The B1 H + -ATPase ( Atp6v1b1 ) Subunit in Non-Type A Intercalated Cells is Required for Driving Pendrin Activity and the Renal Defense Against Alkalosis.

SIGNIFICANCE STATEMENT: In the kidney, the B1 H + -ATPase subunit is mostly expressed in intercalated cells (IC). Its importance in acid-secreting type A ICs is evident in patients with inborn distal renal tubular acidosis and ATP6V1B1 mutations. However, the protein is also highly expressed in alkali-secreting non-type A ICs where its function is incompletely understood. We demonstrate in Atp6v1b1 knock out mice that the B1 subunit is critical for the renal response to defend against alkalosis during an alkali load or chronic furosemide treatment. These findings highlight the importance of non-type A ICs in maintaining acid-base balance in response to metabolic challenges or commonly used diuretics. BACKGROUND: Non-type A ICs in the collecting duct system express the luminal Cl - /HCO 3- exchanger pendrin and apical and/or basolateral H + -ATPases containing the B1 subunit isoform. Non-type A ICs excrete bicarbonate during metabolic alkalosis. Mutations in the B1 subunit (ATP6V1B1) cause distal renal tubular acidosis due to its role in acid secretory type A ICs. The function of B1 in non-type A ICs has remained elusive. METHODS: We examined the responses of Atp6v1b1-/- and Atp6v1b1+/+ mice to an alkali load and to chronic treatment with furosemide. RESULTS: An alkali load or 1 week of furosemide resulted in a more pronounced hypokalemic alkalosis in male ATP6v1b1-/- versus Atp6v1b1+/+ mice that could not be compensated by respiration. Total pendrin expression and activity in non-type A ICs of ex vivo microperfused cortical collecting ducts were reduced, and ß2 -adrenergic stimulation of pendrin activity was blunted in ATP6v1b1-/- mice. Basolateral H + -ATPase activity was strongly reduced, although the basolateral expression of the B2 isoform was increased. Ligation assays for H + -ATPase subunits indicated impaired assembly of V 0 and V 1 H + -ATPase domains. During chronic furosemide treatment, ATP6v1b1-/- mice also showed polyuria and hyperchloremia versus Atp6v1b1+/+ . The expression of pendrin, the water channel AQP2, and subunits of the epithelial sodium channel ENaC were reduced. CONCLUSIONS: Our data demonstrate a critical role of H + -ATPases in non-type A ICs function protecting against alkalosis and reveal a hitherto unrecognized need of basolateral B1 isoform for a proper H + -ATPase complexes assembly and ability to be stimulated.

Effects of furosemide, acetazolamide and amiloride on renal cortical and medullary tissue oxygenation in non-anaesthetised healthy sheep.

It has been proposed that diuretics can improve renal tissue oxygenation through inhibition of tubular sodium reabsorption and reduced metabolic demand. However, the impact of clinically used diuretic drugs on the renal cortical and medullary microcirculation is unclear. Therefore, we examined the effects of three commonly used diuretics, at clinically relevant doses, on renal cortical and medullary perfusion and oxygenation in non-anaesthetised healthy sheep. Merino ewes received acetazolamide (250 mg; n = 9), furosemide (20 mg; n = 10) or amiloride (10 mg; n = 7) intravenously. Systemic and renal haemodynamics, renal cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ , and renal function were then monitored for up to 8 h post-treatment. The peak diuretic response occurred 2 h (99.4 ± 14.8 mL/h) after acetazolamide, at which stage cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ were not significantly different from their baseline levels. The peak diuretic response to furosemide occurred at 1 h (196.5 ± 12.3 mL/h) post-treatment but there were no significant changes in cortical and medullary tissue oxygenation during this period. However, cortical tissue P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ fell from 40.1 ± 3.8 mmHg at baseline to 17.2 ± 4.4 mmHg at 3 h and to 20.5 ± 5.3 mmHg at 6 h after furosemide administration. Amiloride did not produce a diuretic response and was not associated with significant changes in cortical or medullary tissue oxygenation. In conclusion, clinically relevant doses of diuretic agents did not improve regional renal tissue oxygenation in healthy animals during the 8 h experimentation period. On the contrary, rebound renal cortical hypoxia may develop after dissipation of furosemide-induced diuresis.

Furosemide attenuates tubulointerstitial injury and allows functional testing of porcine kidneys during normothermic machine perfusion.

