Angiotensin II Stress Test (TENSINTEST) is an investigator-initiated, multicenter observational study on renin kinetics before and 6 hours after starting an infusion of AT II in patients with vasoplegic shock.

TENSINTEST will be carried out in 8 different ICUs (surgical and medical) in Slovenia. Multimedia Appendix 1 provides details on the included ICUs.

All patients aged 18 years or older who are admitted to the ICU because of vasodilatory (distributive) shock regardless of etiology and who fail to achieve an adequate MAP of 65 to 85 mm Hg despite optimal fluid resuscitation and receiving norepinephrine base at a dose of 0.3 mcg per kilogram per minute or higher and vasopressin at 0.03 IU per minute will be enrolled.

Additional inclusion criteria will be the absence of AT II administration before enrollment, an expected survival of more than 24 hours, and no predefined limitations to active treatment.

The exclusion criteria are listed in Textbox 1.

The intervention is designed to test which patients could benefit from AT II infusion. The decision to treat patients with AT II infusion will be made by a treating clinical intensivist as per institutional protocol. The only intervention will be to obtain a sample of blood immediately before and 6 hours after starting AT II infusion to determine the renin concentration. Two additional venous blood samples for measurement of direct (active) renin concentration will be collected into ethylenediaminetetraacetic acid (EDTA) tubes in accordance with the specimen requirements of the LIAISON Direct Renin assay (DiaSorin S.p.A) [23]. Patients will be treated according to established guidelines and protocols in all other aspects of their ICU stay.

Patients who fulfill the inclusion criteria, meet none of the exclusion criteria, and provide informed consent will be enrolled in the study.

Patients who fail to achieve adequate MAP despite optimal fluid resuscitation, a norepinephrine base of 0.3 mcg per kilogram per minute or higher and vasopressin of 0.03 IU per minute will receive AT II infusion initiated at 20 ng per kilogram per minute as per the institutional protocol for initiation of a third vasopressor when adequate MAP is not achieved (Figure 1).

Adequate fluid resuscitation will include patients who have received an initial crystalloid bolus of approximately 30 mL per kilogram when clinically appropriate, followed by individualized fluid management based on repeated assessment of fluid responsiveness, clinical evaluation, and the treating physician’s judgment.

Following initiation of AT II at 20 ng per kilogram per minute, if the target MAP (65‐85 mm Hg) is not achieved within 15 minutes, the AT II dose will be increased in 5 ng per kilogram per minute increments every 15 minutes up to a maximum dose of 40 ng per kilogram per minute. If the target MAP is still not achieved, norepinephrine and vasopressin may be titrated as needed up to a maximum norepinephrine dose of 1 μg per kilogram per minute and a maximum vasopressin dose of 0.04 IU per minute. If MAP remains insufficient, further adjustments of vasopressors and adjunctive therapies (eg, higher doses of norepinephrine base, methylene blue, etc) will be made at the discretion of the treating physician. The decision to initiate hydrocortisone prior to AT II infusion will also be left to the clinical judgment of the treating physician based on the underlying disease.

In the event of hypertension, norepinephrine base and vasopressin will be reduced and discontinued if necessary. If both are discontinued, then AT II will be reduced in decrements of 5 ng per kilogram per minute to achieve target MAP and discontinued if necessary.

Blood samples for serum renin determination will be obtained immediately prior to and 6 hours after the start of AT II infusion. Renin analyses will be performed at the central laboratory of University Medical Centre (UMC) Maribor. Samples obtained outside UMC Maribor will be centrifuged immediately at the local laboratory, frozen to –85 °C to await transport, and transported at –20 °C to UMC Maribor. All other laboratory analyses will be performed at the local laboratories of participating centers.

