Postoperative acute kidney injury (PO-AKI) is a common complication of major surgery that is strongly associated with short-term surgical complications and long-term adverse outcomes, including increased risk of chronic kidney disease, cardiovascular events and death. Risk factors for PO-AKI include older age and comorbid diseases such as chronic kidney disease and diabetes mellitus. PO-AKI is best defined as AKI occurring within 7 days of an operative intervention using the Kidney Disease Improving Global Outcomes (KDIGO) definition of AKI; however, additional prognostic information may be gained from detailed clinical assessment and other diagnostic investigations in the form of a focused kidney health assessment (KHA). Prevention of PO-AKI is largely based on identification of high baseline risk, monitoring and reduction of nephrotoxic insults, whereas treatment involves the application of a bundle of interventions to avoid secondary kidney injury and mitigate the severity of AKI. As PO-AKI is strongly associated with long-term adverse outcomes, some form of follow-up KHA is essential; however, the form and location of this will be dictated by the nature and severity of the AKI. In this Consensus Statement, we provide graded recommendations for AKI after non-cardiac surgery and highlight priorities for future research. The development of acute kidney injury (AKI) after major non-cardiac surgery is associated with substantial long-term morbidity and mortality. This joint Consensus Statement from the Acute Disease Quality Initiative and the PeriOperative Quality Initiative provides recommendations for the definition, prevention and management of postoperative AKI. AKI can be considered a sentinel postoperative complication that is strongly associated with increased risk of death, co-occurrence of other postoperative complications, increased length of hospital stay and the development of progressive chronic kidney disease (CKD), which results in a considerable health-care and societal burden7.The development of postoperative complications after major non-cardiac surgery — particularly acute kidney injury (AKI) — has consistently been associated with substantial long-term morbidity and mortality8. However, the role of AKI after other forms of major surgery, particularly gastrointestinal surgery, has received less attention, even though such procedures are associated with higher 30-day mortality than many forms of cardiac surgery9. Despite the importance of AKI as a complication after all forms of major surgery, lack of consensus exists with regard to the definition, prevention and treatment of postoperative AKI (PO-AKI) and the pathophysiology of PO-AKI in the non-cardiac setting has not been well described. Furthermore, the understanding of and approach to this condition can differ between specialists in nephrology, anaesthesia and/or perioperative medicine.Considerable research and previous consensus meetings have focused on AKI after cardiac surgeryIn 2019, a joint meeting of the Acute Disease Quality Initiative (ADQI-24) and the PeriOperative Quality Initiative (POQI-7) was convened to address PO-AKI after major non-cardiac surgery. Here, we present our recommendations for clinical practice as well as for further research in this important area.10. The broad objective of ADQI 24 was to produce expert-based statements and a summary of current knowledge pertaining to the definition and management of PO-AKI in the setting of non-cardiac surgery for use by clinicians and researchers, based on review of evidence by and professional judgment of the faculty as well as to identify evidence gaps to establish research priorities.The Conference Chairs of the 24th ADQI/7th POQI consensus committee  convened a diverse panel of clinicians and researchers representing relevant disciplines  from Europe, North America and Australia, to discuss the issues relating to PO-AKI. The consensus meeting was held at Emmanuel College, Cambridge, UK, on 4–7 September 2019, and followed the established ADQI process and POQI protocols, including use of a modified Delphi method to achieve consensus11. A number of ungraded research recommendations were also identified in areas where high-quality evidence is lacking. Strength of recommendations and aggregated conclusions were established by consensus of all of the participants, who formally voted and approved the consensus recommendations.Conference participants were divided into five work groups: group 1 was tasked with the epidemiology and pathophysiology of PO-AKI. Group 2 was tasked with developing a definition for PO-AKI. Group 3 developed recommendations for the prevention of PO-AKI. Group 4 focused on treatment options for patients with PO-AKI, and group 5 explored outcomes after PO-AKI . Members13  is a clinical syndrome rather than