BackgroundSGLT2 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.MethodsMale 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.ResultsEMPA-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.ConclusionsPrevention 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.

Dipeptidyl peptidase IV (DPPIV) inhibitors are antidiabetic agents that exert renoprotective actions independently of glucose lowering. Cardiac dysfunction is one of the main outcomes of chronic kidney disease (CKD); however, the effects of DPPIV inhibition on cardiac impairment during CKD progression remain elusive. This study investigated whether DPPIV inhibition mitigates cardiac dysfunction and remodeling in rats with a 5/6 renal ablation and evaluated if these effects are associated with changes in the cardiac renin-angiotensin system (RAS). To this end, male Wistar rats underwent a 5/6 nephrectomy (Nx) or sham operation, followed by an 8-week treatment period with the DPPIV inhibitor sitagliptin (IDPPIV) or vehicle. Nx rats had lower glomerular filtration rate, overt albuminuria and higher blood pressure compared to sham rats, whereas CKD progression was attenuated in Nx + IDPPIV rats. Additionally, Nx rats exhibited cardiac hypertrophy and fibrosis, which were associated with higher cardiac DPPIV activity and expression. The sitagliptin treatment prevented cardiac fibrosis and mitigated cardiac hypertrophy. The isovolumic relaxation time (IRVT) was higher in Nx than in sham rats, which was suggestive of CKD-associated-diastolic dysfunction. Sitagliptin significantly attenuated the increase in IRVT. Levels of angiotensin II (Ang II) in the heart tissue from Nx rats were higher while those of angiotensin-(1-7) Ang-(1-7) were lower than that in sham rats. This cardiac hormonal imbalance was completely prevented by sitagliptin. Collectively, these results suggest that DPPIV inhibition may delay the onset of cardiovascular impairment in CKD. Furthermore, these findings strengthen the hypothesis that a crosstalk between DPPIV and the renin-angiotensin system plays a role in the pathophysiology of cardiorenal syndromes.

Cell therapy repair strategies using adult mesenchymal stromal cells have shown promising evidence to prevent cardiac deterioration in rodents even in the absence of robust differentiation of the cells into cardiomyocytes. We tested whether increasing doses of porcine adipose-tissue derived mesenchymal stem cells (pASCs) increase cardiac tissue perfusion in pigs post-myocardial infarction (MI) receiving angiotensin-converting-enzyme inhibitor (ACE inhibitors) and Beta-blockers similarly to patients. Female pigs were subjected to MI induction by sponge permanent occlusion of left circumflex coronary artery (LCx) generating approximately 10% of injured LV area with minimum hemodynamic impact. We assessed tissue perfusion by real time myocardial perfusion echocardiography (RTMPE) using commercial microbubbles before and following pASCs treatment. Four weeks after the occlusion of the left circumflex artery, we transplanted placebo or pASCs (1, 2 and 4x106 cells/Kg BW) into the myocardium. The highest dose of pASCs increased myocardial vessel number and blood flow in the border (56% and 3.7-fold, respectively) and in the remote area (54% and 3.9-fold, respectively) while the non-perfused scar area decreased (up to 38%). We also found an increase of immature collagen fibers, although the increase in total tissue collagen and types I and III was similar in all groups. Our results provide evidence that pASCs-induced stimulation of tissue perfusion and accumulation of immature collagen fibers attenuates adverse remodeling post-MI beyond the normal beneficial effects associated with ACE inhibition and beta-blockade.

Sodium-glucose cotransporter 2 (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. We tested the hypothesis that the SGLT2 inhibitor empagliflozin inhibits proximal tubule (PT) NHE3 activity and improves renal salt and water handling in nondiabetic rats with HF. Male Wistar rats were subjected to myocardial infarction or sham operation. After four weeks, rats that developed HF and sham rats were treated with empagliflozin (EMPA) or untreated for an additional four weeks. EMPA-treated HF rats displayed lower levels of serum BNP 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 normalized the glomerular filtration rate and proteinuria 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 function and protein and mRNA abundance were upregulated in the PT of HF rats. Collectively, our data show that the prevention of HF progression by empagliflozin is associated with inhibition of PT NHE3 activity and restoration of euvolemia. Moreover, we propose that the dysregulation of PT SGLT2 may be involved in the pathophysiology of nondiabetic HF.

Neonatal cardiomyocytes are instrumental for disease modeling, but the effects of different cell extraction methods on basic cell biological processes remain poorly understood. We assessed the influence of two popular methods to extract rat neonatal cardiomyocytes, Pre-plating (PP), and Percoll (PC) on cell structure, metabolism, and function. Cardiomyocytes obtained from PP showed higher gene expression for troponins, titin, and potassium and sodium channels compared to PC. Also, PP cells displayed higher levels of troponin I protein. Cells obtained from PC displayed higher lactate dehydrogenase activity and lactate production than PP cells, indicating higher anaerobic metabolism after 8 days of culture. In contrast, reactive oxygen species levels were higher in PP cells as indicated by ethidium and hydroxyethidium production. Consistent with these data, protein nitration was higher in PP cells, as well as nitrite accumulation in cell medium. Moreover, PP cells showed higher global intracellular calcium under basal and 1 mM isoprenaline conditions. In a calcium-transient assessment under electrical stimulation (0.5 Hz), PP cells displayed higher calcium amplitude than cardiomyocytes obtained from PC and using a traction force microscope technique we observed that PP cardiomyocytes showed the highest relaxation. Collectively, we demonstrated that extraction methods influence parameters related to cell structure, metabolism, and function. Overall, PP derived cells are more active and mature than PC cells, displaying higher contractile function and generating more reactive oxygen species. On the other hand, PC derived cells display higher anaerobic metabolism, despite comparable high yields from both protocols.