Background: Non-intubated thoracic surgery has not achieved widespread acceptance despite its potential to improve postoperative outcomes. To ensure airway safety, our institute has developed a technique combining spontaneous ventilation with double-lumen tube intubation (SVI). This study aimed to verify the feasibility and limitations of this SVI technique. Methods: For the SVI method, anesthesia induction involves fentanyl and propofol target-controlled infusion, with mivacurium administration. Bispectral index monitoring was used to ensure the optimal depth of anesthesia. Short-term muscle relaxation facilitated double-lumen tube intubation and early surgical steps. Chest opening preceded local infiltration, followed by a vagal nerve blockade to prevent the cough reflex and a paravertebral blockade for pain relief. Subsequently, the muscle relaxant was ceased. The patient underwent spontaneous breathing without coughing during surgical manipulation. Results: Between 10 March 2020 and 28 October 2022, 141 SVI surgeries were performed. Spontaneous respiration with positive end-expiratory pressure was sufficient in 65.96% (93/141) of cases, whereas 31.21% (44/141) required pressure support ventilation. Only 2.84% (4/141) of cases reversed to conventional anesthetic management, owing to technical or surgical difficulties. Results of the 141 cases: The mean maximal carbon dioxide pressure was 59.01 (34.4–92.9) mmHg, and the mean lowest oxygen saturation was 93.96% (81–100%). The mean one-lung, mechanical and spontaneous one-lung ventilation time was 74.88 (20–140), 17.55 (0–115) and 57.73 (0–130) min, respectively. Conclusions: Spontaneous ventilation with double-lumen tube intubation is safe and feasible for thoracic surgery. The mechanical one-lung ventilation time was reduced by 76.5%, and the rate of anesthetic conversion to relaxation was low (2.8%). Thoracic surgery and anesthesia have undergone a paradigm shift over the last few decades, and specialists have focused on a minimally invasive approach. Video-assisted thoracic surgery (VATS) is the gold standard for minor and major pulmonary resections and thymectomies ,2,3,4. TAs a spontaneous breathing anesthetic approach, the SVI method is a novel alternative to previously reported techniques. In our case series, we applied the SVI method in a prospective, nonconsequential manner when patients met the inclusion and exclusion criteria , and a dThis study was approved by the Ethical Committee of the Human Investigation Review Board at the University of Szeged .For patient selection, we applied our previously published criteria for non-intubated thoracic procedures (NITS)  15]. Ho. Ho15]. Preoperative pulmonological and anesthesiologic examinations were the same as those in the normal or NITS cases. From a surgical perspective, we included all cases of SVI that would also be suitable for normal VATS according to consensus meeting recommendations. We included patients with no advanced lung cancer  .Between 10 March 2020 and 28 October 2022, 141 surgeries were performed by our thoracic surgery team using the SVI approach for general anesthesia. All surgeries were performed by a single surgeon, and the patients were anesthetized by three anesthetists using the same procedural algorithm. Initially, we intended to perform 144 SVI procedures. Three cases were excluded from statistical analyses because spontaneous breathing did not return by the end of the surgery.2) and invasive blood pressure measurements were performed. The depth of anesthesia was monitored using the bispectral index . Anesthesia was induced with fentanyl (1–1.5 µg/kg) and propofol using target-controlled infusion (Schnider model) with effect-site targeting. Considering the induction effect, the site target concentration was generally set between 4 and 6 µg/mL, depending on individual patient characteristics. Subsequently, the target concentration was modified to keep the BIS value between 40 and 60.Three-lead ECG, oxygen saturation (SpOMivacurium chloride (0.1–0.15 µg/kg), a short-acting non-depolarizing muscle relaxant, was used to ensure optimal conditions for intubation. Although the dose of mivacurium that was used was below the recommended dose for intubation, we found that, 180 s after drug administration, the conditions for intubation were good or excellent.Similar to the gold standard approach, fiberoptic equipment  was used to confirm the proper position of the DLT. Confirmation of the proper tube position was crucial because further manipulation of the tube after the effects of the muscle relaxant diminished would not be well tolerated.Muscle relaxation facilitated the DLT insertion and helped in early surgical steps. After the thoracic cavity was opened, a paravertebral nerve blockade for pain relief and a vagal blockade to prevent the cough reflex were performed. Spontaneous breathing returned after the muscle relaxant effect was eliminated. In unexpected cases, when spontaneous breathing was unsatisfactory , temporary pressure support ventilation with a low-flow trigger (1.0 L/min) was used until the muscle relaxant effect was fully eliminated.2 (40–100%) and applying 3–5 H2O cm positive end-expiratory pressure (PEEP) to the dependent lung helped keep the SpO2 and PaCO2 within normal or close-to-normal ranges. Severe hypercapnia or hypoxia was also prevented or managed by applying pressure support ventilation to the dependent lung.Intraoperatively, altering the FiOIn case of necessity, the intraoperative evaluation of air leakage involved conducting a water submersion test (WST). After filling the thoracic cavity with saline, manual positive pressure ventilation was synchronized with the patient’s spontaneous breathing activity. Furthermore, if it was necessary, we were able to apply pressure support ventilation to perform the leak test.According to our intraoperative hemodynamic management protocol, when the mean arterial pressure was <60 mmHg, the systolic blood pressure was <90 mmHg or decreased by more than 25%, and ephedrin (5–10 mg) or phenylephrine (50–100 µg) was administered in divided doses.2 < 92% or PaO2 < 60 mmHg, 3–5 H2O cm PEEP administration and FiO2 alteration were used to increase oxygenation. In the cases of PaCO2 > 75 mmHg or pH < 7.15, the non-dependent lung was considered to be re-inflated for a short period to eliminate carbon dioxide and improve oxygenation. The effect of reinflation is partial and temporary. Thus, if the improvement was not satisfactory, or as an initial step, we administered pressure support ventilation with 3–5 H2O cm PEEP and 8–14 H2O cm pressure support to assist in gas exchange. If hypercapnia and/or hypoxia were persistent, anesthetic conversion with muscle relaxation (0.05–0.1 µg/kg of mivacurium) and volume-controlled mechanical ventilation  were applied  was administered at the site of incision at the fifth intercostal space in the mid-axillary line. After opening the thoracic cavity under thoracoscopic guidance, vagal and paravertebral blocks were performed. For the vagal nerve blockade, 3–5 mL of bupivacaine (0.5%) was administered close to the nerve . A deep intercostal or paravertebral blockade was achieved by administering 4–5 mL of bupivacaine (0.5%) close to each intercostal nerve . The maximum amount of bupivacaine used was 0.5 mL/kg (2.5 mg/kg). In cases of open SVI, an intercostal nerve blockade was guaranteed by administering 4–5 mL of bupivacaine (0.5%) between the third and sixth intercostal spaces.We performed the same VATS uniportal method during the SVI procedures that we published in our NITS study ,18, with2 postoperatively to achieve a SpO2 of >94% or >88% in patients with chronic obstructive pulmonary disease. None of the patients required a higher level of oxygen or a higher degree of respiratory support (non-invasive ventilation) during the PACU stay or in the later postoperative period. None of the patients experienced fever or required bronchial secretion removal. Postoperatively, chest radiography was performed before and after the chest tube removal. Any pneumothorax, atelectasia/dystelectasia, infiltration or pleural fluid observed in the radiography were considered abnormal findings. The pain intensity during hospitalization was assessed using a numeric pain rating scale (NPRS). An NPRS > 3 was the intervention point, and a minor analgesic agent prescribed by the anesthetist was administered orally as soon as possible. For patients who underwent thoracotomy, a pleural catheter was inserted for continuous local anesthetic (0.1 mL/h/kgbw bupivacain 0.33%) administration.Every patient was observed in the post-anesthesia care unit (PACU) for at least 2 h or until they met the criteria for leaving the PACU . Oxygen was administered to all patients via a face mask with 4–6 L/min O2 of 0.21. For T2, steady-state blood samples were collected 15 min after the vagal nerve blockade. For T3, blood samples were collected 15 min after anatomical resection . For T4, postoperative blood samples were collected 30 min after the patient arrived in the recovery room and at a FiO2 of 0.5.In cases of major pulmonary resection, blood samples were collected four times  and personal patient documentation. Personal patient data were also collected. Descriptive statistics were performed using R statistical software, version 4.2.2 , and SPSS for Windows, version 26.0 . Descriptive statistics are presented as the mean ± standard deviation (SD) for continuous variables and as the count and percentage for categorical variables.A total of 67 (47.52%) of the patients were men, and 74 (52.48%) were female patients. The mean age was 62.13 years (19–83), with a mean BMI of 25.82 (15.79–38.54) . A total2O cm PEEP administration, produced satisfactory gas exchange. In 44 cases , temporary or permanent PSV administration was necessary for supportive oxygenation and carbon dioxide removal. In four cases , repeated muscle relaxation and a return to a conventional anesthetic pathway were necessary. In one patient, high-amplitude mediastinal movement was intolerable despite the provision of applied pressure support ventilation. In two cases, vagal blockade ineffectiveness resulted in coughing under surgical manipulation, and muscle relaxation was necessary. In one patient, endotracheal tube malposition was confirmed, and anesthetic conversion was necessary for correction , spontaneous respiration, with or without 3–5 Hrrection .1 and DLCO values were lower in the PSV group (75.71% ± 22.75% and 65.89% ± 22.04%) than in the non-PSV group (83.77% ± 18.35% and 76.55% ± 16.04%). In the PSV group, thymectomy was performed in five cases , in which pressure support ventilation was necessary due to a bilateral surgical pneumothorax.We compared the possibly relevant factors influencing the necessity of pressure support ventilation during an SVI procedure . The meaAfter anesthetic induction and 5–10 min after the vagal nerve blockade, hypotension was common. Of the 141 patients, 65 (46.1%) required phenylephrine or ephedrine due to hypotension. Ephedrin or phenylephrin administration was necessary in 49 cases  in patients with hypertension or other cardiovascular diseases (CV group), and it was administered for 15 patients  without any cardiovascular disease (non-CV group). The systolic blood pressure reduction was 33.45 mmHg ± 18.71 mmHg in the CV group, and it was 28.67 mmHg ± 19.55 mmHg in the non-CV group. Moreover, the diastolic blood pressure reduction was 17.64 mmHg ± 10.68 mmHg and 15.49 mmHg ± 11.55 mmHg in the CV and non-CV groups, respectively . HoweverThe mean one-lung ventilation time was 74.88 min (20–140 min). The mean mechanical and spontaneous OLV times were 17.55 min (0–115 min) and 57.73 min (0–100 min), respectively. The mechanical OLV time was reduced by 76.5%. The respiratory rate was altered by between 4 and 36 min. The mean minimum respiratory rate was 12.19 (4–30), whereas the mean maximum respiratory rate was 19.19 (6–36) .At least one blood gas test result was available for 94 patients. The demographic comparison between the patient populations is