BACKGROUND: Normothermic machine perfusion (NMP) is a promising pretransplant kidney quality assessment platform, but it remains crucial to increase its diagnostic potential while ensuring minimal additional injury to the already damaged kidney. Interventions that alter tubular transport can influence renal function and injury during perfusion. This study aimed to determine whether furosemide and desmopressin affect renal function and injury during NMP. METHODS: Eighteen porcine kidneys (n = 6 per group) were subjected to 30 min of warm ischemia and 4 h of oxygenated hypothermic perfusion before being subjected to 6 h of NMP. Each organ was randomized to receive no drug, furosemide (750 mg), or desmopressin (16 µg) during NMP. RESULTS: Compared with the other groups, the addition of furosemide resulted in significantly increased urine output, fractional excretion of sodium and potassium, and urea clearance during NMP. Urinary neutrophil gelatinase-associated lipocalin levels decreased significantly with furosemide supplementation compared with the other groups. The addition of desmopressin did not result in any significantly different outcome measurements compared with the control group. CONCLUSIONS: This study showed that the addition of furosemide affected renal function while attenuating tubulointerstitial injury during NMP. Therefore, furosemide supplementation may provide renal protection and serve as a functional test for pretransplant kidney viability assessment during NMP.

Phototoxic Reactions Inducted by Hydrochlorothiazide and Furosemide in Normal Skin Cells-In Vitro Studies on Melanocytes and Fibroblasts.

Hypertension is known to be a multifactorial disease associated with abnormalities in neuroendocrine, metabolic, and hemodynamic systems. Poorly controlled hypertension causes more than one in eight premature deaths worldwide. Hydrochlorothiazide (HCT) and furosemide (FUR), being first-line drugs in the treatment of hypertension, are among others the most frequently prescribed drugs in the world. Currently, many pharmacoepidemiological data associate the use of these diuretics with an increased risk of adverse phototoxic reactions that may induce the development of melanoma and non-melanoma skin cancers. In this study, the cytotoxic and phototoxic potential of HCT and FUR against skin cells varied by melanin pigment content was assessed for the first time. The results showed that both drugs reduced the number of metabolically active normal skin cells in a dose-dependent manner. UVA irradiation significantly increased the cytotoxicity of HCT towards fibroblasts by approximately 40% and melanocytes by almost 20% compared to unirradiated cells. In the case of skin cells exposed to FUR and UVA radiation, an increase in cytotoxicity by approximately 30% for fibroblasts and 10% for melanocytes was observed. Simultaneous exposure of melanocytes and fibroblasts to HCT or FUR and UVAR caused a decrease in cell viability, and number, which was confirmed by microscopic assessment of morphology. The phototoxic effect of HCT and FUR was associated with the disturbance of redox homeostasis confirming the oxidative stress as a mechanism of phototoxic reaction. UVA-irradiated drugs increased the generation of ROS by 10-150%, and oxidized intracellular thiols. A reduction in mitochondrial potential of almost 80% in melanocytes exposed to HCT and UVAR and 60% in fibroblasts was found due to oxidative stress occurrence. In addition, HCT and FUR have been shown to disrupt the cell cycle of normal skin cells. Finally, it can be concluded that HCT is the drug with a stronger phototoxic effect, and fibroblasts turn out to be more sensitive cells to the phototoxic effect of tested drugs.

Combination of furosemide, gold, and dopamine as a potential therapy for breast cancer.

Breast cancer is one of the leading causes of death in women worldwide. Initially, it develops in the epithelium of the ducts or lobules of the breast glandular tissues with limited growth and the potential to metastasize. It is a highly heterogeneous malignancy; however, the common molecular mechanisms could help identify new targeted drugs for treating its subtypes. This study uses computational drug repositioning approaches to explore fresh drug candidates for breast cancer treatment. We also implemented reversal gene expression and gene expression-based signatures to explore novel drug candidates computationally. The drug activity profiles and related gene expression changes were acquired from the DrugBank, PubChem, and LINCS databases, and then in silico drug screening, molecular dynamics (MD) simulation, replica exchange MD simulations, and simulated annealing molecular dynamics (SAMD) simulations were conducted to discover and verify the valid drug candidates. We have found that compounds like furosemide, gold, and dopamine showed significant outcomes. Furthermore, the expression of genes related to breast cancer was observed to be reversed by these shortlisted drugs. Therefore, we postulate that combining furosemide, gold, and dopamine would be a potential combination therapy measurement for breast cancer patients.

Il-6 and UGT1A1 variations may related to furosemide resistance in heart failure patients.

Furosemide is a diuretic and is used for the treatment of patients with heart failure (HF). It has been found that in some HF patients, the drug does not treat patients efficiently. This condition is named as furosemide resistance. In this study, it is aimed to investigate the relationship between UDP-glucuronosyltransferase 1 (UGT1A1) and interleukine-6 (IL-6) variations with furosemide resistance in HF patients. Sixty HF patients using furosemide (patient group) and 30 healthy individuals (control group) were enrolled in this study. Patients were divided into two subgroups as non-responders (furosemide resistant) group (n = 30) and the responders (non-resistant) group (n = 30) according to the presence of furosemide resistance (n = 30). Variations in the first exon of UGT1A1 and rs1800795 and rs1800796 variations in IL-6 were analyzed by direct sequencing and real-time polymerase chain reaction (RT-PCR), respectively. The effects of newly detected mutations on 3-D protein structure were analyzed by in silico analysis. At the end of the study, 11 variations were detected in UGT1A1, of which nine of them are novel and eight of them cause amino acid change. Also, rs1800795 and rs1800796 variations were detected in all the groups. When patient and control groups were compared with each other, rs1800796 mutation in IL-6 was found statistically high in the patient group (p = 0.027). When the three groups were compared with each other, similarly, rs1800796 mutation in IL-6 was found statistically high in the non-responders group (p = 0.043). When allele distributions were compared between the patient and control groups, the C allele of rs1800795 mutation in IL-6 was found statistically high in the patient group (p = 0.032). When allele distributions were compared between the three groups, 55T-insertion in UGT1A1 was found statistically high in the non-responders group (p = 0.017). According to in silico analysis results, two variations were found deleterious and six variations were detected as probably damaging to protein functions. Our study may contribute to the elucidation of pharmacogenetic features (drug response-gene relationship) and the development of individual-specific treatment strategies in HF patients using furosemide.

The loop diuretic torasemide but not azosemide potentiates the anti-seizure and disease-modifying effects of midazolam in a rat model of birth asphyxia.

Loop diuretics such as furosemide and bumetanide, which act by inhibiting the Na-K-2Cl cotransporter NKCC2 at the thick ascending limb of the loop of Henle, have been shown to exert anti-seizure effects. However, the exact mechanism of this effect is not known. For bumetanide, it has been suggested that inhibition of the NKCC isoform NKCC1 in the membrane of brain neurons may be involved; however, NKCC1 is expressed by virtually all cell types in the brain, which makes any specific targeting of neuronal NKCC1 by bumetanide impossible. In addition, bumetanide only poorly penetrates the brain. We have previously shown that loop diuretics azosemide and torasemide also potently inhibit NKCC1. In contrast to bumetanide and furosemide, azosemide and torasemide lack a carboxylic group, which should allow them to better penetrate through biomembranes by passive diffusion. Because of the urgent medical need to develop new treatments for neonatal seizures and their adverse outcome, we evaluated the effects of azosemide and torasemide, administered alone or in combination with phenobarbital or midazolam, in a rat model of birth asphyxia and neonatal seizures. Neither diuretic suppressed the seizures when administered alone but torasemide potentiated the anti-seizure effect of midazolam. Brain levels of torasemide were below those needed to inhibit NKCC1. In addition to suppressing seizures, the combination of torasemide and midazolam, but not midazolam alone, prevented the cognitive impairment of the post-asphyxial rats at 3 months after asphyxia. Furthermore, aberrant mossy fiber sprouting in the hippocampus was more effectively prevented by the combination. We assume that either an effect on NKCC1 at the blood-brain barrier and/or cells in the periphery or the NKCC2-mediated diuretic effect of torasemide are involved in the present findings. Our data suggest that torasemide may be a useful option for improving the treatment of neonatal seizures and their adverse outcome.

Drug Repurposing Approach in Developing New Furosemide Analogs as Antimicrobial Candidates and Anti-PBP: Design, Synthesis, and Molecular Docking.

Multidrug-resistant microorganisms have become a global health problem, prompting research into new antimicrobials. Drug repurposing is a new technique in drug discovery used to improve drug development success. As a well-studied medication with a sulfonamide moiety, furosemide was chosen to study its antimicrobial effect on different microbial strains. In addition, a new family of furosemide analogs was investigated for their antimicrobial efficacy. According to the obtained results, the majority of the examined molecules exhibited potential antimicrobial activity. Compounds 3b and 4a had the best anti-MRSA results, with an MIC = 7.81 µg/mL. They also demonstrated potent anti-gram-negative activity against E. coli (MIC = 1.95 µg/mL and 3.91 µg/mL, respectively). A time-killing kinetics study against E. coli and MRSA showed bactericidal actions of 3b and 4a within 120-150 min. Moreover, an anti-PBP activity and an in vitro cytotoxicity evaluation were performed. Furosemide decreased the PBP2a levels in MRSA by 21.5% compared to the control. However, the furosemide analogs 3b and 4a demonstrated superior anti-PBP activity (55.9 and 57.1 % reduction in the expression of PBP2a, respectively). In addition, compound 4a was nearly nontoxic to normal WI-38 cells (IC50 = 248.60 µg /mL) indicating its high safety profile. Finally, the ability of furosemide and compounds 3b and 4a to bind to the target PBP2a enzyme has also been supported by molecular docking research.

Impact of a combination of pimobendan, furosemide, and enalapril on heart rate variability in naturally occurring, symptomatic, myxomatous mitral valve degeneration dogs.

BACKGROUND: Pimobendan, diuretics, and an angiotensin-converting enzyme inhibitor (ACEi) are widely used for the management of chronic valvular heart disease in dogs; however, the effects of that combination on heart rate variability (HRV) are unknown. The purpose of this study was to assess the HRV of symptomatic myxomatous mitral valve degeneration (MMVD) dogs in response to therapy with a combination of pimobendan, diuretics, and ACEi. RESULTS: MMVD stage C (n = 17) dogs were enrolled and a 1-hour Holter recording together with echocardiography, blood pressure measurement, and blood chemistry profiles were obtained before and 1, 3, and 6 months after oral treatment with pimobendan (0.25 mg/kg), enalapril (0.5 mg/kg), and furosemide (2 mg/kg) twice daily. The results revealed that MMVD stage C dogs at the baseline had lower values of time-domain indices, low frequency (LF), high frequency (HF), and total power, as well as higher value of LF/HF. Triple therapy significantly increases these parameters in MMVD stage C dogs (P < 0.05). A positive moderate correlation was observed between time domain parameters and a left ventricular internal diastole diameter normalized to body weight (P < 0.05). CONCLUSIONS: It can be concluded that MMVD stage C dogs possess low HRV due to either the withdrawal of parasympathetic tone or enhanced sympathetic activation, and a combination therapy was shown to enhance cardiac autonomic modulation inferred from the increased heart rate variability. Therefore, a combination therapy may be useful for restoring normal autonomic nervous system activity in dogs with MMVD stage C.

The diuretic effect of adding aminophylline or theophylline to furosemide in pediatric populations: a systematic review.

The diuretic effect of the combined furosemide and aminophylline/theophylline among pediatric patients remains unclear. The primary aim of this systematic review was to examine the clinical diuretic effects (urine output and fluid balance) of co-administration of furosemide and aminophylline/theophylline as compared to furosemide alone in pediatric population. Ovid MEDLINE, CENTRAL, and EMBASE were searched from its inception until March 2022 for observational studies and randomized controlled trials (RCTs) comparing the administration of furosemide versus furosemide and aminophylline/theophylline in pediatric population. Case reports, case series, commentaries, letters to editors, systematic reviews, and meta-analyses were excluded. Five articles with a total sample population of 187 patients were included in this systematic review. As compared to the furosemide alone, our pooled data demonstrated that co-administration of furosemide and aminophylline/theophylline was associated with higher urine output (mean difference: 2.91 [90% CI 1.54 to 4.27], p < 0.0001, I2 = 90%) and a more negative fluid balance (mean difference - 28.27 [95% CI: - 46.21 to - 10.33], p = 0.002, I2 = 56%) than those who received furosemide alone. CONCLUSION: This is the first paper summarizing the evidence of combined use of furosemide with aminophylline/theophylline in pediatric population. Our systematic review demonstrated that the co-administration of furosemide and aminophylline/theophylline could potentially yield better diuretic effects of urine output and negative fluid balance than furosemide alone in pediatric patients with fluid overload. Given the substantial degree of heterogeneity and low level of evidence, future adequately powered trials are warranted to provide evidence regarding the combined use of aminophylline/theophylline and furosemide as diuretic in the pediatric population. WHAT IS KNOWN: • Fluid overload is associated with poor prognosis for children in the intensive care unit. • The ineffective result of furosemide alone, even at high dose, as diuretic agent for children with diuretic resistant fluid overload in the intensive care unit. WHAT IS NEW: • This is the first systematic review that compares furosemide alone and co-administration of furosemide and aminophylline/theophylline. • This paper showed potential benefit of co-administration of furosemide and aminophylline/theophylline promoting urine output and negative fluid balance compared to furosemide alone.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

NHE3 in the thick ascending limb is required for sustained but not acute furosemide-induced urinary acidification.

Na+/H+ exchanger isoform 3 (NHE3) facilitates Na+ reabsorption and H+ secretion by the kidneys. Despite stronger NHE3 abundance in the thick ascending limb (TAL) compared with the S1 and S2 segments of the proximal tubule, the role of NHE3 in the TAL is poorly understood. To investigate the role of NHE3 in the TAL, we generated and phenotyped TAL-specific NHE3 knockout (NHE3TAL-KO) mice. Compared with control mice, NHE3TAL-KO mice did not show significant differences in body weight, blood pH, or plasma Na+, K+, or Cl- levels. Fluid intake trended to be higher and urine osmolality was significantly lower in NHE3TAL-KO mice. Despite a similar glomerular filtration rate, NHE3TAL-KO mice had a greater urinary K+-to-creatinine ratio. One proposed role of NHE3 relates to furosemide-induced urinary acidification. Acute bolus treatment with furosemide under anesthesia did not result in differences in the dose dependence of urinary flow rate, Cl- excretion, or maximal urinary acidification between genotypes; however, in contrast with control mice, urinary pH returned immediately toward baseline levels in NHE3TAL-KO mice. Chronic furosemide treatment reduced urine osmolality similarly in both genotypes but metabolic alkalosis, hypokalemia, and calciuresis were absent in NHE3TAL-KO mice. Compared with vehicle, chronic furosemide treatment resulted in greater Na+-K+-2Cl- abundance regardless of genotype. Na+-phosphate cotransporter 2a abundance was also greater in furosemide-treated control mice compared with vehicle treatment, an effect that was absent in NHE3TAL-KO mice. In summary, NHE3 in the TAL plays a role in the sustained acidification effect of furosemide. Consistent with this, long-term treatment with furosemide did not result in metabolic alkalosis in NHE3TAL-KO mice.NEW & NOTEWORTHY Na+/H+ exchanger isoform 3 (NHE3) is very abundant in the thick ascending limb (TAL) compared with the proximal tubule. Much has been learned about the role of NHE3 in the proximal tubule; however, the function of NHE3 in the TAL remains elusive. A novel mouse model that lacks NHE3 selectively in the TAL not only shows a phenotype under baseline conditions but also identifies that NHE3 is required for sustained but not acute furosemide-induced urinary acidification.

SGLT2 inhibitor and loop diuretic induce different vasopressin and fluid homeostatic responses in nondiabetic rats.

Loop diuretics are commonly used diuretics in the treatment of fluid retention but induce hypovolemia-related renal dysfunction. Na+-glucose cotransporter 2 (SGLT2) inhibitors induce osmotic diuresis, but body fluid volume is maintained by stimulating vasopressin-induced fluid intake and collecting duct water reabsorption as previously reported in diabetic rats. We aimed to test the hypothesis that unlike SGLT2 inhibitors, loop diuretics lack activation of similar fluid homeostatic mechanisms. Nondiabetic male Sprague-Dawley rats were treated daily by oral gavage with vehicle, the SGLT2 inhibitor ipragliflozin (5 mg/kg), or the loop diuretic furosemide (50 mg/kg) and monitored in metabolic cages for 2 or 7 days. Ipragliflozin and furosemide similarly increased urine volume on day 2. This was associated with increased serum Na+ concentration, urine vasopressin excretion, fluid intake, and solute-free water reabsorption in response to ipragliflozin but not to furosemide. Ipragliflozin maintained fluid balance (fluid intake - urine volume) on day 2 and total body water measured by bioimpedance spectroscopy and serum creatinine on day 7. In comparison, furosemide decreased fluid balance on day 2 and decreased total body water and increased serum creatinine on day 7. Furosemide, but not ipragliflozin, increased plasma renin activity, and systolic blood pressure was similar among the groups. In conclusion, the osmotic diuresis of the SGLT2 inhibitor increased serum Na+ concentration and the vasopressin-related stimulation of fluid intake and renal water retention maintained fluid balance, whereas the loop diuretic did not engage the compensatory vasopressin system. The data suggest differences in vasopressin and fluid homeostatic responses between SGLT2 inhibitors and loop diuretics.NEW & NOTEWORTHY In nondiabetic rats, the Na+-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin increased vasopressin-related stimulation of fluid intake and free water reabsorption and maintained fluid balance and serum creatinine, whereas the loop diuretic furosemide reduced vasopressin and induced a negative fluid balance followed by a subsequent increase in serum creatinine. This study suggests that differences in vasopressin secretion in response to a SGLT2 inhibitor or loop diuretic may contribute to differences in body fluid status and subsequent renal function.

Sodium Glucose Co-Transporter 2 Inhibitors Improve Renal Congestion and Left Ventricular Fibrosis in Rats With Hypertensive Heart Failure.

BACKGROUND: Elevated central venous pressure (CVP) in heart failure causes renal congestion, which deteriorates prognosis. Sodium glucose co-transporter 2 inhibitor (SGLT2-i) improves kidney function and heart failure prognosis; however, it is unknown whether they affect renal congestion. This study investigated the effect of SGLT2-i on the kidney and left ventricle using model rats with hypertensive heart failure.Methods and Results: Eight rats were fed a 0.3% low-salt diet (n=7), and 24 rats were fed an 8% high-salt diet, and they were divided into 3 groups of untreated (n=6), SGLT2-i (canagliflozin; n=6), and loop diuretic (furosemide; n=5) groups after 11 weeks of age. At 18 weeks of age, CVP and renal intramedullary pressure (RMP) were monitored directly by catheterization. We performed contrast-enhanced ultrasonography to evaluate intrarenal perfusion. In all high-salt fed groups, systolic blood pressure was elevated (P=0.287). The left ventricular ejection fraction did not differ among high-salt groups. Although CVP decreased in both the furosemide (P=0.032) and the canagliflozin groups (P=0.030), RMP reduction (P=0.003) and preserved renal medulla perfusion were only observed in the canagliflozin group (P=0.001). Histological analysis showed less cast formation in the intrarenal tubule (P=0.032), left ventricle fibrosis (P<0.001), and myocyte thickness (P<0.001) in the canagliflozin group than in the control group. CONCLUSIONS: These results suggest that SGLT2-i causes renal decongestion and prevents left ventricular hypertrophy, fibrosis, and dysfunction.

Natural phenylethanoid glycosides diuretics derived from Lagopsis supina: Biological activity, mechanism, molecular docking, and structure-activity relationship.

Aquaporins (AQPs) and vasopressin type 2 receptor (V2R) play a crucial role in urine excretion and are widely used to explore novel diuretics. In this study, three phenylpropanoids including stachysoside A (L1), acteoside (L2), and glucopyranosyl (1 â†’ 6) martynoside (L3) were isolated from Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. Their diuretic activity, mechanism, molecular docking, and structure-activity relationships were explored. The results suggest that L1, L2, and L3 exhibit acute (6 h) and prolonged (6 d) activities including increased urinary excretion volume, diuretic action, and diuretic activity, without affecting the urinary pH and minor altering the electrolyte balance in saline-loaded rats. Further, L1, L2, and L3 significantly reduced the levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), and aldosterone (ALD), AQPs 1-4 and 7, and V2R, and remarkably elevated the atriopeptin (ANP) level. Besides, L1, L2, and L3 obviously suppressed mRNA and protein levels of AQPs 1-4 and 7, and V2R. The hypothetical binding modes of L1, L2, and L3 with these proteins were determined by molecular docking, and a tight structure-activity relationship was also proposed. Collectively, L1, L2, and L3 represent three natively novel phenylethanoid glycoside diuretics, which inhibit AQP and V2R-mediated molecular mechanisms. They are superior to furosemide as long-term diuretics.

Effects of Combined Gentamicin and Furosemide Treatment on Cochlear Macrophages.

Combining aminoglycosides and loop diuretics often serves as an effective ototoxic approach to deafen experimental animals. The treatment results in rapid hair cell loss with extended macrophage presence in the cochlea, creating a sterile inflammatory environment. Although the early recruitment of macrophages is typically neuroprotective, the delay in the resolution of macrophage activity can be a complication if the damaged cochlea is used as a model to study subsequent therapeutic strategies. Here, we applied a high dose combination of systemic gentamicin and furosemide in C57 BL/6 and CBA/CaJ mice and studied the ototoxic consequences in the cochlea, including hair cell survival, ribbon synaptic integrity, and macrophage activation up to 15-day posttreatment. The activity of macrophages in the basilar membrane was correlated to the severity of cochlear damage, particularly the hair cell damage. Comparatively, C57 BL/6 cochleae were more vulnerable to the ototoxic challenge with escalated macrophage activation. In addition, the ribbon synaptic deterioration was disproportionately limited when compared to the degree of outer hair cell loss in CBA/CaJ mice. The innate and differential otoprotection in CBA/CaJ mice appears to be associated with the rapid activation of cochlear macrophages and a certain level of synaptogenesis after the combined gentamicin and furosemide treatment.

Association of Antihypertensive Effects of Esaxerenone with the Internal Sodium Balance in Dahl Salt-Sensitive Hypertensive Rats.

BACKGROUND: The nonsteroidal mineralocorticoid receptor blocker esaxerenone is effective in reducing blood pressure (BP). OBJECTIVE: In this study, we investigated esaxerenone-driven sodium homeostasis and its association with changes in BP in Dahl salt-sensitive (DSS) hypertensive rats. METHODS: In the different experimental setups, we evaluated BP by a radiotelemetry system, and sodium homeostasis was determined by an approach of sodium intake (food intake) and excretion (urinary excretion) in DSS rats with a low-salt diet (0.3% NaCl), high-salt diet (HSD, 8% NaCl), HSD plus 0.001% esaxerenone (w/w), and HSD plus 0.05% furosemide. RESULTS: HSD-fed DSS rats showed a dramatic increase in BP with a non-dipper pattern, while esaxerenone treatment, but not furosemide, significantly reduced BP with a dipper pattern. The cumulative sodium excretion in the active period was significantly elevated in esaxerenone- and furosemide-treated rats compared with their HSD-fed counterparts. Sodium content in the skin, skinned carcass, and total body tended to be lower in esaxerenone-treated rats than in their HSD-fed counterparts, while these values were unchanged in furosemide-treated rats. Consistently, sodium balance tended to be reduced in esaxerenone-treated rats during the active period. Histological evaluation showed that esaxerenone, but not furosemide, treatment attenuated glomerulosclerosis, tubulointerstitial fibrosis, and urinary protein excretion induced by high salt loading. CONCLUSIONS: Collectively, these findings suggest that an esaxerenone treatment-induced reduction in BP and renoprotection are associated with body sodium homeostasis in salt-loaded DSS rats.

Changes in Proximal Tubular Reabsorption Modulate Microvascular Regulation via the TGF System.

This review paper considers the consequences of modulating tubular reabsorption proximal to the macula densa by sodium-glucose co-transporter 2 (SGLT2) inhibitors, acetazolamide, and furosemide in states of glomerular hyperfiltration. SGLT2 inhibitors improve renal function in early and advanced diabetic nephropathy by decreasing the glomerular filtration rate (GFR), presumably by activating the tubuloglomerular feedback (TGF) mechanism. Central in this paper is that the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy can only be partially explained by TGF activation, and there are alternative explanations. The sustained activation of TGF leans on two prerequisites: no or only partial adaptation should occur in reabsorption proximal to macula densa, and no or only partial adaptation should occur in the TGF response. The main proximal tubular and loop of Henle sodium transporters are sodium-hydrogen exchanger 3 (NHE3), SGLT2, and the Na-K-2Cl co-transporter (NKCC2). SGLT2 inhibitors, acetazolamide, and furosemide are the most important compounds; inhibiting these transporters would decrease sodium reabsorption upstream of the macula densa and increase TGF activity. This could directly or indirectly affect TGF responsiveness, which could oppose sustained TGF activation. Only SGLT2 inhibitors can sustainably activate the TGF as there is only partial compensation in tubular reabsorption and TGF response. SGLT2 inhibitors have been shown to preserve GFR in both early and advanced diabetic nephropathy. Other than for early diabetic nephropathy, a solid physiological basis for these effects in advanced nephropathy is lacking. In addition, TGF has hardly been studied in humans, and therefore this role of TGF remains elusive. This review also considers alternative explanations for the renoprotective effects of SGLT2 inhibitors in diabetic patients such as the enhancement of microvascular network function. Furthermore, combination use of SGLT2 inhibitors and angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs). in diabetes can decrease inflammatory pathways, improve renal oxygenation, and delay the progression of diabetic nephropathy.

Impact of Albumin Binding Function on Pharmacokinetics and Pharmacodynamics of Furosemide.