Renin concentration in plasma samples will be measured as direct (active) renin using the chemiluminescent immunoassay method on a LIAISON fully automated analyzer platform. EDTA plasma is the validated specimen type for this assay. In the LIAISON Direct Renin assay, renin is sandwiched between a capture antibody adsorbed on magnetizable beads and a second isoluminol-linked antibody that detects the reaction. The relative light units developed by the mixture when exposed to an essential environment are proportional to the concentration of renin and allow its quantification. Renin concentration is expressed in mIU per liter (μIU/mL), with a normal range of 4 to 50 mIU/L and an upper reported limit at a value of 500 mIU/L. For the purpose of the study, the sample will be additionally diluted, and a higher value will be determined if the first result of the analysis is above 500 mIU/L to capture potential renin kinetics above this normally used upper limit. In the results, the values of renin will be given as a relative renin ratio (Multimedia Appendix 1). As renin secretion is influenced by sodium and extracellular volume status, these factors represent potential biological sources of variability in renin concentrations [24]. However, all patients will be enrolled only after adequate fluid resuscitation, and fluid balance will be prospectively recorded during the study period.

In the event of an allergic reaction or other serious adverse event (thrombosis or severe ischemia of the extremities or gastrointestinal tract), AT II will be immediately discontinued, and the patient will receive appropriate treatment in accordance with established clinical guidelines. Mechanical ventilation, renal replacement therapy, antibiotics, and all other routine intensive care treatment will be provided outside of the parameters of the study intervention as per international guidelines and protocols.

Participants will not be excluded from the study after enrollment except in the case of withdrawal of informed consent. In the event of an adverse event, in which treatment with AT II would be discontinued, the reason for treatment discontinuation and the adverse event will be documented in detail, participants will remain in the study, and their available data will be included in the analyses.

All included patients will be monitored daily for adverse events. As many of the potential adverse events are common complications of vasoplegia or critical illness, additionally, we will monitor prespecified adverse reactions listed in the European Medicines Agency prescribing information for AT II that occur only after recruitment: tachycardia, lactic acidosis, new venous or arterial thrombosis, thrombocytopenia, peripheral digital or limb ischemia, gut ischemia, and new arrhythmia [20].

Due to the nature of the study, investigators and other ICU staff cannot be blinded. All analyses are preplanned and will be performed by an independent investigator.

Patients with vasoplegia and an infusion of norepinephrine base of 0.3 mcg per kilogram per minute or higher and vasopressin of 0.03 IU per minute will be screened for eligibility by study investigators.

The characteristics of screened patients meeting the inclusion criteria will be recorded as follows: date of screening, age, possible cause of vasoplegia, and the reasons for not being enrolled. All recorded data will be stored in a screening database maintained by the research coordinators and stored on a dedicated and secured computer according to the principles of the General Data Protection Regulation.

For enrolled patients, we will collect demographic data as follows: age, gender, height, weight, comorbidities, therapy before critical illness (including the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), therapy during the ICU stay, and cause of vasoplegia. We will use the results of standard daily laboratory examinations, including complete blood count, electrolytes, C-reactive protein, procalcitonin, interleukin-6, troponin, creatinine, and liver function tests. Additionally, we will collect data on daily fluid balance for the first 48 hours after starting AT II infusion; the need for mechanical ventilation (invasive or noninvasive) and its duration; the need for renal replacement therapy and potential use of cytokine removal membranes; and the need for antimicrobial therapy, results of microbiological cultures, and source of infection.

A schedule of enrollment, interventions, and assessments is shown in Table 1.

After completion and validation of data collection, the norepinephrine-equivalent dose (NED) will be calculated from the recorded doses of norepinephrine, vasopressin, and AT II at each predefined time point (0, 1, and 6 hours) using the conversion outlined in Table 2. In the case of the use of an additional vasopressor administered (eg, methylene blue), its use and NED using the conversion scale by Kotani et al [25] will be recorded. The NED will be used in the statistical analyses.

The principal investigator, the coordinating investigator, local investigators, and an international expert are part of the steering committee, by whom the study was designed and all protocol revisions were made.

Access to the documents and data will be granted to the principal investigator, coordinating investigators, auditors, and monitors.

Daily screening for eligible patients will be conducted by local investigators and dedicated intensivists. Data will be collected by the principal investigator, coordinating investigators, and local investigators, and strict adherence to the trial protocol and relevant regulatory requirements will be ensured.

Protocol violations (deviations) will be reported to the principal investigator and discussed with the principal investigator and the coordinating investigators.

Patients will be treated according to established guidelines in all other aspects of treatment outside the 6-hour study intervention.

Patients or their legal representatives will be provided, within the informed consent form, with the contact details of a physician who can address any questions that may arise during the course of the study or in the event of a serious adverse reaction to vasopressor therapy. All adverse effects related to the use of vasopressor agents (including AT II) will be managed individually according to the internal protocol of each hospital.

Findings will be disseminated to health care professionals, participants, and the public via publication in peer-reviewed journals, conference presentations, and social media without any publication restrictions. The final manuscript will be drafted by the primary investigators.

The TENSINTEST study is designed to determine whether an early decrease in the relative renin ratio within 6 hours of AT II initiation identifies patients with distributive shock who are most likely to benefit from AT II therapy (Textbox 2). We hypothesize that a negative trend in the relative renin ratio will be associated with more 28-day organ failure–free days, more ICU-free days, reduced vasopressor requirements, and lower mortality. We further hypothesize that early changes in renin will predict mortality more accurately than lactate alone and that the combined renin-lactate biomarker will provide superior prognostic performance compared with either biomarker alone. The study aims to identify patients who are likely to benefit from AT II while outlining nonresponders in whom continued AT II treatment is unlikely to improve outcomes, allowing earlier discontinuation of AT II and consideration of alternative therapies.

A post hoc ATHOS-3 analysis showed lower renin in AT II vs placebo groups [19], mirrored in post–cardiac surgery studies [18] and a prospective feasibility trial comparing AT II to norepinephrine assessing consent, adherence, infusion duration, MAP maintenance, and adverse outcomes [28]. Patients exhibited varied AT II responses, with different phenotypes for hyperresponders, responders, or nonresponders, probably linked to RAAS dysfunction and hemodynamic changes [7,8]. The 6-hour interval is derived from a study by Gleeson et al [21], who determined that renin change rates over 6 hours differed significantly between survivors and nonsurvivors, enabling rapid laboratory assessment. The RAAS underpins circulatory homeostasis; renin levels increase with poor perfusion, increased sympathetic drive, and hypoxia, outperforming lactate in tracking hemodynamic changes [21,22]. As lactate and renin capture distinct pathologies, their product may superiorly classify responders vs nonresponders over either alone.

This is an emerging and clinically relevant area of research, with an increasing number of studies investigating biomarkers that may predict response to AT II therapy. However, our study has a larger planned sample size and is being carried out in more centers than other ongoing studies [29,30]. These other studies randomize patients to AT II use, whereas this study will only evaluate patients to whom AT II is prescribed as part of routine clinical care as a third-line vasopressor, which better matches the current clinical use, and will also evaluate AT II use in all types of vasodilatory shock, not only septic shock. If our hypotheses are confirmed, TENSINTEST could support a biomarker-guided approach to AT II therapy, avoiding unnecessary treatment in nonresponders, optimizing vasopressor use, improving organ recovery, and shortening ICU length of stay.

Due to the relatively small sample size and exclusion of patients with a component of cardiogenic shock, the results of our study might not be useful for a general population of patients with shock who are severely ill. As no similar prospective study has been conducted so far, sample size estimation could not be optimal.

A further limitation is the absence of a control arm, which prevents us from determining whether the prognostic significance of changes in renin is specific to AT II exposure or would also apply to patients treated with other vasopressors.

An additional limitation is that patients are enrolled only after reaching the institutional threshold for third-line vasopressor therapy (norepinephrine ≥0.3 μg per kilogram per minute plus vasopressin of 0.03 IU per minute). Consequently, our findings may not be directly applicable to patients with earlier stages of vasodilatory shock, in whom earlier initiation of AT II is increasingly being investigated. An additional consequence of AT II being used as a third-line vasopressor in patients who are severely ill is that we expect to observe high mortality and a potential beneficial effect of AT II in patients who are less severely ill, and use of AT II at lower cumulative doses of vasopressors might not be observed. Future studies should determine whether the predictive value of early renin kinetics extends to patients receiving AT II at lower vasopressor requirements.