shown in 2 level at time T2 was 115.97 mmHg (50.4–472.6 mmHg) and 143.831 mmHg (59.9–425.6 mmHg) at T3 (2 level at T2 was 59.05 mmHg (37.1–92.9 mmHg), with a mean pH of 7.27 (7.1–7.41). The mean PaCO2 level at T3 was 58.17 mmHg (34.4–90.9 mmHg), accompanied by a mean pH of 7.27 (7.14–7.44). Hypercapnia and the acid–base discrepancy diminished in the early postoperative period. The mean PaCO2 level at T4 was 47.44 mmHg (36.7–66.7 mmHg)  at T3 , and it g) at T3 . Hyperca.7 mmHg) . Consequ.7 mmHg) . The meaUpon completion of the surgery, all patients were extubated. After a mean of 68.18 min (30–170 min) of observation in the recovery room, all patients were transported back to the thoracic surgical ward. None of the patients required a higher flow of oxygen therapy, non-invasive ventilation, reintubation or intensive care unit admission. In the absence of contraindications, our patients routinely received 75 mg of diclofenac in the operating room as part of our multimodal pain management strategy. In the PACU, 25 of 141 patients did not require further analgesics. Metamizole  and paracetamol  were the most commonly administered analgesics postoperatively. A total of 94  patients received metamizole, and 83 of the 141 patients (58.87%) received paracetamol. In half of the patients, when metamizol was necessary, it was co-administered with paracetamol . Approximately, 10–75 µg of fentanyl administration was necessary in 8 (5.67%) patients to achieve a VAS score <4. Tranexamic acid administration was necessary in 1 patient  because of the high rate of bleeding drained from the chest tube .A total of 3 of the 141 surgeries were intended for open SVI procedures. In 12 additional cases , conversion to thoracotomy was necessary, without anesthetic conversion . Of thes2 rebreathing (the pendelluft phenomenon) is a possible consequence of chest opening. Hypoxia and hypercapnia can develop as a result of pendelluft and paradoxical mediastinal shifting, which are the main obstacles to the widespread acceptance of NITS among anesthetists and surgeons [The main concern with the non-intubated technique is airway safety. Without lung isolation, COsurgeons ,23,24,25surgeons , the nonSVI combines the beneficial effects of spontaneous ventilation thoracic procedures with airway safety via tracheal intubation using a double-lumen tube. The essential components of SVI are double-lumen tube intubation for maximal airway safety, short-acting non-depolarizing muscle relaxants for the early recovery of spontaneous ventilation, paravertebral blockades as part of multimodal pain management and vagal nerve blockades to prevent the cough reflex. Infiltration of the vagal nerve with 2–5 mL of bupivacain (0.25–0.5%) into the thoracic cavity is a widely accepted method ,15,26,27Double-lumen endotracheal tube placement provides an opportunity for all anesthetic interventions (recruitment maneuvers and fiberoptic control) with complete lung isolation. Therefore, if a surgical or anesthetic complication occurs, conversion to the conventional anesthetic pathway is easy and safe. The most risky part of the NITS procedure is the conversion process under aggravated circumstances (2–11% of cases) ,29,30,31Furthermore, the incidence of unforeseen difficult airways and difficult intubation can reach up to 20%. According to Langiano et al., the overall incidence of difficult airways is 16% in the thoracic surgical patient population, whereas the frequency of unexpected difficult airways is 5.2% . Corso e2, PEEP administration or applying pressure support. However, it is important to emphasize that the peak airway pressure is lower during spontaneous ventilation, with or without pressure support, than that during controlled ventilation [2 was 47.44 mmHg (36.7–66.7 mmHg), and the pH was 7.332 (7.275–7.401). However, hypercapnia itself may result in better ventilation–perfusion matching and reduce intraoperative lung injury by suppressing inflammatory responses [2 level were found in the non-intubated group (vs. the SVI group) [Gas exchange imbalance is common during spontaneous ventilation surgery. Hypoxia and hypercapnia have also been observed during NITS . In SVI,tilation . Hypercatilation ,24,35,36esponses ,38. FuráI group) .In our SVI series, hypotension commonly occurred after anesthesia induction and vagal nerve blockades. In total, 46.1% of our patients needed temporary pharmacological hemodynamic support. For the rest of the patients 53.9%), reductions in blood pressure were below our hemodynamic management cut-off value, and thus, self-regulation was sufficient to normalize blood pressure. The incidence of hypotension and the extent of intraoperative blood pressure reduction were higher among patients in the cardiovascular disease (CV) group. The elevated occurrence of hypotension can be attributed to the medications routinely used by patients in the CV group. Among these patients, 52  were taking beta-blockers, 75 75/95, 78.95%) were on antihypertensive medications , 30  were on combinations of multiple antihypertensive drugs, and 15 patients  were regularly taking antidiuretic agents. As described earlier, SVI shows better hemodynamic stability than that of the non-intubated technique . The neg.95% were%, reductFurák and Szabó previously published their results on SVI  and SVI There are only a few established exclusion criteria for SVI, and patients indicated for VATS are also candidates for SVI. NITS has several exclusion criteria, such as potentially difficult airways or intubation, coagulation disorders or mental conditions, which are not exclusion reasons for SVI ,40,43. IOur study has several limitations. More detailed perioperative monitoring  and postoperative outpatient follow-ups would provide more comprehensive physiological and pathophysiological data related to SVI. Having compared the conventional and non-intubated methods, our findings suggest that future investigations are required to further evaluate the advantages and disadvantages of the SVI method.The maintenance of spontaneous respiration during thoracic procedures is a well-known anesthetic strategy ,16,44,45


Blood pressure goals in the intensive care unit (ICU) have been extensively investigated in large datasets and have been associated with various harm thresholds at or greater than a mean pressure of 65 mm Hg. While it is difficult to perform interventional randomized trials of blood pressure in the ICU, important evidence does not support defense of a higher pressure, except in retrospective database analyses. Perfusion pressure may be a more important target than mean pressure, even more so in the vulnerable patient population. In the cardiac ICU, blood pressure targets are tailored to specific cardiac pathophysiology and patient characteristics. Generally, the goal is to maintain adequate blood pressure within a certain range to support cardiac function and to ensure end organ perfusion. Individualized targets demand the use of both invasive and noninvasive monitoring modalities and frequent titration of medications and/or mechanical circulatory support where necessary. In this review, we aim to identify appropriate blood pressure targets in the ICU, recognizing special patient populations and outlining the risk factors and predictors of end organ failure. The distinction between hypotension and hypoperfusion is particularly critical in the cardiac intensive care unit (CICU), specifically for patients in the perioperative period. The perfusion of an organ is dependent on the mean arterial pressure, which is a function of cardiac output and systemic vascular resistance. A shift in thought process around the definition of shock may be the presence of hypoperfusion, even in the setting of normotension, which is generally thought of as “pre-shock” or compensated shock. Unsurprisingly, outcomes, including mortality, are worse in patients with evidence of both hypotension and hypoperfusion.2 This goal MAP is recommended as the lowest pressure to target initial resuscitation in patients with septic shock by the guidelines of the Surviving Sepsis Campaign.3 The extrapolation of the same threshold to all patient types, pathologies, and comorbidities is a challenge and certainly deserves more investigation.Historically, mean arterial pressure (MAP) has defined blood pressure target thresholds in the critical care unit. Every unit of time-weighted average (TWA) spent at MAP < 65 mm Hg has been strongly associated with myocardial infarction, acute kidney injury, and mortality in the critical care unit.4 Importantly, pressures were about 5 mm Hg higher in each group, and this was likely a trial of two higher pressures, albeit with adequate intergroup separation.Randomized trials of blood pressure targets are inherently difficult. Asfar and colleagues performed the largest such trial in septic shock patients and compared mean pressures of 80 mm Hg to 85 mm Hg (high target group) with current recommendations of 65 to 70 mm Hg (low target group) in patients with septic shock undergoing resuscitation. The primary outcome was mortality at 28 and 90 days, and they found no difference.5 Estimated change-points for the risk of ICU mortality in septic patients were 69 mm Hg for mean, 100 mm Hg for systolic, 60 mm Hg for diastolic, and 57 mm Hg for pulse pressure.5 Other observational data derived from large datasets show that higher thresholds of MAP closer to 80 to 85 mm Hg may be the inflection point for increasing harm in both the medical and surgical ICU population.7 While it is difficult to separate intraoperative hypotension from postoperative hypotension in the ICU, even previously normotensive operating room patients suffer harm with new-onset hypotension in the critical care unit.8 Thresholds at 75 mm Hg or higher may be associated with decreased delirium and improved arousal levels as measured by sedation scales.9 In the SEPSISPAM  trial, patients with chronic hypertension had significantly more renal injury if they were exposed to a MAP of 70 to 75 mm Hg  while more rhythm abnormalities occurred in the higher target group at a MAP of 85 to 90 mm Hg.4 Lamontagne and colleagues in the 65-trial showed no difference in survival when patients were randomized to a MAP of at least 65 mm Hg versus permissive hypotension at a MAP of 60 mm Hg.10A recent retrospective analysis from a well-validated electronic ICU dataset with nearly 80,000 patients found that components of blood pressure, namely mean, systolic, and diastolic pressures, were comparable in their strength of association with organ system injury and ICU mortality.11 Critically ill patients with cardiogenic shock and a 24-hour average MAP < 65 mm Hg have a rapidly increasing rate of mortality, exceeding 70% with a 24-hour average MAP < 60 mm Hg.12 Further, patients with sustained (duration > 10 minutes), MAP < 64 mm Hg before, during, and after cardiopulmonary bypass (CPB), particularly those with sustained MAP < 55 mm Hg, have a strong association with acute perioperative stroke.13 Other investigators have confirmed that duration of hypotension per 10 minutes of a MAP < 65 mm Hg during an entire cardiac surgery and after CBP is equally important.14In the critically ill cardiac patient population, diastolic blood pressure (DBP) is an important determinant of coronary blood flow, and low DBP, particularly < 60 mm Hg, has been associated with increased risk of myocardial injury.16 Almost 10 years after the impressive work by the SEPSISPAM group, we need to think of trials with appropriate patient populations, targets, and outcomes. Certainly, the concept of perfusion pressure being the difference of MAP and central venous pressure or in some cases intra-abdominal pressure is a critically important area of focus.17 Perfusion pressure may be extrapolated to renal perfusion and a necessary threshold of at minimum 60 mm Hg. Analysis comparing perfusion pressure deficits and MAP deficits has been seen to have a nearly similar and strong association with ICU mortality at two weeks.19The conundrum, therefore, is whether we should target higher