 in the renal proximal tubule.

Isoform 3 of the Na+/H+ exchanger (NHE3) is responsible for the majority of the reabsorption of NaCl, NaHCO3, and, consequently, water in the renal proximal tubule. As such, this transporter plays an essential role in acid-base balance and extracellular fluid volume homeostasis and determining systemic arterial blood pressure levels. NHE3 activity is modulated by a number of mechanisms, including the redistribution of the transporter between the body of the microvilli (where NHE3 is active) and the base of the microvilli (where NHE3 is less active). Although the physiological, pathophysiological, and pharmacological importance of the subcellular distribution of NHE3 has been well established, the exact mechanism whereby NHE3 is translocated along microvilli microdomains of the proximal tubule apical membrane is unknown. Nonmuscle myosin IIA and unconventional myosin VI move cargoes in anterograde and retrograde directions, respectively, and are known to redistribute along with NHE3 in the proximal tubule in response to a variety of natriuretic and antinatriuretic stimuli, including stimulation or inhibition of the renin-angiotensin system, high dietary Na+ intake, and high blood pressure. Therefore, this review aims to discuss the current evidence that suggests a potential role of myosin IIA and myosin VI in mediating the subcellular distribution of NHE3 along the kidney proximal tubule microvilli and their possible contribution in modifying NHE3-mediated Na+ reabsorption under both physiological and pathophysiological conditions.

Signaling pathways involved in the rapid biphasic effect of aldosterone on Na+/H+ exchanger in rat proximal tubule cells.

The receptors and signaling pathways for nongenomic effects of aldosterone (Aldo) on the proximal Na+/H+ exchanger are still unknown; therefore, the aim of this study was to investigate the mineralocorticoid receptor (MR) and/or glucocorticoid receptor (GR) participation in rapid Aldo effects on NHE1 (basolateral Na+/H+ exchanger isoform) and cytosolic calcium concentration ([Ca2+]i). In addition, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase kinase (MEK) involvement in signaling pathways of such effects was evaluated, using immortalized proximal tubule cells of rat (IRPTC) as an experimental model. MR and GR expression was investigated using reverse transcription polymerase chain reaction and immunoblotting. The intracellular pH recovery rate (after acid loading) and [Ca2+]i were determined by the probes BCECF-AM and FURA 2-AM, respectively. Aldo (10-12 M) promoted a moderate increase in [Ca2+]i and stimulation of NHE1, whereas Aldo (10-6 M) greatly increased the [Ca2+]i, but inhibited the NHE1. BAPTA-AM (a calcium chelator), GR antagonism and inhibition of PLC, PKC and MEK pathway abolished the biphasic and dose-dependent effect of Aldo on NHE1 and decreased the [Ca2+]i; whereas MR do not appear to participate in this rapid signaling in IRPTC cells. The reduction of GR content, by gene silencing, abolished the Aldo effect on NHE1, in low concentration, confirming the importance of this receptor in the rapid modulation of proximal sodium and hydrogen transports.

Angiotensin II counteracts the effects of cAMP/PKA on NHE3 activity and phosphorylation in proximal tubule cells.

Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na+/H+ exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.

Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling.

Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na(+)/H(+) exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na(+)-dependent intracellular pH recovery. We found that 10(-7) M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization.