Impact of tadalafil on cardiovascular and organ dysfunction induced by experimental sepsis

Marcelo Almeida Nakashima; Gabrielle Delfrate; Lucas Braga Albino; Gustavo Ferreira Alves; Junior Garcia Oliveira; Daniel Fernandes

doi:10.4266/acc.002904

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Original Article Basic science and research Impact of tadalafil on cardiovascular and organ dysfunction induced by experimental sepsis: Marcelo Almeida Nakashima^*, Gabrielle Delfrate^*, Lucas Braga Albino, Gustavo Ferreira Alves, Junior Garcia Oliveira, Daniel Fernandes; Acute and Critical Care 2025;40(1):46-58.
DOI: https://doi.org/10.4266/acc.002904
Published online: February 12, 2025

Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, Brazil

Corresponding author: Daniel Fernandes Department of Pharmacology, Universidade Federal de Santa Catarina, Block D/CCB, Campus Trindade, Florianopolis - SC - 88049-900, Brazil Tel: +55-48-3721-7142, Email: fernandes.d@ufsc.br

*These authors contributed equally to this work as co-first authors.

• Received: June 18, 2024 • Revised: November 1, 2024 • Accepted: November 2, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
KEY MESSAGES
NOTES
REFERENCES

Abstract

Background
Sepsis is a life-threatening condition that affects the cardiovascular and renal systems. Severe hypotension during sepsis compromises tissue perfusion, which can lead to multiple organ dysfunction and death. Phosphodiesterase 5 (PDE5) degrades intracellular cyclic guanosine monophosphate (cGMP) levels which promotes vasodilatation in specific sites. Our previous studies show that inhibiting cGMP production in early sepsis increases mortality, implying a protective role for cGMP production. Then, we hypothesized that cGMP increased by tadalafil (PDE5 inhibitor) could improve microcirculation and prevent sepsis-induced organ dysfunction.
Methods
Rats were submitted to cecal ligation and puncture (CLP) sepsis model and treated with tadalafil (2 mg/kg, s.c.) 8 hours after the procedure. Hemodynamic, inflammatory and biochemical assessments were performed 24 hours after sepsis induction. Moreover, the effect of tadalafil on the survival of septic rats was evaluated for 5 days.
Results
Tadalafil treatment improves basal renal blood flow during sepsis and preserves it during noradrenaline infusion. Sepsis induces hypotension, impaired response to noradrenaline, and increased cardiac and renal neutrophil infiltration, in addition to increased levels of plasma nitric oxide and lactate. None of these dysfunctions were changed by tadalafil. Additionally, tadalafil treatment did not increase the survival rate of septic rats.
Conclusions
Tadalafil improved microcirculation of septic animals; however, no beneficial effects were observed on macrocirculation and inflammation parameters. Then, the potential benefit of tadalafil in the prognosis of sepsis should be evaluated within a therapeutic strategy covering all sepsis injury mechanisms.
Key Words: cecal ligation and puncture; multiple organ failure; phosphodiesterase 5; phosphodiesterase inhibitors; renal blood flow; septic shock

INTRODUCTION

Sepsis is characterized as an organ dysfunction due to a dysregulated host response to an infection [1]. It can progress to septic shock, a state of acute circulatory failure. These conditions of high global incidence are potentially fatal. It is estimated that one in four patients diagnosed with sepsis does not survive their hospitalization [2]. Despite the severity of these conditions, there are still no specific therapies for sepsis and septic shock.

Severe and persistent hypotension has a pivotal role in sepsis injury mechanism, and much of this condition is caused by the up-regulation of the enzyme inducible nitric oxide synthase (NOS-2) rising nitric oxide (NO) levels [3]. At physiological levels, NO promotes vasodilatation via soluble guanylate cyclase activation and cyclic guanosine monophosphate (cGMP) production and has an important role in controlling vascular tone. However, the exacerbated release of NO via NOS-2 during sepsis contributes to the sustained drop in blood pressure and, thus decreased perfusion and organ dysfunction [4-6].

On the other hand, previous studies of our group have demonstrated that the mortality rate rises when inhibiting the soluble guanylate cyclase enzyme within the initial hours following the induction of experimental sepsis [7], suggesting that cGMP production may have a protective role in the early stages of sepsis. Moreover, increasing cGMP levels through the inhibition of phosphodiesterase 5 (PDE5) by sildenafil reduces lactate levels and improves renal blood flow (RBF) in septic rats [8]. Thus, increasing cGMP levels via PDE5 inhibitors during early stages of sepsis was suggested to improve tissue oxygenation and prevent ischemic damage.

Among the PDE5 inhibitors available in clinical practice, tadalafil has a characteristic chemical scaffold and the longest half-life (about 17.5 hours) [9]. Although sildenafil pioneered the field of treating erectile dysfunction as the first PDE5 inhibitor, tadalafil is opening new avenues by potentially offering perspectives for treating other diseases. Tadalafil is already approved for erectile dysfunction and pulmonary hypertension treatment and has been clinically tested for others cardiovascular disorders, such as heart failure [10,11]. In addition, it has been shown that tadalafil has renoprotective effects in animal models [12,13]. However, the effect of tadalafil in the sepsis scenario remains not elucidated. Then, considering that their pharmacokinetic and pharmacodynamics may provide benefits during sepsis, this study aimed to evaluate the effect of increasing cGMP levels via tadalafil on cardiovascular, inflammatory, and organ dysfunctions induced by sepsis.

MATERIALS AND METHODS

This study was approved by Ethics Committee on Animal Use of the Federal University of Santa Catarina (CEUA/UFSC) for animal use in research (protocol 6005071118) and are in accordance with the Brazilian government guidelines for animal use in research (CONCEA) and the National Institutes of Health guide for the care and use of laboratory animals (NIH).

Animals

Male Wistar rats (90 days of age; 250–350 g) were supplied by the animal facility of the university. Animals were kept in vertical ventilated racks (Tecniplast; five animals per cage, wood shavings bedding) with free access to water and commercial rodent chow (Puro Trato) in a temperature/light-controlled room (22 ± 2 ºC; 12-hour light/dark cycle, lights on at 7 am).

Cecal Ligation and Puncture

Sepsis was induced by cecal ligation and puncture (CLP) as previously described, with minor modifications [14]. Analgesia was induced with tramadol hydrochloride (10 mg/kg; i.p.). Thus, animals were anesthetized with ketamine-xylazine (90 mg/kg and 10 mg/kg, i.p., respectively) and accommodated in a heating pad (37 °C). The abdomen was shaved, decontaminated with iodized alcohol (0.1% iodine; 50% ethyl alcohol, v/v), and the peritoneum was opened. Cecum was exposed and partially occluded with cotton silk near the ileocecal valve. A transfixing puncture was made (22-G needle), squeezing approximately 5 mm of intestinal contents in the peritoneal cavity. The abdominal incision was then closed with a silk-needled suture thread (Lamedid). Sham-operated group was submitted to laparotomy without CLP. Immediately after, all animals received volume replacement with saline solution (50 mL/kg, 37 °C, s.c.), and 12 hour-after surgery, tramadol (5 mg/kg; i.p.) was re-administered.

Noninvasive Systolic Arterial Pressure Measurement

Systolic arterial pressure (SAP) was measured in awake rats using a specialized tail cuff and pulse transducer to intermittently measure blood pressure based on the periodic occlusion of tail blood flow. In a 30 °C room, animals were immobilized in containment tubes. The tails were heated by a blanket for 5 minutes, and the cuff was placed with a pulse transducer on the animals’ tails. Animals underwent 3 days adaptation before the experiment in which SAP was registered. Signal was processed by a data acquisition system (PowerLab, ADInstruments) and registered by a LabChart 7 Pro integration software (ADInstruments). SAP data were expressed in mm Hg.

Invasive Hemodynamical Measurement

Rats were anesthetized by intramuscular injection of ketamine and xylazine (90 and 15 mg/kg, respectively) and were placed in a heating pad (37 °C). A heparinized polyethylene catheter (PE-50) was inserted into the left carotid artery and connected to a pressure transducer coupled to a Powerlab 8/30 (ADInstruments Pty Ltd.). MAP and HR were registered by a LabChart 7 Pro integration software (ADInstruments). A PE-20 catheter (saline-heparin 50 UI/mL) was inserted in the jugular vein for drug administration. Simultaneously, a probe (model VP1T) was positioned on the surface of the left kidney to record the RBF by a Laser Doppler monitor (VMS-LDF2, Moor Instruments) coupled to PowerLab system (ADInstruments).

Experimental Protocols

Tadalafil has been administered orally in animal models in a range of 2 to 10 mg/kg [15-18], which corresponds to approximately 20 to 100 mg in a 60 kg adult and approaches clinical dosage [19]. Considering that sepsis most occurs in hospitalized patients; the oral route may be impractical. Therefore, intravenous (i.v.), intraperitoneal (i.p.), or subcutaneous (s.c.) routes offer a more feasible and clinically relevant option. The i.p. route is usually avoided in the CLP model, as infection and associated edema at the peritoneum can alter drug absorption. The i.v. route is more invasive and difficult to perform in animals that are already weakened due to the sepsis procedure. Then, the s.c. route is a more appropriate option for drug administration. Given that the bioavailability of tadalafil is higher via the subcutaneous route compared to the oral route, where only 36% is absorbed [20], we selected a dose of 2 mg/kg s.c. as well as higher doses could excessively increase the absorption and effect of the drug, increasing the risk of hypotension.

Initially, we evaluated the effect of tadalafil on cardiovascular parameters of naïve rats. Six animals without any intervention were treated with tadalafil (2 mg/kg, s.c.) diluted in DMSO (0.2 mL/kg, s.c.) and monitored over time. SAP was registered at 1, 8, 12, and 24 hours post-treatment in the same awake animals. Additionally, 30 rats treated with tadalafil (2 mg/kg, s.c.) were anesthetized for invasive measurement of mean arterial pressure at 1, 2, 6, 8 or 16 hours after treatment (n=6). Nine rats treated with vehicle (DMSO, 0.2 mL/kg, s.c.) were used as control. DMSO is used as a drug solvent, widely administered in animal models. Despite the fact that we used a secure dose, control animals received this vehicle in order to eliminate any differences between groups caused by DMSO administration itself. MAP and the response duration of the hypotensive response to sodium nitroprusside administration (0.1; 0.3; 1; 3; 10; 30; 100 and 300 nmol/kg) were recorded (Figure 1A).

In another set of experiments, rats were submitted to CLP (n=28) or sham procedure (n=12). Eight hours after CLP, animals were randomly divided to receive either tadalafil (2 mg/kg, s.c.) or vehicle (DMSO 0.2 mL/kg, s.c.). Sham animals received vehicle (DMSO 0.2 mL/kg, s.c.) and were used as control. Twenty-four hours after surgery, animals were anesthetized for invasive measurement of mean arterial pressure MAP (in mm Hg), HR (in bpm) and RBF (in perfusion units, PU) recorded after 10 minutes of stabilization. Noradrenaline was intravenously administered by using an infusion pump (0.03 µmol/kg/minutes, Bonther), and the response to noradrenaline on these parameters was recorded over 30 minutes of infusion. Blood and tissues samples were collected for subsequent analyzes (Figure 1B).

In the last set of experiments, 40 animals were submitted to CLP procedure. Animals received a single dose of 24,000 UI/kg, i.m. of a long-lasting antibiotic (70% benzylpenicillin+30% streptomycin) 6 hours after CLP. After 8 hours, animals were randomized to receive tadalafil (2 mg/kg, s.c.) or vehicle (DMSO 0.2 mL/kg, s.c.). One animal died before treatment and were excluded from analysis. Survival was monitored by 5 days (Figure 1C).

Myeloperoxidase

Neutrophils recruitment in cardiac tissue was determined indirectly by myeloperoxidase (MPO) enzyme activity quantification. Heart (left ventricle) and kidney (cortex) tissue were homogenized in 1 mL of sodium phosphate buffer solution (20 mM, pH 7.4) and centrifuged at 13,000 g for 10 minutes. Pellets were resuspended in 1 mL of sodium phosphate buffer (50 mM, pH 6.0) with 0.5% (w/v) hexadecyltrimethylammonium bromide detergent. After centrifugation (13,000 ×g, 10 minutes), 30 µL of the supernatant was incubated (10 minutes, 37 °C) with tetramethylbenzidine (1.6 mM) and H2O2 (0.3 mM, in 80 mM sodium phosphate buffer, pH 5.4) in a final volume of 230 µL. Absorbance was measured in a plate reader (Infinite M200) at 650 nm every 2 minutes. Protein total was estimated by the bicinchoninic acid method. MPO activity was expressed as optical density O.D./mg of protein.

Nitrate and Nitrite

Plasma NO levels were estimated from the quantification of nitrate and nitrite (NOx) metabolites. Briefly, zinc sulfate-deproteinized plasma was incubated with Escherichia coli (37 °C, 3 hours) to nitrate-nitrite conversion. Samples were centrifuged (2,250 g, 15 minutes) to remove bacteria, and the enzymatic conversion reaction was revealed by adding 100 μL of Griess reagent (1% sulfanilamide in 10% phosphoric acid and 0.1% alpha-naphthyl ethylenediamine in Milli-Q ultrapure water) to the supernatant. Absorbance was determined in a plate reader (Infinite M200, Tecan) at 540 nm. Values of µM of NOx were obtained after linear regression.

Plasma Biomarkers

Plasma levels of creatinine (mg/dl), urea (blood urea nitrogen, mg/dl), aspartate aminotransferase (AST, U/L) and alanine aminotransferase (ALT, U/L) were measured in heparin collected blood. Lactate level (mmol/L) was measured in plasma from ethylenediaminetetraacetic acid/sodium fluoride collected blood. All the measurements were performed by commercial Labtest kits (LabTest Diagnóstica S.A.) according to the manufacturer’s instructions.

Survival Rate

In order to explore the effect of tadalafil on survival rate, animals were submitted to sepsis by CLP procedure as previously described. Six hours after the surgery to induce sepsis, both groups received a single dose of a long-lasting antibiotic composed by 70% of benzylpenicillin (benzathine, procaine and potassium) and 30% of streptomycin and dihydrostreptomycin (sulfate; 24,000 UI/kg, i.m., Zoetis). Eight hours after the surgery, animals were randomized into two treatment groups: CLP+vehicle (0.2 ml/kg, s.c.) and CLP+tadalafil (2 mg/kg, s.c.). Survival rate was followed up every 12 hours over 5 days.

Sample Size

The sample calculation of in vivo analysis was based on the standard deviation (SD) and the magnitude of difference between sham, CLP and CLP+4-aminopyridine groups obtained in the analysis of mean arterial pressure (mm Hg, primary outcome) from our previous studies [21]. The expected difference in mean arterial pressure was 17±8 mm Hg between sham and CLP groups. Thus, considering three experimental groups, α=0.05 and a power of 80%, six animals in each group are required for statistical significance. However, CLP were performed on at least 14 animals per group due to the 24-hour mortality rate associated with sepsis and the loss of animals during anesthesia for invasive experimentation. We also used 12 animals in the control group to ensure at least 6 valid records during in vivo measurement. We consider valid records those in which the animal did not present significant blood loss during surgery and a stable blood pressure record. Animals that died during surgery had also blood and tissue samples collected for analysis, then analyses performed with blood and tissue samples were performed based on sample availability. The G*Power 3.1.1 software (Heinrich-Heine-Universität Düsseldorf) was used for sample size calculation [22].

In the survival analysis, a sample size of 20 animals per group was calculated based on an expected mortality reduction rate of 45%, with a power of 80% and α=0.05. The Primer of Statistics version 7 software was used for sample size calculation of survival experiments. One animal from the CLP tadalafil group died before treatment and was excluded from the analysis. The total number used in each analysis was shown in graphs and figures captions.

Statistical Analysis

Data were expressed as mean±SD of the number of animals used in each experiment. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was assessed using the Bartlett test. The heart MPO, NOx, and lactate data underwent a logarithmic transformation to ensure normality and homogeneity of variance. Subsequently, they were analyzed using one-way analysis of variance (ANOVA), followed by the Tukey post-test. The analysis of SAP through the tail-cuff procedure was performed using repeated measures one-way ANOVA and Dunns’ post hoc. Response duration of hypotensive response to sodium nitroprusside and mean arterial pressure, heart rate, and RBF response over the time after norepinephrine infusion were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s test. The survival rate of groups was analyzed by a Kaplan-Meier curve. Statistical analyses were performed using the GraphPad Prism 9 software (GraphPad Software). A P-value less than 0.05 was considered significant.

RESULTS

Tadalafil Does Not Induce Hypotension to Healthy Animals But Potentiates Nitric Oxide Response

Systolic arterial blood pressure remained unaffected over time by a single dose of tadalafil in awake animals (Figure 2A). Moreover, the mean arterial pressure, measured through an invasive procedure in anesthetized animals, showed no discernible change in response to tadalafil (Figure 2B). However, there was a potentiation in the response to higher doses of sodium nitroprusside (100 and 300 nmol/Kg), a NO donor, in 8 hours and 16 hours after a single dose of tadalafil (Figure 2C).

Tadalafil Protects against Sepsis-Induced Renal Poor Perfusion without Any Effect on Mean Arterial Pressure or Heart Rate

Septic animals exhibited hypotension 24 hours after sepsis induction, with no differences between vehicle or tadalafil administrations to CLP groups (Figure 3A). The infusion of noradrenaline induced a rapid elevation in blood pressure, reaching its peak within 2 minutes and subsequently stabilizing around 10 minutes following the initiation of the infusion (Figure 3B). Nevertheless, blood pressure levels consistently remained lower in the septic groups compared to control group. Mainly, after the first 10 minutes post-infusion, the blood pressure values from the septic group fell down below the baseline of the control group (Figure 3B). There were no discernible differences in baseline heart rates among the groups, and following noradrenaline infusion, the heart rates of all groups increased uniformly and comparably (Figure 3C). After 30 minutes, there was a 78%, 70% and 69% increase in heart rate in sham, CLP, and CLP-tadalafil, respectively (Figure 3D). Baseline RBF was decreased in CLP vehicle and CLP tadalafil, but there was an increase in CLP tadalafil compared to CLP vehicle (Figure 3E). Additionally, tadalafil administration prevented decreasing RBF after NOR infusion compared to the CLP Vehicle group (Figure 3F).

Tadalafil Failed to Reduce Inflammatory Parameters during Experimental Sepsis

MPO activity increased in cardiac and renal tissues at 24 hours after CLP procedure, indicating neutrophil infiltration (Figure 4A and B, respectively). However, those increases were not changed by tadalafil treatment. Concomitantly, plasma levels of NOx increased in septic rats compared to control. Administration of tadalafil attenuated 32% NOx levels compared to the vehicle-treated septic group (CLP vehicle, 96.9±48.5 vs. CLP tadalafil, 65.8±28.8 µM), but with no significant difference (Figure 4C).

Tadalafil Reduces Sepsis-Induced Biochemical Markers of Organ Dysfunction

Concerning plasma biomarkers of renal dysfunction, experimental sepsis resulted in a slight increase in both creatinine and urea levels, albeit without reaching statistical significance. (Figure 5A). Among CLP-induced septic rats, tadalafil treatment reduces urea levels compared to septic rats treated with vehicle (Figure 5B). There was a significant increase in AST levels in CLP vehicle compared to control, but not in CLP tadalafil group (Figure 5C). ALT levels were reduced in CLP tadalafil group compared to sham and CLP vehicle (Figure 5D). In addition, lactate levels showed similarly increased in both septic groups (Figure 5E).

The Role of Tadalafil in the Survival Rate of Sepsis

Within 5 days of follow-up, the survival rate of septic rats treated with a single dose of vehicle was 50% (10/20), a percentage compatible with a mid-grade cecal ligation of CLP model as described by Rittirsch et al. [23]. On the other hand, septic rats treated with tadalafil showed lightly higher survival rate, about 68.4% (13/19) (Figure 6). However, the comparison between the curves by log-rank test showed no significant difference (P=0.26).

DISCUSSION

NO plays a pivotal role in sepsis. NO overproduction in response to infection is a substantial contributor to severe hypotension and vasoplegia [6]. However, global inhibition of NO synthesis was associated with increased mortality in septic patients [24]. Although large amounts of NO generated by inducible NOS-2 have known deleterious effects in sepsis, endothelial NOS-3 has an important role in maintaining microcirculation perfusion [25,26]. This embraces the dual role of NO in sepsis. Indeed, NOS-3 is uncoupled in sepsis, decreasing the bioavailability of NO to the microcirculation [27]. In addition, there is a large increase in the catecholamines release in response to hypotension during sepsis onset, which can induce vasoconstriction in tissues and ischemic injury [28]. Then, increasing NO signaling at specific times and locations could improve microperfusion and prevent multiple organ failure in sepsis.

Therefore, several preclinical studies show improved microcirculatory blood flow in animals with sepsis treated with an NO donor [29]. However, the volatile property of NO and the associated risk of hypotension present significant obstacles to the effective therapeutic utilization of NO donors in sepsis [30]. In contrast to the administration of NO donors [31], tadalafil did not reduce blood pressure in healthy animals nor exacerbate hypotension in septic animals. Differently to NO which acts indistinctly on the smooth muscle [32], PDE5 inhibitors act in specific regulatory domains where PDE5 is expressed, such as corpus cavernosum, platelets, kidneys, heart, lungs, pancreas, and smooth muscle [9,11] which may be associated with a lower systemic vasodilatory effect.

It is particularly important to note that tadalafil potentiates the response of sodium nitroprusside, a NO donor, at 8 and 16 hours after its administration in healthy animals, consistent with the prolonged half-life of the drug [9]. These data demonstrate that the tadalafil effect at the chosen dose effectively persisted throughout the entirety of the experiment. Therefore, inhibition of PDE5 appears to be more viable for prolonging NO signaling in specific sites than NO itself.

Then, we further investigated tadalafil effects in sepsis, where there is an exacerbated vasodilation in the peripheral beds along with vasoconstriction in the renal microvasculature, reducing RBF. In this context, the renal vascular bed emerges as a potential target for cGMP-mediated vasodilation. Moreover, the expression and activity of PDE5 may diverge accordingly in the vascular bed [33]. Interestingly, PDE5 plays a pivotal role in kidney vascular function as demonstrated by several studies in which tadalafil preserves RBF during acute kidney injury [34]. Our findings demonstrate that, with the appropriate dosing, it is possible to increase RBF without causing a reduction in blood pressure. Similar results were already observed by our research group in which increased serum levels of cGMP was associated with an increase in the RBF of septic animals treated with sildenafil [8]. Additionally, Pofi et al. [35] showed that the mechanism involved in the renal function protection exerted by the PDE5 inhibitor (sildenafil) during diabetic kidney disease was through modulation of intraglomerular hemodynamics and not secondary to a reduction in systemic hypertension.

Next, we evaluated whether tadalafil improved the sepsis-induced impaired vasoconstrictor response. Noradrenaline is used in clinical practice in order to increase blood pressure, avoiding multiple organ failure [36]. However, most septic patients show impaired response to vasoconstrictor, which is associated with a higher mortality rate [37,38]. Although the vasopressor response to noradrenaline in mean arterial pressure was not improved by tadalafil, RBF was preserved over the vasoconstrictor infusion. Some studies have raised concerns regarding the utilization of vasoconstrictors in sepsis [39,40]. One noteworthy consideration is their potential to diminish renal perfusion. Therefore, our data now suggest a protective effect of tadalafil in renal microcirculation during noradrenaline infusion [41]. This has clinical relevance as well as kidney injury is associated with a worse prognosis in sepsis.

In addition, we showed that sepsis has induced an increase in the plasma NOx levels, corroborating several studies that identify the increased NO production in the experimental sepsis model [8,42]. Notable, tadalafil treatment induces a slight decrease in NOx values of septic animals (without statistical significance). This may result from reduced NO production or, more likely, from increased clearance of its metabolites by the renal system. Additional experiments, such as evaluating NOS-2 protein expression by Western Blotting, would be valid to verify whether the inhibition of PDE5 affects the NO production by this enzyme.

Along with its role in regulating vascular tone, cGMP exhibits anti-inflammatory properties [43]. Furthermore, tadalafil has been demonstrated to effectively reduce inflammation [44,45]. In this work, the inhibition of PDE5 with tadalafil did not reduce neutrophil migration to the heart and kidney during sepsis. However, we did not investigate other inflammatory markers. Benli et al. [46] administered 5 and 10 mg/kg of tadalafil via gavage immediately after the CLP procedure and the kidney was harvested after 16 hours. Tadalafil showed reduced histopathological inflammation scores in the kidney as well as decreased tubular dilatation, vacuolization and epithelial flattening in septic animals. We did not perform histological analysis to confirm these results, which can be considered a limitation of this study. Therefore, the role of the anti-inflammatory effects of tadalafil in sepsis, especially in kidney tissue, is a topic that needs to be further explored in subsequent studies.

Renal dysfunction is a common find in sepsis clinical practice [47]. Despite this, creatinine and urea markers did not significantly increase in septic animals in the determined time point (24 hours). Concerning liver injury markers, sepsis elevated AST but not ALT levels. Tadalafil prevents AST elevation and significantly reduces ALT levels. However, sepsis has induced an increase in lactate, and tadalafil could not change it. This anaerobic respiration marker suggests hypoperfusion, which can lead to organic dysfunction. Therefore, other studies with longer experimental times (48 or 72 hours) may be interesting in evaluating the effect of tadalafil on organic protection during sepsis.

Although tadalafil preserves the RBF and early liver injury, administering a single dose of tadalafil failed to increase survival. The data highlights the intricate nature of sepsis, unveiling its multifaceted complexity. It becomes evident that improving a few specific parameters may not be enough to generate substantial benefits in the most critical outcome, such as mortality. There are no studies that used multiple doses of tadalafil in the treatment of sepsis in the CLP model. The potential benefits of tadalafil might be better demonstrated in a continuous treatment and when administered in conjunction with other supportive therapies, such as the concurrent use of vasopressors. However, confirming this would necessitate additional studies.

This study has some limitations. The tadalafil dose used was not calculated using the conventional dose conversion between species [19], as allometric scaling is applicable only for oral administration. Since our study employed subcutaneous administration rather than oral, the interspecies dosage equivalency may not be directly comparable. This distinction should be considered when interpreting the translational relevance of our findings. Moreover, the results cover a specific time course of the disease, as well as sepsis is a progressive condition, different outcomes could be found at other experimental times. Additionally, we chose to administer antibiotics only in survival analysis, however, the treatment of tadalafil and antibiotics together could be interesting on cardiovascular evaluation for mimicking clinical practice. Therefore, the results presented here should be carefully interpreted.

Therefore, we concluded that tadalafil improved RBF and may have improved microcirculation of septic animals, preventing liver injury. However, no beneficial effect was observed on macrocirculation and inflammation parameters. Nevertheless, the possible protective effects of tadalafil on mortality associated with kidney dysfunction in sepsis could be further evaluated.

KEY MESSAGES

▪ Tadalafil reduces hepatic injury markers in septic rats.

▪ Tadalafil preserves renal blood flow during noradrenaline infusion in sepsis.

▪ Tadalafil does not improve hemodynamic and inflammatory parameters, failing to improve survival in sepsis.

NOTES

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

FUNDING

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil; grant No. 405538/2021-9 and No. 403615/2023-2), by Coordenação de Aperfeiçoamento de Pessoal de Nível (CAPES, Brazil), and by Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC; grant No. 2021TR000442). Marcelo Almeida Nakashima, Lucas Braga Albino, and Gabrielle Delfrate were supported by a CNPq fellowship.

ACKNOWLEDGMENTS

The technical assistance of Mrs. Adriane Madeira is acknowledged. We are grateful to the Multiuser Laboratory of Biology Studies at the Universidade Federal de Santa Catarina (LAMEB/UFSC) and to Hospital Universitário Polydoro Ernani São Thiago (HU-UFSC/EBSERH) for providing infrastructure for carrying out some experimental tests. Cristália Pharmaceutical Industries (São Paulo, Brazil) is also gratefully acknowledged for the gift of heparin.

AUTHOR CONTRIBUTIONS

Conceptualization: MAN, DF. Data curation: MAN, GD, LBA, GFA, JGO. Formal analysis: MAN. Funding acquisition: MAN, DF. Methodology: MAN, GD, LBA, GFA, JGO. Project administration: DF. Visualization: DF. Writing – original draft: GD, LBA. Writing – review & editing: GD, LBA, GFA, JGO, DF. All authors read and agreed to the published version of the manuscript.

Figure 1.

Experimental protocols of the study. (A) Characterization of the effect of tadalafil on blood pressure over time in healthy animals. (B) Evaluation of the effect of tadalafil 24 hours after sepsis induction by cecal ligation and puncture. (C) Investigation of the effect of tadalafil on survival of septic animals. See the text for more details.

Figure 2.

Effect of tadalafil in naïve animals. (A) Systolic arterial pressure of naïve animals was measured by tail plethysmography at (0 hour), then after 1 hour, 8 hours, 12 hours, and 24 hours of tadalafil administration (2 mg/kg, s.c.). Statistical analysis was performed by repeated measures one-way analysis of variance (ANOVA) followed by Dunnett post hoc. (B) Baseline mean arterial pressure was measured invasively in anesthetized animals at 1 hour, 2 hours, 4 hours, 8 hours, and 16 hours after tadalafil administration. Statistical analysis was performed by one-way ANOVA followed by Tukey post hoc test. (C) After recording baseline mean arterial pressure in anesthetized animals, increasing non-cumulative doses of sodium nitroprusside (0.1, 0.3, 1, 3, 10, 30, 100, and 300 nmol/kg; i.v.) was injected. The time (in seconds) for mean arterial pressure returning to baseline was recorded. Significance differences were determined by two-way repeated measures ANOVA followed by Bonferroni’s test. a) Represents the difference between vehicle vs. tadalafil 8 hours (P=0.008 and P=0.02, respectively; b) Compares vehicle vs. tadalafil 16 hours (P=0.01). Values are represented as mean±standard deviation of 6–9 animals per group as indicated by dot plots and numbers in the bars.

Figure 3.

Effect of tadalafil on mean arterial pressure (MAP), heart rate (HR) and renal blood flow (RBF) at baseline and after noradrenaline infusion in septic rats. Eight hours after cecal ligation and puncture (CLP) procedure, rats were treated with vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.). Sham group received vehicle at the same time point. Baseline MAP (A), HR (C), and RBF (E) were recorded after 24 hours. Significance differences were determined by one-way analysis of variance (ANOVA) and Tukey post hoc. MAP (B), HR (D), and RBF (F) variation after noradrenaline infusion (0.03 µmol/kg/min) over time. Significance differences were determined by two-way repeated measures ANOVA followed by Bonferroni’s test. a) Represents Sham Vehicle vs. all CLP groups (P<0.05); b) Represents CLP vehicle vs. CLP tadalafil (P=0.006; P=0.002; P=0.033, and P=0.029, respectively); c) Represents sham vehicle vs. CLP vehicle (P=0.037 and P=0.018, respectively). Values are presented as mean±standard deviation of 6–8 animals per group as indicated by dot plots and numbers in the bars.

Figure 4.

Effect of tadalafil on inflammatory markers and nitrate+nitrite production in sepsis. Rats received vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.) administration 8 hours after cecal ligation and puncture (CLP) procedure. Sham group received vehicle at the same time point. Twenty-four hours after sepsis induction, myeloperoxidase (MPO) activity was evaluated in cardiac (A) and kidney tissue (B). Plasma nitrate plus nitrite (NOx) was also measured (C). Significance differences were determined by one-way analysis of variance with Tukey post hoc. Values are presented as mean±standard deviation of 6–8 animals per group as indicated by dot plots and numbers in the bars.

Figure 5.

Effect of tadalafil on plasma biomarkers of tissue damage during sepsis. Eight hours after sepsis induction, animals received vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.). Twenty-four hours after surgery, plasma was obtained to measure creatinine (A), urea (B), aspartate aminotransferase (AST; C), alanine aminotransferase (ALT; D), and lactate (E). Significance differences were determined by one-way analysis of variance with Tukey post hoc. Values are presented as mean±standard deviation of 9–12 animals per group as indicated by dot plots and numbers in the bars. CLP: cecal ligation and puncture.

Figure 6.

Survival rate of sepsis-induced animals by cecal ligation and puncture (CLP) treated or not with tadalafil. Six hours after the CLP procedure, both groups received a broad-spectrum veterinary antibiotic administration (24,000 IU/kg, i.m.). Animals received treatment with a single dose of tadalafil (2 mg/kg, s.c.) or vehicle (DMSO 0,2 mL/kg, s.c.) 8 hours after CLP and were follow-up by 5 days.

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Impact of tadalafil on cardiovascular and organ dysfunction induced by experimental sepsis

Acute Crit Care. 2025;40(1):46-58. Published online February 12, 2025

DOI: https://doi.org/10.4266/acc.002904

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Impact of tadalafil on cardiovascular and organ dysfunction induced by experimental sepsis

Figure 1. Experimental protocols of the study. (A) Characterization of the effect of tadalafil on blood pressure over time in healthy animals. (B) Evaluation of the effect of tadalafil 24 hours after sepsis induction by cecal ligation and puncture. (C) Investigation of the effect of tadalafil on survival of septic animals. See the text for more details.

Figure 2. Effect of tadalafil in naïve animals. (A) Systolic arterial pressure of naïve animals was measured by tail plethysmography at (0 hour), then after 1 hour, 8 hours, 12 hours, and 24 hours of tadalafil administration (2 mg/kg, s.c.). Statistical analysis was performed by repeated measures one-way analysis of variance (ANOVA) followed by Dunnett post hoc. (B) Baseline mean arterial pressure was measured invasively in anesthetized animals at 1 hour, 2 hours, 4 hours, 8 hours, and 16 hours after tadalafil administration. Statistical analysis was performed by one-way ANOVA followed by Tukey post hoc test. (C) After recording baseline mean arterial pressure in anesthetized animals, increasing non-cumulative doses of sodium nitroprusside (0.1, 0.3, 1, 3, 10, 30, 100, and 300 nmol/kg; i.v.) was injected. The time (in seconds) for mean arterial pressure returning to baseline was recorded. Significance differences were determined by two-way repeated measures ANOVA followed by Bonferroni’s test. a) Represents the difference between vehicle vs. tadalafil 8 hours (P=0.008 and P=0.02, respectively; b) Compares vehicle vs. tadalafil 16 hours (P=0.01). Values are represented as mean±standard deviation of 6–9 animals per group as indicated by dot plots and numbers in the bars.

Figure 3. Effect of tadalafil on mean arterial pressure (MAP), heart rate (HR) and renal blood flow (RBF) at baseline and after noradrenaline infusion in septic rats. Eight hours after cecal ligation and puncture (CLP) procedure, rats were treated with vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.). Sham group received vehicle at the same time point. Baseline MAP (A), HR (C), and RBF (E) were recorded after 24 hours. Significance differences were determined by one-way analysis of variance (ANOVA) and Tukey post hoc. MAP (B), HR (D), and RBF (F) variation after noradrenaline infusion (0.03 µmol/kg/min) over time. Significance differences were determined by two-way repeated measures ANOVA followed by Bonferroni’s test. a) Represents Sham Vehicle vs. all CLP groups (P<0.05); b) Represents CLP vehicle vs. CLP tadalafil (P=0.006; P=0.002; P=0.033, and P=0.029, respectively); c) Represents sham vehicle vs. CLP vehicle (P=0.037 and P=0.018, respectively). Values are presented as mean±standard deviation of 6–8 animals per group as indicated by dot plots and numbers in the bars.

Figure 4. Effect of tadalafil on inflammatory markers and nitrate+nitrite production in sepsis. Rats received vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.) administration 8 hours after cecal ligation and puncture (CLP) procedure. Sham group received vehicle at the same time point. Twenty-four hours after sepsis induction, myeloperoxidase (MPO) activity was evaluated in cardiac (A) and kidney tissue (B). Plasma nitrate plus nitrite (NOx) was also measured (C). Significance differences were determined by one-way analysis of variance with Tukey post hoc. Values are presented as mean±standard deviation of 6–8 animals per group as indicated by dot plots and numbers in the bars.

Figure 5. Effect of tadalafil on plasma biomarkers of tissue damage during sepsis. Eight hours after sepsis induction, animals received vehicle (DMSO, 0.2 mL/kg, s.c.) or tadalafil (2 mg/kg, s.c.). Twenty-four hours after surgery, plasma was obtained to measure creatinine (A), urea (B), aspartate aminotransferase (AST; C), alanine aminotransferase (ALT; D), and lactate (E). Significance differences were determined by one-way analysis of variance with Tukey post hoc. Values are presented as mean±standard deviation of 9–12 animals per group as indicated by dot plots and numbers in the bars. CLP: cecal ligation and puncture.

Figure 6. Survival rate of sepsis-induced animals by cecal ligation and puncture (CLP) treated or not with tadalafil. Six hours after the CLP procedure, both groups received a broad-spectrum veterinary antibiotic administration (24,000 IU/kg, i.m.). Animals received treatment with a single dose of tadalafil (2 mg/kg, s.c.) or vehicle (DMSO 0,2 mL/kg, s.c.) 8 hours after CLP and were follow-up by 5 days.

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Impact of tadalafil on cardiovascular and organ dysfunction induced by experimental sepsis