Background and Objectives: Albumin binding of the loop diuretic furosemide forms the basis for its transport to the kidney and subsequent tubular secretion, which is a prerequisite for its therapeutic effects. Accordingly, high albumin concentrations should result in higher efficacy of furosemide. However, study results on the combination of furosemide in conjunction with albumin, and on the efficacy of furosemide in hypoalbuminemia, did not confirm this hypothesis. The aim of this study was to determine the efficacy of furosemide not only in relation to albumin concentration, but also taking albumin function into account. Materials and Methods: In a prospective and non-interventional clinical observational trial, blood and urine samples from 50 intensive care patients receiving continuous intravenous furosemide therapy were evaluated. Albumin binding capacity (ABiC) determination allowed conclusions to be drawn about the binding site-specific loading state of albumin, by quantifying the unbound fraction of the fluorescent marker dansylsarcosine. In addition, assessment of the total concentration of furosemide in plasma and urine, as well as the concentration of free furosemide fraction in plasma, was performed by HPLC−MS. The efficacy of furosemide was evaluated by the ratio of urine excretion to fluid intake. Results: In patients with an ABiC ≥ 60% free furosemide fraction was significantly lower compared to patients with a lower ABiC (p < 0.001), urinary furosemide concentration was higher (p = 0.136), and a significantly higher proportion of infused furosemide was excreted renally (p = 0.010). ABiC was positively correlated (r = 0.908, p = 0.017) with increase in the urine excretion to fluid input ratio after initiation of furosemide therapy. Conclusions: ABiC could serve as a marker for individual response to furosemide and could be used to generate patient-specific therapeutic regimens. In view of the relatively low number of patients in this study, the relationship between furosemide efficacy and albumin function should be investigated in larger studies in the future.

[Evaluation of the results of sodium fumarate, furosemide, and mannitol on the initiation and outcome of renal warm ischemia in an experimental study].

While performing surgical treatment of the localized form of renal cell cancer by means of open or laparoscopic partial nephrectomy, renal warm ischemia is an important issue. Using renal warm ischemia allows to prevent parenchymal bleeding, to optimize conditions for resection of the tumor and to increase significantly the efficiency of hemostasis. However, an important problem is the probability of ischemic hypoxic damage of the remaining part of the kidney tissue during renal warm ischemia and renal functional impairment in the postoperative period. AIM: To compare nephroprotective activity of sodium fumarate, mannitol and furosemide using experimental model of 30- and 60-minute renal warm ischemia in rabbits. MATERIALS AND METHODS: The experiments were carried out on 360 conventional male-rabbits of the "Chinchilla" breed weighed 2,6+/-0,3 kg which were allocated into 10 groups. The control group No1 included intact animals, the control group No2 included the rabbits in which renal artery was not clamped. For the animals from the trial groups (No3-No10) the experimental model of 30- and 60-minute renal warm ischemia was used. In groups No3 and No4 no drugs were provided. Other rabbits undergone renal warm ischemia with a protection by sodium fumarate (groups No5 and No6 - 1,5 ml/kg IV), lasix (groups No7 and No8 - 3,0 mg/kg IV) and mannitol (No9 and No10 - 1,0 g/kg IV). The influence of renal warm ischemia on the renal tissue ultrastructure and the levels of NGAL, Cystatin-C and creatinine in blood and urine were studied. RESULTS: During experimental pharmacologically uncorrected 30-minute renal warm ischemia in animals, edema of the terminal part of microvilli of the proximal tubules epithelium, an increase of lysosome number in the hyaloplasm of epithelial cells, appearance of flaky content of medium electronic density in the lumens of distal tubules and collecting tubules, as well as sharp peak-like increase of NGAL and cystatin-C in blood and urine were observed. Increasing the time of ischemia up to 60 minutes was accompanied by more severe disturbances. In groups where sodium fumarate, lasix and mannitol were used the observed ultrastructural disturbances were expressed to lesser extent, whereas sodium fumarate demonstrated the best nephroprotective activity. After using mannitol the severity of disturbances was less than in the groups where mannitol, lasix or sodium fumarate were not given. Lasix and sodium salt of fumaric acid showed a higher nephroprotective activity. The best results were received in the animals received sodium fumarate. CONCLUSIONS: The studied drugs provided a nephroprotective effect regarding ischemia of rabbit kidney. The effect of sodium fumarate was the most pronounced, followed by furosemide and, to a lesser extent, mannitol. Use of sodium fumarate allows to protect and stimulate the kidney tissue effectively during oxygen deprivation under ischemic state.