pressures in all patients or in more vulnerable ones. Furthermore, it seems that the mechanisms to achieve higher pressures may be as important as the goal itself. Exposure to supratherapeutic doses of catecholamines may not be desirable.Individualization of MAP goals is important in the post-cardiac surgery patient, as different comorbidities, operative interventions, and the presence of mechanical circulatory support, among other criteria, may influence the MAP required to maintain adequate perfusion .20 Mechanistically, a worsened microcirculation in the chronically hypertensive and the need for a higher perfusion pressure to maintain end-organ perfusion contribute to the underlying pathophysiology.21Most commonly, patients with chronic uncontrolled hypertension are presumed to have a rightward shift in their cerebral autoregulation curve, thereby requiring a higher average MAP goal to maintain cerebral blood flow and perfusion. A frequently referenced threshold for these patients is a minimum MAP no less than 20% from their baseline blood pressure, usually averaged over several previous measurements.22After post-acute myocardial infarction, higher MAP goals are associated with smaller infarct sizes, and thus use of vasopressor and inotropic support to obtain higher mean pressures is recommended in this population.23Patients with uncorrected valvular lesions and physiology that requires higher afterload may also require higher MAP goals. These specifically include patients with clinically significant aortic stenosis, left ventricular (LV) hypertrophy, and those with septal anterior motion of the anterior leaflet of the mitral valve due to hypertrophic obstructive cardiomyopathy or asymmetric septal hypertrophy. Finally, patients who develop tamponade physiology postoperatively require higher afterload until the pericardium can be decompressed. Given ventricular interdependence, maintenance of normal to slightly increased systemic blood pressure is critical to maintaining the transseptal pressure gradient and thus normal wall motion of both the left and right ventricles. Consequently, those with significant right ventricular dysfunction, including those post-orthotopic heart transplantation, will require higher MAP goals.24 patients with a prolonged CPB run or cross-clamp time, and those who have difficulty weaning from CPB, which may be associated with relative coronary ischemia. Finally, poor perioperative urine output may indicate end-organ hypoperfusion and increased risk of acute kidney injury, so such patients may benefit from higher perfusion pressure.25 In contrast, patients with hemodynamically significant aortic insufficiency and mitral regurgitation require reduced afterload. Lower MAP goals within limits and without compromising on coronary or another end-organ perfusion may be necessary.Other specific populations that may benefit from higher MAP goals include patients with coronary air embolism during weaning from CPB,26Aside from patients with valvular or other pathology, a small subset of patients may have indications for lower MAP goals. Predominantly, this group includes patients who have undergone aortic surgery, those with complex congenital repairs (including fistulas), and those with ventricular septal defects—all cases in which increased MAP leads to tension on critical suture lines, causing possible disruption that can lead to catastrophic and potentially irreparable bleeding. However, recent emerging evidence suggests that this may be an exaggerated association of lower clinical significance.21 Appropriate MAP targets in this setting remain a particular enigma since increasing pressure with afterload most surely compromises forward flow in most of these patients.Another subset of patients in whom decreased systemic blood pressure may be appropriate is those with mechanical circulatory support devices, including temporary and durable LV assist devices, as well as extracorporeal membranous oxygenation through which flow may be compromised in high afterload states.If relative hypotension is deemed appropriate in a specific patient, close monitoring of end-organ function is mandatory, and indications should be repeatedly addressed to liberalize toward normal MAP goals as soon as it is safe.27 Cardiogenic shock (CS) is a continuum of hemodynamic instability, including isolated hypotension with preserved organ perfusion, isolated organ hypoperfusion despite preserved blood pressure, and the combination of hypotension and hypoperfusion.128While initial presentation of shock includes evidence of hypoperfusion and hypotension, not all such states are equal .27 Cardi1 However, the equation of MAP with markers of tissue perfusion and microcirculation per se is still up for debate.29 Capillary microvascular density and flow at high dose vasopressors to maintain MAP compared to normotension with no vasopressors is very different. Capillary refill time has been the subject of recent exploration and was found to be no different from lactate in guiding resuscitation in septic shock.30 A recent meta-analysis reported a very weak negative correlation between capillary refill time and MAP.31Hypotension and hypoperfusion are both associated with increased mortality in CICU patients. Hospital mortality is higher with isolated hypoperfusion or concomitant hypotension and hypoperfusion (classic shock); therefore, patients with hypoperfusion can be considered to have shock irrespective of blood pressure.The specific effects on flow and pressure can vary depending on the underlying cause and severity of the shock. In general, CS is characterized by a decrease in cardiac output, which can lead to a decrease in blood pressure. However, the relationship between flow and pressure in CS can be complex as systemic vascular resistance increases to compensate for the decreased cardiac output; this can help maintain blood pressure but may further decrease cardiac output. The goal is to optimize cardiac output and tissue perfusion by targeting a specific flow in addition to blood pressure . This ca32 In the ICU, and especially in the cardiac critical care unit, use of an “invasive continuous arterial blood measurement” is widely considered the gold standard. However, blood pressure alone is simply not enough in these patients. Therefore we are seeing a consistent move away from static toward dynamic measurements.33 Beyond knowing that a patient is hypotensive, a critical care physician needs to determine whether the patient is fluid responsive or needs vasopressor or inotropic support. To this end, dynamic variables such as plethysmography variability index, pulse pressure variation, and stroke volume variation are helpful and increasingly used as easy bedside tools.33Arterial blood pressure is determined primarily by cardiac output, systemic vascular resistance, vascular compliance, and blood volume.33 In a prospective observational study of mechanically ventilated patients undergoing noncardiac surgery, PVI was measured and a fluid bolus given. Patients with a PVI > 10.5% were noted to have a decrease in heart rate with concurrent increase in cardiac index following the bolus.34 Forget et al. likewise showed that during major abdominal surgery, use of PVI to guide fluid therapies led to decreased lactate levels when compared with standard care both during surgery and 48 hours postoperatively.35Plethysmography variability index (PVI) utilizes changes in pulse oximetry waveforms over the entire respiratory cycle to predict fluid responsiveness.36 This examination of 22 studies found PPV had a pooled sensitivity of 0.88 and pooled specificity of 0.89 for fluid responsiveness.36Pulse pressure variation (PPV) has been a popular marker of fluid responsiveness since it requires no additional equipment beyond an invasive blood pressure monitor. To be accurate, it does require appropriately large tidal volumes in a mechanically ventilated patient using volume control ventilation in normal sinus rhythm. Yang and Du undertook a recent meta-analysis to evaluate PPV in the critical care population.37 In a meta-analysis of 24 studies utilizing SVV in the surgical and critical care population, Zhang et al. found SVV highly correlated with fluid responsiveness (r = 0.72) and with a sensitivity and specificity of 0.81 and 0.80, respectively.37 The correlation was found to be even stronger in the ICU population, which the authors postulated was due to the relative hypovolemic state of these patients compared with those in the operating room when evaluated.37Stroke volume variation (SVV) can be evaluated by one of several commercially available monitors that differ in their method of estimating aortic impedance.39In addition, an important and easy to use bedside tool in the ICU is the tidal volume challenge that uses the change of tidal volume from the typical lung protective 6 mL/kg to a larger target of 8 mL/kg. This change in SVV and PPV obtained by transiently increasing tidal volume  was superior to PPV and SVV in predicting fluid responsiveness during low tidal volume ventilation. Myatra and colleagues demonstrated this in a study in 20 patients in 2017, and these findings were validated in various larger external cohorts in subsequent analyses.40 In an extensive review of the literature, Prager et al. noted that utilization of TEE in the critical care setting resulted in a change of diagnosis in 52% to 78% of patients and a change in management in 32% to 79% of patients examined.41 They additionally showed that TEE was able to successfully distinguish the etiology of cardiac arrest in 25% to 35% of cases when it was used for this purpose.41The important role of echocardiography in the critical care setting cannot be overstated. Regardless of modality, echocardiography can be a useful tool for examining hemodynamic failure, assessing etiology of cardiopulmonary arrest, and determining volume status via superior vena cava (with transgastric TEE) or inferior vena cava (with transthoracic echocardiogram) collapsibility.As shown in 46 Specifically, intraoperative hypotension is associated with postoperative acute kidney injury (AKI), myocardial injury, stroke, delirium, and mortality.48 Interestingly, this relationship and risk is no different for an absolute MAP < 65 mm Hg or a relative drop from baseline by about 25%.49 Patients who are hypotensive in the operating room are more likely to remain hypotensive in the post-surgical ICU and possibly when they recover on the hospital ward, where it may be undetected for lack of adequate monitoring.51 An analysis by Khanna et al. showed that a lowest MAP reading less than that compared to a population median MAP of 87 mm Hg in the surgical ICU was significantly associated with the risk of a composite of myocardial injury or mortality. However, intraoperative hypotension had a significant interaction with the primary outcome.52Intraoperative hypotension with even brief exposure to a MAP of < 65 mm Hg is known to be associated with an increased risk of organ system failure and mortality.8 Unsurprisingly, in patients undergoing cardiac surgery with CPB, the total duration of intraoperative hypotension per 10-minute exposure of MAP < 65 mm Hg throughout the surgery was statistically significantly associated with the composite primary outcome of stroke, AKI, or death.53 Hypotension pre-CPB can result from decreased venous return, arrythmias due to surgical manipulation, depressed cardiac function, decreased systemic vascular resistance, or induced hypotension for aortic cannulation.54 Despite much emphasis placed on perfusion pressure, there are no guidelines for target perfusion pressure during CPB. According to the 2019 European Adult Cardiac Surgery Guidelines for CPB, it is safe to maintain MAP targets between 50 and 70 mm Hg during CPB.55 However, studies have shown that higher MAP targets during CPB were associated with lower post-bypass lactate levels,56 lower incidence of stroke,13 and decreased risk of AKI.53Even patients with normotension in the operating room may suffer harm associated with trivial new-onset hypotension in the post-surgical ICU.It may be prudent to add monitors of cerebral perfusion and oxygenation in patients undergoing cardiac surgery, particularly in those with a history of a cerebrovascular event. A good clinical practice may be to use baseline cerebral oximetry (placed before induction of anesthesia) to monitor trends in the oximeter values with changes in blood pressure and to establish a baseline MAP goal for these patients. Subsequently, this more personalized approach could be used as a targeted postoperative MAP in the ICU to optimize cerebral perfusion pressure.57 Additionally, renal resistive index (RRI) calculated using TEE has shown that an RRI > 0.68 is a good predictor of AKI in patients undergoing cardiac surgery on CPB.58 An important analysis showed significant associations of intraoperative post-CPB RRI elevation with subsequent development of AKI in a cohort of 99 adult cardiac surgery patients.59 However, there remains a lack of evidence to support a higher MAP target in patients with increased baseline RRI, who are presumably at high risk for postoperative AKI and mortality.Transesophageal echocardiography is a good guide to establish volume status intraoperatively and to estimate the patient’s blood pressure goals, cardiac function, and need for pressors versus inotropes.The latest iteration of blood pressure evaluation pivots from dynamic measurements to prediction and proactive management of hypotension. This is a step beyond our traditional management of hypotension based on reactive responses, which result in time delays of various lengths and may not be adequate to prevent downstream effects.61 One such algorithm can predict arterial hypotension with high sensitivity and specificity.61 Davies et al. analyzed this algorithm in 255 patients undergoing major noncardiac surgery and determined a sensitivity and specificity of about 80% 15 minutes preceding hypotension and 85% five minutes prior.62 A recent systematic review by Li et al. evaluated a total of five trials of the hypotension predictive index and determined that it has the potential to reduce frequency and severity of hypotensive episodes when strictly adhering to the protocol.60Machine-learning-derived algorithms that predict hypotension are based on arterial wave form analysis using millions of waveforms from various shock pathophysiological states in model development and external validation.64 Randomized trials have reported conflicting evidence for benefits, which may be due to variable adherence to protocol and directions from technology.66 Ongoing trials, including the HPI (Hypotension Prediction Index) CARE Trial, are currently underway to examine this technology in cardiac surgery and cardiac critical care.67 Other trials, such as the REACT-SHOCK group in Australia and New Zealand, will examine targeted BP management in the ICU based on baseline (pre-morbid) pressures. Previously, important data suggested that we tend to keep patients with lower pre-morbid pressures on a longer duration of vasopressors, with consequently longer ICU stays, all of which may be unnecessary.68 This and the use of perfusion pressure targets for the future of large interventional pressure targeted trials will be necessary as we grow the evidence base.Most data surrounding these technologies come from the operating room and support the thought that different varieties of intraoperative hypotension need different treatment, an idea which also could be extrapolated to the ICU within limits.We have yet to establish a definitive target for hypotension in different clinical settings. This should prompt us to move away from using a single predefined number for a hypotensive threshold for all patients and towards using a more individualized approach based on patient characteristics. Further, we should consider integrating emerging innovative technologies to assess and monitor an individual patient’s hemodynamic profile, using parameters in addition to blood pressure, before and during admission to a critical care unit. The goal is to optimize tissue perfusion and oxygen delivery rather than targeting a single blood pressure goal as we advance and personalize care for the critically ill.Individualization of mean arterial pressure (MAP) goals is important in the post-cardiac surgery patient, as different comorbidities, operative interventions, and the presence of mechanical circulatory support, amongst other criteria, may influence the MAP required to maintain adequate perfusion.Almost 10 years after the impressive work by the SEPSISPAM  group, we need to think of trials with appropriate patient populations, targets, and outcomes, as studies have shown that certain vulnerable patient populations may need a higher blood pressure target.Other blood pressure components, namely mean, systolic, and diastolic pressures, are comparable in their strength of association with organ system injury and intensive care unit (ICU) mortality.Hospital outcomes, including mortality, are worse in patients with evidence of both hypotension and hypoperfusion.Analysis comparing perfusion pressure deficits and MAP deficits have been seen to have a nearly similar and strong association with ICU mortality. This is a critical area of focus.As intensivists, we should move away from a single predefined blood pressure target for all patients and toward a more individualized approach based on patient characteristics.Houston Methodist is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians.1 AMA PRA Category 1 Credit™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.Houston Methodist designates this Journal-based CME activity for a maximum of learn.houstonmethodist.org/MDCVJ-19.4.Click to earn CME credit:


The objective of this analysis was to evaluate the effect of a diet rich in animal protein and low in glycemic index on blood pressure during pregnancy.post hoc, secondary data analysis of a randomized controlled trial, evaluated blood pressure in pregnant participants who were randomized either to an ad libitum diet with high protein and low glycemic index, rich in dairy and seafood, or an ad libitum control diet according to national recommendations.This The study occurred in pregnant women in Copenhagen, Denmark.A total of 279 pregnant females with overweight or obesity were enrolled.Blood pressure was measured at 5 timepoints during pregnancy from gestational week 15 through week 36, and blood pressure between groups was compared.p = 0.02).There were no differences between diet arms in systolic or diastolic blood pressure over time. There were also no differences in most blood-pressure-related pregnancy complications, including the prevalence of premature birth, preeclampsia, or hypertension, but the frequency of total cesarean sections was lower in the active than the control group (16 out of 104 vs. 30 out of 104) (Increased animal protein intake was not associated with changes in blood pressure in pregnant women with overweight or obesity.ClinicalTrials.gov], identifier [NCT01894139].[ During pregnancy, women are confronted with an onslaught of new and sometimes conflicting information about how best to maintain health through this period of metabolic adaptations . Differi2 or ≥ 30 kg/m2, respectively], because excess body weight and body fat during pregnancy are associated with many health risks, including increased risk of cesarean section, premature delivery, excess birthweight, abnormal glucose and lipid metabolism, hypertensive disorders, postpartum hemorrhage and neonatal asphyxia (This concern is particularly relevant for women with overweight or obesity .Maternal height was measured to the nearest 0.5 cm at screening by using a wall mounted stadiometer . Pre-pregnancy weight was obtained either through self-report or through the participants’ medical records. Subsequent maternal body weights were measured to the nearest gram at screening, and at each of the 9 dietary counseling sessions throughout the intervention using a medical scale  with the female being barefoot and in light clothing or undergarments. Gestational weight gain (GWG) was calculated as  to its vitamin D receptor and is diminished when vitamin D status is impaired (Obesity is associated with higher serum parathyroid hormone (PTH) levels derived from increased vitamin D deposition in adipose tissue. This reduces systemic vitamin D bioavailability and subsequently impairs the negative feedback mechanism between vitamin D and PTH , 20. Theimpaired . AccordiThe United States Endocrine Society recommends a 25-hydroxyvitamin D level of around 75 nmoL/L as normal during pregnancy, though some literature suggests a level of 100 nmoL/L for optimal maternal and offspring outcomes , 28. HowAnother potential mechanism involves obesity and the inhibition of magnesium and potassium’s role in nitric oxide production and endothelial relaxation and vasodilation . In obesDespite the high consumption of dairy foods by the participants in the experimental group, intakes of some of the blood pressure-associated micronutrients found in dairy was—in both groups—below the Nordic Nutrition Recommendations  . Also, aad libitum diet with high protein and low glycemic index, which was rich in dairy, seafood, and meat, and those in the control group to an ad libitum diet with moderate protein and moderate glycemic index.The pregnancies in the present study were largely uncomplicated, particularly in regard to outcomes that might have been influenced by obesity and blood pressure. The consumption of similar amounts of fat, but with larger differences in carbohydrates, glycemic index, and protein, had no impact on the blood pressure responses during pregnancy in this at-risk group of women. Furthermore, the women consumed relatively high amounts of saturated fat, above the recommendations of <10% of daily intake, and close to the upper limit of the recommendation for sodium (2.4 g/d) with no adverse impact on their health during pregnancy, or on the health of their offspring . Both diThe study had several strengths. The combination of several FFQ and 24-HR has been shown to increase precision of food intake estimates—crucial in nutrition and diet studies . FurtherHowever, the study also had several limitations. Blood pressure was not recorded after gestational week 37 and until birth, when a potential spike in blood pressure might have occurred. The relatively small sample size might have increased the likelihood of type 2 error, particularly regarding detecting blood-pressure related pregnancy complications. Lastly, a challenge of secondary data analysis is the inability to control the data collection process retrospectively. Though there is a standard for blood pressure measurement in Denmark, the protocol had no standard operating procedure for blood pressure, and there was no case report form confirming details which may alter blood pressure reading, including type of cuff used , placement of cuff over clothing, talking during the measurement, body positioning during measurement, and amount of time resting beforehand , 41. TheUltimately, the two diet patterns made little difference on blood pressure physiology, contradicting the 2021 American Heart Association’s guidelines for cardiovascular health which provide counsel on appropriate protein types for optimal health . These rnge@cslt.dk.The data analyzed in this study is subject to the following licenses/restrictions: data may be requested from the authors of the original paper. Requests to access these datasets should be directed to The studies involving humans were approved by Ethics Committee of the Capital Region of Denmark (H-3-2013-119). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.EL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Writing – original draft, Writing – review & editing. FM: Data curation, Methodology, Software, Supervision, Writing – review & editing. HZ: Writing – review & editing. JS: Writing – review & editing. AA: Writing – review & editing. NG: Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing – review & editing.


The primary aim was to determine the association of intracranial hemorrhage lesion type, size, mass effect, and evolution with the clinical course during neurointensive care and long-term outcome after traumatic brain injury (TBI).In this observational, retrospective study, 385 TBI patients treated at the neurointensive care unit at Uppsala University Hospital, Sweden, were included. The lesion type, size, mass effect, and evolution (progression on the follow-up CT) were assessed and analyzed in relation to the percentage of secondary insults with intracranial pressure > 20 mmHg, cerebral perfusion pressure < 60 mmHg, and cerebral pressure autoregulatory status (PRx) and in relation to Glasgow Outcome Scale-Extended.p < 0.05) and acute subdural hematoma (p < 0.001) volume, greater midline shift (p < 0.001), and compressed basal cisterns (p < 0.001) correlated with craniotomy surgery. In multiple regressions, presence of traumatic subarachnoid hemorrhage (p < 0.001) and intracranial hemorrhage progression on the follow-up CT (p < 0.01) were associated with more intracranial pressure-insults above 20 mmHg. In similar regressions, obliterated basal cisterns (p < 0.001) were independently associated with higher PRx. In a multiple regression, greater acute subdural hematoma (p < 0.05) and contusion (p < 0.05) volume, presence of traumatic subarachnoid hemorrhage (p < 0.01), and obliterated basal cisterns (p < 0.01) were independently associated with a lower rate of favorable outcome.A larger epidural hematoma (The intracranial lesion type, size, mass effect, and evolution were associated with the clinical course, cerebral pathophysiology, and outcome following TBI. Future efforts should integrate such granular data into more sophisticated machine learning models to aid the clinician to better anticipate emerging secondary insults and to predict clinical outcome. Traumatic brain injury (TBI) leads to a significant burden of mortality and morbidity worldwide , 30. TheThis was a retrospective analysis of prospectively collected data, conducted at the Department of Neurosurgery at the Uppsala University Hospital, Sweden. There were 514 TBI patients aged 16 or older who received ICP-monitoring at our NIC unit between 2008 and 2018. Four hundred seventy-two of these had long-term outcome data, 409 patients had at least 12 h of available ICP data the first 7 days post-injury, and those 385 that also had two available computed tomography (CT) scans (and the 2nd within 48 h from the first scan) made up the final study cohort.2 ≥ 12 kPa, arterial glucose 5–10 mM, hemoglobin ≥ 10 g/dL, electrolytes within normal ranges, normovolemia, and body temperature < 38 °C.The patients were treated according to our standardized ICP- and CPP-oriented TBI management protocol, which has been described in detail in previous studies . Treatme2 4.0–4.5 kPa). Neurological wake-up tests were done three times per day. In situations of increased ICP, despite basic treatment and if no mass lesion was present, an EVD was used to drain cerebrospinal fluid. If ICP was still refractory elevated, the patients were held continuously sedated and stress was treated with increased sedation, pain relief, β1-antagonists, and/or α2-agonists. If the patients did not respond to these treatments, last-tier treatments included thiopental infusion (if there was no midline shift and uncus herniation) and/or decompressive craniectomy (DC) [Unconscious (GCS M 1–5) patients were intubated, mechanically ventilated, and received propofol and morphine for sedation and analgesia, respectively. ICP was monitored with an external ventricular catheter  or an intraparenchymal sensor device  in all unconscious patients. Surgical evacuation was done in case of significant intracranial mass lesions. Basal ICP-lowering treatments included head elevation to 30° and mild hyperventilation (pCOomy (DC) .The physiological variables  and ICP) were recorded at 100 Hz using the Odin software . ABP wasThe initial post-traumatic CT scan of the brain was evaluated according to the Marshall classification  for eachClinical outcome was assessed by specially trained personnel with structured telephone interviews at 6 months post-injury using the Extended Glasgow Outcome Scale (GOS-E), which has eight categories of global outcome, from death to upper good recovery , 32. TheThe primary aim was (i) to determine the association between intracranial hemorrhage lesion type, size, mass effect, and evolution in relation to the clinical course (ICP-lowering treatments and NIC variables) and secondarily (ii) in relation to clinical outcome.10-transformed due to skewness of data. Similarly, a multiple logistic regression analysis for favorable outcome was done with age, GCS M, pupillary status, EDH (mL), ASDH (mL), tSAH (yes), IVH (mL), contusion (mL), midline shift (mm), basal cisterns (open/compressed/obliterated), and intracranial hemorrhage evolution (progression and emergency neurosurgery) as independent variables. Missing data were rare and these few cases were excluded from the analyses. A p-value < 0.05 was considered statistically significant. The statistical analyses were performed in SPSS version 28 .Demography, admission status, treatments, radiological measures, NIC variables, and clinical outcome were described as median (interquartile range) or number (proportion), depending on the type of data. The association between EDH (mL), ASDH (mL), tSAH (yes/no), IVH (mL), contusions (mL), midline shift (mm), and basal cisterns  with ICP-lowering treatments , NIC variables , and clinical outcome (GOS-E) was analyzed with the Spearman correlation coefficient. The hematoma volume was denoted as 0 mL in the analyses if the patient did not have that type of intracranial hemorrhagic lesion type. The association between hemorrhage evolution  was analyzed in relation to the same variables by using the chi-square or Kruskal-Wallis test, depending on the data . Separate multiple linear regression with ICP > 20 mmHg, CPP < 60 mmHg, and PRx, respectively, as dependent variables and age, GCS M, pupillary status, EDH (mL), ASDH (mL), tSAH (yes), IVH (mL), contusion (mL), midline shift (mm), basal cisterns (open/compressed/obliterated), and intracranial hemorrhage evolution (progression and emergency neurosurgery) as independent variables were done. In these separate regressions, the percentage of ICP > 20 mmHg and CPP < 60 mmHg were logIn this cohort of 385 TBI patients Table , median n = 186 (48%)) of the cohort exhibited a stable hemorrhage on the follow-up CT, whereas 108 (28%) showed significant hemorrhage progression, and 91 (24%) required emergency neurosurgery after the first CT.On any of the first two CT scans, 34 (9%) exhibited an EDH, 170 (44%) an ASDH, 279 (72%) tSAH, 94 (24%) IVH, and 272 (71%) contusions. The maximal size of the hemorrhage as well as the extent of midline shift and compression of the basal cisterns on any of the two first CT scans is described in Table During the first 7 days post-injury, ICP was in median 12 (IQR 9–15) mmHg and the percentage of monitoring time with ICP above 20 mmHg was in median 3 (IQR 1–10) %. During the same period, median CPP was 75 (IQR 71–82) mmHg, whereas the percentage of monitoring time of CPP below 60 mmHg was in median 4 (IQR 1–9) %. PRx was in median 0.06 (IQR − 0.03–0.14).r = 0.10, p < 0.05), ASDH , greater midline shift , and more compressed basal cisterns  were more often operated with craniotomy, whereas presence of tSAH  and larger IVH size  showed the opposite association. Greater contusion size  correlated with the need for thiopental , whereas tSAH , contusion size , and more compressed basal cisterns  were associated with DC surgery. The same variables  were also associated with more ICP insults. Greater EDH volume  and no/smaller ASDH  were associated with more CPP insults below 60 mmHg. Larger ASDH volume , midline shift , and compressed basal cisterns  were associated with higher PRx.In univariate analyses Table , patientp < 0.01; Table p < 0.01).Those with a stable follow-up CT were operated with a craniotomy and DC to a lesser extent than those with hemorrhage progression or emergency neurosurgery after the first CT (p < 0.001), no/smaller volumes of IVH , and hemorrhage progression on follow-up CT  together with younger age  were independently associated with a greater burden of ICP insults above 20 mmHg. Younger age , no/smaller volumes of ASDH , hemorrhage progression on the second CT , and emergency neurosurgery  after the first CT were independently associated with a higher burden of CPP < 60 mmHg. Lastly, higher age , lower GCS M at admission , and more compressed/obliterated basal cisterns  were associated with higher PRx.In multiple linear regression analyses Table , presencr = 0.20, p < 0.001) was associated with higher GOS-E, whereas larger ASDH , presence of tSAH , larger IVH , larger contusions , greater midline shift , and more compressed basal cisterns  were associated with lower GOS-E (Table p < 0.05), presence of tSAH (OR (95% CI) = 0.38 (0.20–0.74), p < 0.01), greater contusion size (OR (95% CI) = 0.99 (0.98–1.00), p < 0.05) and obliterated basal cisterns (OR (95% CI) = 0.05 (0.01–0.41), p < 0.01) in addition to older age (OR (95% CI) = 0.97 (0.95–0.98), p < 0.01), and lower GCS M at admission (OR (95% CI) 1.55 (1.22–1.96), p < 0.001) were associated with a lower rate of favorable outcome  and therefore more likely tolerated larger hematomas due to certain brain atrophy, which could also explain why larger ASDH size did not independently translate into more ICP problems. Also, if brain swelling occurred during evacuation of the ASDH, the bone flap was usually not repositioned, an action that likely reduced postoperative ICP problems in many severe ASDH cases. Interestingly, larger ASDHs were independently associated with less episodes of low CPP, even after adjustment for age as a potential confounder for high ABP and low ICP. Furthermore, we only found a univariate association between larger ASDH size and higher PRx, but not in multiple linear regressions. The association was likely attenuated after adjustment by higher age which correlated with both high PRx and large ASDHs. ASDHs have been associated with higher PRx in some [Not surprisingly, those with larger ASDH volumes more often required craniotomy, but, in similarity with larger EDHs, they were not more prone to require last-tier ICP treatments or develop more ICP insults. Patients with larger ASDHs were generally older  without midline shift and also that total volume was based on the sum of potentially multiple, scattered contusions more resembling a diffuse injury. In addition, for the cases with more diffuse spread of contusions involving extensive areas of the brain, DC might have been preferred over evacuation . Also, eNot surprisingly, indicators of significant mass effect, i.e., midline shift and compressions of basal cisterns, were associated with a worse clinical trajectory. Both correlated with a higher frequency of craniotomy and DC surgery. These measures of mass effect were not independently associated with more ICP insults, most likely since such malignant early signs of brain herniation required aggressive emergency neurosurgery (craniotomy and DC) which alleviated ICP post-operatively when it was monitored and analyzed. Still, compressed/obliterated basal cisterns were independently associated with disturbances in cerebral pressure autoregulation during the first 7 days post-injury, which was also found in a previous study . This haFurthermore, intracranial hemorrhage progression was independently associated with more ICP and CPP insults. This probably both reflected that these patients experienced greater intracranial hemorrhagic volumes leading to higher ICP, but also implied an evolving process between the CTs while ICP monitoring might have already been started and therefore captured in this data. However, hemorrhage progression and emergency neurosurgery were not independent predictors of worse outcome, as this was rather explained by specific hemorrhagic lesion types and mass effect.On balance, classifications of intracranial hemorrhagic lesion types, sizes, mass effect, and evolution from early CT scans post-injury may be of value for predicting the clinical trajectories and development of secondary insults of important NIC variables such as ICP, CPP, and PRx. In future efforts, it may be of value to integrate such data into machine learning models for more granular predictions of these secondary insults during NIC  and longThe strengths of this study was the relatively large patient cohort with a granular methodology including both very accurate radiological volume estimations of traumatic intracranial hemorrhages and high-frequency data for the main NIC variables of cerebral physiology. Although intracranial lesion features in relation to clinical management has been investigated previously, by, e.g., Wintermark et al. , 33, thoThe main limitations were the retrospective and single-center design of this study, which limits the external validity. Furthermore, although we strived for a high adherence to the local treatment protocol within our center, there was most likely some variation in clinical decision-making among the colleagues. This probably had some effect on the association between intracranial hemorrhage features and the treatment variables, NIC course, and outcome. Due to technical difficulties, we abstained from further analyses of tSAH volume. The analysis of IVH may to some extent be unreliable since the hemorrhage could have been diluted to various extent in the cerebrospinal fluid and we did not control for any potential difference in Hounsfield units of the IVH volume between the scans. Third, in further studies, more elaborate analyses on specific injury patterns of isolated hemorrhagic lesions and various mixed injury combinations may yield a deeper understanding on the clinical course during NIC and outcome. Similarly, it would be interesting to proceed with more granular studies of hemorrhagic volumes in relation to cerebral physiology in relation to specific interventions such as hematoma evacuation, based on pre- and postoperative CT scans. Lastly, significant hemorrhage evolution has previously been defined in many different ways . We defined it as an absolute hemorrhagic increase with 6 mL, similar to Shin et al. and a previous study by our group , 25. ThiTBI is a heterogeneous disease with several different types of intracranial hemorrhagic lesions of various sizes, mass effect, and evolution. It is generally challenging to predict the clinical course and outcome. This study provides granular radiological analyses of lesion specific variables  in relation to high-frequency variables of cerebral physiology  and long-term clinical outcome. Future efforts should to integrate such granular data into more sophisticated machine learning models to aid the clinician to better anticipate emerging secondary insults and to predict clinical outcome.


N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied “explainable” machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.The host-microbiota co-metabolite trimethylamine  TMAO is known to be atherothrombotic. Here the authors show that i) kidney function is the main determinant of serum TMAO, ii) TMAO increases kidney scarring with TGF-β1 signalling and iii) anti-diabetic drugs with reno-protective properties such as GLP1R agonists reduce plasma TMAO. The microbiota can influence host pathophysiology by producing molecules that directly alter metabolism and/or modulate cellular signaling either locally in the gut or systemically via the circulation2.Over the past two decades, the central role of the commensal gut microbiota in pathologies such as atherosclerosis and type-2 diabetes (T2D) has gained prominenceN-oxide (TMAO) is the phase-one liver N-oxide of trimethylamine (TMA). TMA is a product of the microbial6 (predominately Firmicutes) metabolism of phosphatidylcholine8, choline8, and L-carnitine11, components of the high-fat, high red meat western diet. TMA is taken up from the gut via the hepatic portal vein and N-oxidized into TMAO by host flavin mono-oxygenase 312. High circulating levels of TMAO have been linked to increased thrombotic and cardiovascular risk in animal and human studies, even after adjustment for known cardiovascular risk factors13. TMAO is therefore proposed to mediate the higher cardiovascular risk associated with high red meat and fat intake14. Fish, the consumption of which is associated with reduced incidence of cardiovascular disease14, is also a rich source of TMAO15. Dietary TMAO is subject to retro-conversion: i.e., it can undergo microbial reduction to TMA by Enterobacteriaceae followed by hepatic conversion back to TMAO16. Concurrent with diet and microbiota composition, TMAO plasma levels also reflect age17, sex18, kidney function21, and chronic diseases22. To date, the relative contribution of each of these factors to circulating TMAO levels and, therefore, elevated cardiovascular risk remains unclear. Understanding how serum TMAO levels are regulated could uncover host TMAO mechanistic targets and identify modifiable and actionable therapeutic factors to lower circulating TMAO levels.Trimethylamine 23, multivariate and univariate analyses of epidemiological data and mechanistic studies in cultured cells and rodents, we sought to objectively identify variables influencing serum TMAO levels in participants of the European multicenter MetaCardis study. Moreover, by taking advantage of the unique cross-sectional MetaCardis design, we queried how variables influencing circulating TMAO manifest at different stages of cardiometabolic disease. Capitalizing on the ML analysis, we aimed to identify (i) novel host TMAO-related mechanistic targets and (ii) a rationale for future interventions that could reduce circulating TMAO levels and thereby decrease associated excess cardiovascular risk.Here, by using a data-driven “explainable” machine learning (ML) strategy24; GLP-1RAs) with evidenced reno-protective effects25 had lower serum circulating TMAO levels when compared to propensity-score matched controls  comprising obese/overweight individuals presenting with a range of metabolic syndrome features but not overt T2D or ischemic heart disease (IHD) . Moreover, BMIS individuals with hypertension  had higher plasma TMAO  and higher fasting concentrations of uric acid  after adjustment for age, sex, country of recruitment (demographics thereafter) and body mass index (BMI) , did not improve predictions explaining 1% of TMAO variance on average  was contributed by serum metabolomic variables, with biological, dietary and microbiota taxonomic variables adding 5.5%, 4%, and 3.4%, respectively, independently explained variance Fig. . Other v28), we assessed how individual variables drive TMAO-predicting models. SHAP analysis identified 24 variables that contributed more than 4% of the regularized TMAO standard deviation (SD) to model outcomes. Of those, age affected predictions the most, followed by eGFR, urinary betaine, percentage of visceral body fat and serum butyryl-carnitine , were among those affecting model outcomes the most. This analysis suggests that kidney function is a major determinant of circulating TMAO. The impact of eGFR on model outcomes for BMIS individuals was bimodal with values over 90 mL/min/1.73 m2, the clinical cut-off value for normal kidney function in adults25, predicting reduced plasma TMAO and lower values resulting in increased predicted circulating TMAO  improved predictions when compared to the full model , presumably by removing noise, supporting the importance of the variables identified by the SHAP analysis.We next trained algorithms predicting TMAO using the 24 variables identified by our SHAP analysis (“top SHAP” model) and compared it to models trained by traditional clinical risk factorsdel Fig. . The “toN = 582; “Methods”). LASSO explained on average 14.9% of circulating TMAO variance after 100 iterations  as opposed to 18.4% by boosted trees for the full model  models to predict circulating TMAO in the left-out group using again fivefold cross-validation with all the available variables (full model) as input in BMIS  is explained by eGFR we built linear-regression models with each metabolite as the dependent variable and eGFR as the independent variable. Kidney function explained 7% of TMAO variance in BMIS  whilst the explained variance for the other metabolites ranged from 6% to 1.4% for p-cresol and urinary oxaloacetic acid, respectively 3 affected circulating TMAO in BMIS participants with a Food Frequency Questionnaire35 . After correcting for demographics and BMI, with the exception of oily fish , circulating TMAO was neither significantly associated with habitual consumption of red meat, eggs or milk nor with the estimated dietary intake of the micronutrients choline, carnitine or betaine, broadly in agreement with a previous study3 , clearance by the kidney and not dietary intake of TMAO precursors is the major determinant of fasting circulating TMAO levels and therefore of the excess cardiovascular risk associated with elevated TMAO .Our ML models suggested that diet plays a modest role in determining circulating TMAO. To corroborate this, we assessed how consumption of food items rich in TMAO dietary precursorsdy3 Fig. . In addiP = 0.033; Fig. 38 and our ML models, multivariate PERMANOVA analysis uncovered a significant, albeit weak, association between circulating TMAO and microbiota composition , that contributed at least 4% of the TMAO SD in our prediction models and was significant (pFDR <0.05) for both correlation and differential abundance analyses Fig. . CAG0190This analysis corroborated the ML models suggesting a small, albeit significant, contribution of microbiota variations on circulating TMAO and identified a bacterium newly associated with higher serum TMAO in BMIS.N = 561) and IHD sub-cohorts (N = 356). Similar to BMIS, microbiota composition and dietary variables alone performed poorly, explaining on average 2% and 1% of TMAO variance, respectively, in T2D and IHD individuals  related to the ratio of white to red meat intake and the levels of the proinflammatory cytokine IP10, a marker of adverse cardiac remodeling40 were the top variables. As in BMIS, the “top SHAP” models significantly improved predictions . Only three variables  strongly contributed as predictors across all three disease groups  and IHD (51%) disease groups  and circulating TMAO  after adjustment for demographics.In patients with T2D, ML and SHAP analyses revealed an inverse association between fecal MAO Fig. , whilst MAO Fig. . R. timoCollectively, these analyses suggest that the composition of the gut microbiota may only modestly influence circulating levels of TMAO in patients with T2D, and in subjects with IHD or T2D abundance of the CAG01909 taxon is not linked with serum TMAO.43. In BMIS, TMAO modestly, albeit significantly, modulated the inverse relationship between age and eGFR , whilst in T2D and IHD the impact of TMAO on kidney function decline with age strengthened . Accordingly, we next employed preclinical models of kidney injury to gain mechanistic insights into the potential interplay between TMAO and kidney function and the putative molecular nature of the 2nd hit.20 similar to renal fibrosis46, we investigated the impact of TMAO on human primary renal fibroblasts (HRFs). In HRFs, unlike platelets47, TMAO stimulation resulted in rapid [Ca2+]i increase , TMAO stimulation for 48 h had minimal effect on ERK1/2 or SMAD3 phosphorylation - or TMAO (0.12% w/w)-supplemented diet for 6 weeks20. Subsequently, the ureter of one kidney was ligated whilst the other kidney remained unobstructed  expression in injured kidneys of mice receiving TMAO or choline diets  surgery in mice, a model assessing renal fibrosis in the absence of other co-morbiditiesets Fig. . SimilarCollectively, our in vivo and in vitro findings are consistent with TMAO aggravating kidney fibrosis due to ERK1/2 hyperactivation synergistically, probably with activation of the TGF-β1-mediated SMAD3 pathway causing a secondary hit in our disease model.25, patients with T2D receiving GLP-1RAs had significantly lower circulating TMAO , albeit significant, association with serum TMAO levels in the MetaCardis population, in agreement with recent human studies38. We replicated associations of Firmicutes5 with circulating TMAO and uncovered novel associations between an unknown bacterium (CAG01909) and R. timonensis, a marker of less diverse diet in autistic children52. However, taxa associated with TMAO diverged between disease modalities. This could at least, partially, be due to medication, suggesting that targeting TMAO production at the species level would be ineffective.Irrespective of cardiometabolic disease severity, gut microbiota composition had a modest , as opposed to dietary L-carnitine (~71%)53, and therefore may be more available for microbial catabolism in the upper gut and large intestine, leading to overestimating its role in TMA and, thereby, TMAO production. Our observations, similar to the report in ref. 3, suggest that in non-interventional settings where individuals habitually consume meat (75 to 233 g/day in European adults37), this contributes minimally to fasting circulating TMAO variability, possibly limiting the isolated effect of dietary manipulation on TMAO levels in non-interventional settings, aside strict vegans or vegetarians.In agreement with a recent study31. Accumulation of butyryl-carnitine has been associated with abnormal mitochondrial lipid β-oxidation in T2D54, suggesting a link between TMAO and mitochondrial dysfunction consistent with the finding that TMAO binds to protein kinase R-like endoplasmic reticulum kinase and increases mitochondrial stress55.Further, our analyses revealed that the multi-omics signature associated with higher circulating TMAO concentrations and, therefore, elevated cardiovascular risk, shifted from obesity with increased insulin resistance toward overt T2D and IHD with only three variables  consistently being strong contributors across models built with the BMIS, T2D and IHD disease groups. This is in agreement with our recent report that the majority of markers of dysmetabolism manifest early during cardiometabolic disease development46, and contributed to renal fibrosis, a hallmark of kidney damage irrespective of the underlying cause that significantly contributes to eGFR decline46. However, TMAO insult alone was not sufficient to convert renal fibroblasts to myofibroblasts requiring synergy with a preexisting pathological state, i.e., pro-fibrotic signaling (“second-hit”) in the form of TGF-β1 stimulation, the most prominent pro-fibrotic cytokine46. TGF-β1 expression is increased in ligated UOO mouse kidneys56 or kidneys of patients with diabetic nephropathy57 and circulating TGF-β1 is elevated in conditions that are risk factors for CKD including hypertension, dyslipidemia and T2D58. Therefore, our results suggest that TMAO accelerates eGFR decline in concert with existing pro-fibrotic signaling , consistent with observations in animal models of CKD45 and humans21.Far from being a bystander, mediation analysis indicated that TMAO significantly accelerated the rate of kidney function decline by age, with its effect increasing in more severe disease stages. Consistent with this, our preclinical work uncovered that TMAO primed renal fibroblasts for conversion to myofibroblasts, the primary collagen-producing cells in the kidney2 (see ref. 59) and circulating TMAO levels in healthy adults were not indicative of future atherosclerotic burden60.Supporting our assertion that TMAO accelerates cardiovascular disease progression together with existing pathology, at least partially by impacting the kidney, TMAO increased all-cause mortality only in individuals with eGFR <90 mL/min/1.73 m19 with accumulating evidence suggesting direct causal effects47. Our work suggests that reno-protective strategies could potentially lower circulating TMAO and therefore preserve kidney function in individuals with high circulating TMAO in the presence of risk factors known to increase TGF-β1 signaling . Indeed, we observed that patients with T2D prescribed GLP-1RAs, drugs with documented reno-protective and beneficial cardiovascular effects25, had significantly lower fasting plasma TMAO levels than propensity-score matched controls. Differences in microbiota composition were predictive of glycemic responses to GLP-1RA intake61 and further work is required to determine factors influencing GLP-1RA-mediated renoprotection which appear to be independent of improvements in glycemic control62.TMAO is an independent risk factor of cardiovascular morbidity and mortality in patients with established IHD or CKD25.Collectively, our findings demonstrate that eGFR, irrespective of disease stage, is the primary modifiable modulator of circulating TMAO, which then by promoting renal fibrosis in conjunction with established pathophysiology (“second-hit” model) further negatively impacts kidney function. Accordingly, we observe that intake of GLP-1RAs is associated with lower circulating TMAO levels, thereby potentially moderating the adverse effect of TMAO on kidney function and suggesting a putative mechanism for these drugs that observed renoprotection in large pharmaceutical trialsOur work conceptually advances the understanding of how TMAO levels (and therefore associated cardiovascular risk) are regulated in humans with a wide range of cardiometabolic disease burden in non-interventional settings. In addition, we uncover a direct mechanistic link between TMAO and renal fibrosis in conjunction with existing co-morbidities  such as hypertension and T2D. Furthermore, our findings suggest that therapeutic modalities preserving kidney function could markedly benefit and reduce cardiovascular risk in individuals with high circulating TMAO in the presence of risk factors . This merits urgent testing in a longitudinal independent clinical trial.63. The overarching goal of the trial was to investigate the impact of qualitative and quantitative changes in the gut microbiota on the pathogenesis of cardiometabolic diseases (CMDs) and their associated co-morbidities . For the present study, patients were subclassified in three groups: BMI-spectrum patients , encompassing MetaCardis participants presenting with metabolic syndrome-related risk factors or conditions  and patients diagnosed with type-2 diabetes  or ischemic heart disease . The IHD group comprised patients with Acute (<15 days) Coronary Syndrome , Chronic IHD  with normal Left Ventricular Ejection Fraction (LVEF) determined by echocardiography and Heart Failure patients . Cardiometabolic disease is used as an umbrella term for all the above cases, and severity of cardiometabolic disease refers, in this manuscript, to the progression from single risk factors such as obesity to overt T2D and cardiac phenotype (ischemic heart disease and heart failure).MetaCardis is a cross-sectional study that recruited individuals at increasing stages of dysmetabolism and IHD severity , aged 18–75-years old and recruited from Denmark, France and Germany between 2013 and 2015. Patients under care of the participating hospitals meeting the inclusion criteria of the study were invited to enroll. Healthy controls were recruited via public advertisement. Study participants provided written informed consent and the study was undertaken according to Helsinki Declaration-II. Ethical approval was obtained from the Ethics Committee CPP Ile-de France, the Ethical Committees of the Capital Region of Denmark (H-3-2013-145), and Ethics Committee at the Medical Faculty at the University of Leipzig (047-13-28012013). Study design, recruitment and exclusion criteria has been extensively described63. Briefly, blood samples were collected in the morning after overnight fasting, and fecal samples were collected at home by participants, frozen immediately and transferred to study centers on dry ice within 48 h. All samples were stored at −80 °C until use. Clinical history, medication and phenotypic information were acquired as described63 with standardized procedures across centers. Participants reported habitual food intake through a customized Food Frequency Questionnaire (FQQ). The participant’s responses to the FQQs was validated by three web-based dietary recalls for a subset of study participants (N = 324) according to established practices35. Bioclinical variables were measured in a single center according to standard procedures31. Estimated glomerular filtration rate (eGFR) was calculated with the CKD-EPI formula without ethnicity adjustment68.Biofluid and biomatter collection has been described elsewhere31. Absolute quantifications were derived using the “In Vitro Diagnostics for research” (IVDr) quantification BI-Quant-UR algorithm . For some analyses metabolites absolute quantifications were divided by the creatinine concentration, also derived by the IVDr platform from the 1H-NMR spectra.Spectra acquisition, using an Avance spectrometer (Bruker) at 600 MHz; and structural assignments have been extensively described previously41. Briefly, protein was methanol-precipitated, methanol was evaporated to dryness and subsequent to derivatization samples were injected to an Agilent 7890B-5977B Inert Plus GC-MS system. The chromatographic column was an Agilent ZORBAX DB5- MS (30 m × 250 µm  × 0.25 µm + 10 m Duragard). The temperature gradient was 37.5 min long and the mass analyzer was operated in full-scan mode between 50 and 600 m/z. Peaks were annotated with the use of the Fiehn library . Metabolic features with low reproducibility or linearity were removed from the dataset, resulting in 102 annotated metabolic features.Serum samples (100 µl) were prepared, analyzed and processed as described41. TMAO circulating values of all MetaCardis participants were log10-transformed, and subsequently the median was subtracted and divided by the standard deviation23 . MassLynxTM software  was used for data acquisition and analysis.UPLC–MS/MS performed on a Waters Acquity UPLC-Xevo TQ-S UPLC–MS/MS system equipped with an Acquity BEH HILIC  chromatographic column was employed to determine TMA, TMAO, choline, betaine, γ-butyrobetaine, betaine aldehyde, butyryl-carnitine, isovaleryl-carnitine, OH-isovaleryl-carnitine, stearoyl-carnitine, oleoyl-carnitine, linoleoyl-carnitine, myristoyl-carnitine, lauroyl-carnitine, and decanoyl-carnitine as described previously63 using a protocol devised in ref. 69 with modifications. Briefly, total fecal DNA was extracted following the International Human Microbiome Standards (IHMS) guidelines (SOP 07 V2 H) and samples were sequenced using ion-proton technology (ThermoFisher Scientific). Gene abundance profiling was performed using the 9.9 million gene integrated reference catalog of the human microbiome, as described63. The following libraries were used to process metagenomic data: METEOR , Alientrimmer v0.4.0, Bowtie2 v2.3.4, MetaOMineR , Omixer-RPM (v1.0).Phylogenetic microbiota profiles were built after correction for bacterial load as extensively described63.Manually curated customized module sets focusing on anaerobic bacterial and archaeal fermentation processes relevant to the human gut microbiota were assembled as previously extensively describedStatistical analysis was undertaken using R . R: A language and environment for statistical computing. R (v4.03) Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/). For comparing two groups, we used Mann–Whitney U and for multiple group comparisons Kruskal–Wallis tests. Unadjusted Spearman correlations were computed using R, whilst adjusted Spearman correlations with ppcor (v1.1). P values were corrected for multiple comparisons using the Benjamini–Hochberg method.41, number of antibiotic courses in the last 5years and number of antihypertensive, anti-diabetic and lipid-lowering treatments. Dietary parameters included habitual consumption of 37 food items, daily nutrients intake derived from these food items calculated as in35, alternative Healthy Eating70 (aHEI), Dietary Approaches to Stop Hypertension71 (DASH) and Dietary Diversity72 (DDS) scores. Clinical risk factors variables included age, systolic and diastolic blood pressure, glycated hemoglobin levels, fasting cholesterol levels, smoking status and waist circumference as described31. Serum metabolomics comprised the absolute or relative levels of 116 circulating metabolites determined by GC-MS or UPLC–MS/MS41. Urine metabolomics included absolute quantification of 47 urine metabolites from 1H-NMR spectra calculated with the IVDr algorithm. Microbiota variables included an abundance of 699 bacterial species present in at least 20% of MetaCardis patients corrected for microbial load and the first 10 principal components from a PCA of relative microbial gene abundances41. Microbiome (Modules) group included abundance of 116 manually curated bacterial modules31. In all cases, categorical variables were converted into dummy variables using caret (v6.0.86).Patient phenotypic variables were separated into ten groups during the model parameter optimization step, training was stopped if predictions were not improved for ten rounds (early_stopping_rounds = 10).23.We incorporated the regularization parameters lambda and gamma in our models, thus making our models more conservativeWe introduced randomness by using 0.8–0.9 of the available variables in each variable group  that in combination with the introduced randomness and the out-of-sample predictions  makes our models conservative.To minimize the risk of overfitting, we took four steps:https://xgboost.readthedocs.io/en/latest/index.html).Please see also the XGBoost documentation for additional information on model parameters  and averaged over 100 iterations. We assigned relative importance to each variable by computing the mean of the absolute SHAP value for all individuals in each disease group similarly to ref. 23. For swarm plots depicting each individual participants SHAP values, variable values were inverse-normalized using the RNOmni (v1.0.0) package.We interpreted our ML models and assigned relative importance to variables influencing circulating TMAO levels by co-opting SHAP values, as expanded for tree-based ML modelsN = 582) participants using all available variables as input (full model). Initially, we used fivefold cross-validation to determine the best regularization parameter lambda  for predicting circulating TMAO in the left-out group. We next trained five LASSO regression models in 80% of BMIS participants excluding the corresponding left-out group with the optimal lambda parameter. These models were then used to predict regularized TMAO in the left-out groups and subsequently to calculate the coefficient of determination using the rsq function from yardstick (v0.0.7). This process was repeated for 100 iterations, similar to the xgboost analysis.We used the LASSO algorithm as implemented in the glmnet (v4.1) package to predict regularized TMAO values in BMIS  and determined statistical significance with a permutation ANOVA test (999 iterations) using the vegan (v2.5.7) package. Permutation analysis of variance (PERMANOVA) of regularized circulating TMAO versus Bray–Curtis taxonomic dissimilarity matrices with age, sex, and country of recruitment as covariates for 999 iterations were performed with vegan (v2.5.7).74 to assess the putative impact of eGFR on plasma TMAO increase with age and, conversely, the impact of TMAO on eGFR decline with age. We used the Preacher and Hayes bootstrapping method, as implemented in the mediation (v4.5.0) R package75, using general linear models with sex and country of recruitment as covariates. Confidence intervals and Bayesian P values were computed after 999 simulations.We performed mediation analysis76 (v.4.1.0) with age, sex, disease group, and hypertension status as covariates using nearest neighbor matching determined by generalized linear models. All covariates were given equal weight.MetaCardis participants with T2D were propensity-score matched using the R package MatchIt50. Fibroblasts were preincubated with Tramenitib , BAPTA-AM  or vehicle for 30 min before TMAO (Sigma) stimulation. For longer-term (24 h) stimulation, fibroblasts were in serum-free low-glucose DMEM overnight and subsequently stimulated with TMAO, TGF-β1 , or their combination. At the end of the experiment, fibroblasts were lysed and stored at −80 °C until further use, as described50.Primary human adult kidney fibroblasts from a single donor  were cultured in 10% FCS low-Glucose DMEM with 1% Pen/Strep antibiotics (Sigma). For acute (up to 30 min) TMAO stimulation, cells  were serum-starved for 1 h in a physiologic serum-free buffer as previously described2+ and Mg2+. Measurements were obtained on an epifluorescence inverted microscope equipped with a ×20 fluorite objective. Single-cell intracellular Ca2+ ([Ca2+]i) was monitored using excitation at 340 and 380 nm, through a monochromator (Cairn Research). Emitted light was reflected through a 515 nm filter to a QImaging Retiga CCD camera (Cairn Research) and digitized to 12-bit resolution. All imaging data were collected and analyzed using software from Andor.Fibroblasts were serum-starved overnight in low-glucose DMEM and subsequently loaded with 5 µM fura-2-AM (Molecular Probes) in pH 7.4 Hanks Balanced Salt Solution  containing Ca20. Food and water were available ad libitum, and mice were held at 12/12 h light/dark cycle at 20–23 °C and 40–60% humidity in standard individually ventilated cages . Subsequently, Unilateral Ureter Obstruction (UUO) was established as previously described50. Only male mice were used, since this is common practice for this model50. Briefly, mice were anesthetized with isoflurane and the abdominal cavity was exposed using midline laparotomy, the right ureter was isolated and tied off 0.5 cm from the pelvis. The left ureter was left unclamped and served as the sham-operated control. At day 5, mice were euthanized by terminal anesthesia with ketamine and cardiac puncture, and subsequently the kidneys were harvested. Both kidneys from each animal (UUO and sham) were cut in half longitudinally. One-half of each kidney was snap-frozen in liquid nitrogen and subsequently stored at −80 °C for Western blotting. The other half was fixed with 10% Formalin (Sigma) for 16 h at 4 °C, transferred to a 70% v/v ethanol solution for a further 24 h and was then paraffin-embedded for immunohistochemistry.All animal experiments were conducted in accordance with the United Kingdom Home Office Animals 1986 Scientific Procedures, with local ethical committee approval (Project License 70/8356). Male C57BL/6 J mice (Charles River) at 6–8 weeks of age were fed a control, 0.12% w/w TMAO- or 1% w/w choline-containing diets ; 6 animals/group) for 6 weeks49. AmershamTM RainbowTM full-range molecular weight (MW) markers  were used to determine MW and cut membranes for probing with specific antibodies. Proteins were visualized with ECL Prime . Optical densities of bands of interest were determined using ImageJ 1.46r (NIH) and normalized against loading controls. The value of the normalized control sample was arbitrarily set to 1. Membranes were stripped using the Restore Plus reagent (Fisher Scientific), and re-probed with appropriate loading controls . In cases where there was overlap in MW between loading controls and proteins of interest, loading controls were run in parallel gels. The following antibodies were used in the present study with dilutions in parentheses: From Cell Signaling Technology, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (1:3000) #9101, p44/42 MAPK (Erk1//2) Antibody (1:5000) #9102, Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb (1:1000) #9520, Phospho-p70 S6 Kinase (Thr389) Antibody (1:1000) #9205, Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (1:1000) #2211, Phospho-4E-BP1 (Ser65) Antibody (1:1000) #9451, Vimentin (R28) antibody (1:1000) #3932. From Sigma-Aldrich, monoclonal anti-actin, α-Smooth Muscle (clone 1A4) A2547 , monoclonal Anti-α-Tubulin antibody  T9026, anti-β-actin antibody  A2228.We used the NuPAGE electrophoresis and buffer system (Invitrogen) for immunoblot analysis of kidney samples or cellular lysates as described50 with the DISCOVERY XT (Ventana) automated slide processing instrument using the OmniMap reagents (Ventana), according to the manufacturer’s recommendations. Images were captured at ×20 magnification, using a Zeiss AxioPhot microscope with an AxioCam HRc camera. Five kidney cortex images were captured per mouse, and staining was quantified as the percentage of the total area, using ImageJ 1.46r (NIH). The Anti-F4/F80 (Cl:A3-1) antibody (1:50) from Biorad (MCA497) was used.Formalin-fixed kidneys were embedded in paraffin, and 4-μm sections were cut by the Barts Cancer Institute Pathology Unit. Staining for Sirius red, α-Smooth Muscle Actin (αSMA), and F4/F80 were performed as describedFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data