a single disease. The majority of cases of PO-AKI are multifactorial in cause (ungraded).Consensus Statement 1bThe incidence of PO-AKI (defined by changes in serum creatinine) varies with the characteristics and urgency of surgery. The incidence of AKI after day or ambulatory surgery is uncertain (ungraded).Consensus Statement 1cAKI defined by transient oliguria is more common in the intraoperative and postoperative period than AKI defined by serum creatinine. Severe oliguria and anuria, even in the absence of changes in serum creatinine, are associated with increased long-term morbidity and mortality (ungraded).Consensus Statement 1dThe majority of observational studies focus on AKI in the immediate postoperative period. Little evidence is available regarding the epidemiology of acute kidney disease in the perioperative setting (ungraded).Consensus Statement 1e2, diabetes mellitus, heart failure, ascites, hypertension, emergency surgery, intraperitoneal surgery, number of medications, use of angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), high American Society of Anesthesiology Physical Status classification score and albuminuria. Patients with chronic kidney disease (CKD) and/or diabetes are at a particularly high risk of AKI (ungraded).Risk factors for PO-AKI include age >50 years, male sex, glomerular filtration rate <60 ml/min/1.73 m22. Adoption of this consensus definition has resulted in a greater appreciation of the incidence, varied presentation and outcomes of PO-AKI  consensus criteria to define PO-AKI in clinical trials and epidemiological studies35. One study suggested that discrimination for subsequent adverse outcomes could be enhanced by testing for novel biomarkers. In this study, which included some patients with PO-AKI, the sum of the concentration of tissue inhibitor of metalloproteinases 2 (TIMP2) multiplied by that of insulin-like growth factor binding protein 7 (IGFBP7) added prognostic information to AKI defined by urine output alone ([TIMP2]×[IGFBP7] > 2 was associated with progression from stage 1 AKI)36.More is known about PO-AKI defined by change in serum creatinine than that defined by perioperative decrements in urine output. The prognostic implications of intraoperative transient oliguria are controversial, with some studies reporting a higher risk of subsequent adverse outcomes and others suggesting no increase in risk39. In a study of PO-AKI after non-cardiac surgery, the incidence of AKI increased from 8% to 64% if urine output was included in the definition37. Some postoperative oliguria is assumed to be an appropriate physiological response to decreased intravascular volume, vasodilatation or non-osmotic release of arginine vasopressin in response to tissue injury40; however, sustained decrements in urine output, even without accompanying changes in serum creatinine, have been associated with longer-term morbidity and mortality41.Many, but not all, of the available studies suggest that PO-AKI defined by oliguria alone is more common than PO-AKI defined by changes in serum creatinine levels42. A study that evaluated substantial decline in kidney function, defined as a 30% decrease in estimated glomerular filtration rate at 60 days after surgery, suggested that the incidence of this outcome was only 2% among patients without PO-AKI (defined by change in serum creatinine only), but 10%, 17% and 29% among patients who experienced PO-AKI stages 1, 2, and 3, respectively43. The incidence of AKD in the general population is likely lower than in the post-surgery population; in a study of Canadian electronic health records, 5.2% of patients in a given period had AKD with or without AKI44.The concept of acute kidney disease (AKD) is intended to bridge the gap between AKI, which refers to an acute alteration in kidney function over a 7-day period, and CKD, which refers to a sustained alteration in kidney function over a 3-month period. AKD therefore refers to AKI that persists for more than 7 days after the initial insult, or a progressive decline in kidney function that does not meet AKI criteria on a week-by-week basis. Few epidemiological data are available on the incidence and prognosis of postoperative AKD, despite its likely importance as a bridge to development of sustained kidney dysfunction and CKD40. The three validated risk scores for PO-AKI include male sex, age >50 years, diabetes mellitus, hypertension, ascites, heart failure, emergent surgery, intraperitoneal surgery, poly-pharmacy, use of an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB), and increasing American Society of Anesthesiologists physical status classification score46. Albuminuria and hypoalbuminaemia have also been described as risk factors for PO-AKI50. Risk factors for PO-AKI occurring in patients in the ICU more than 48 h after surgery reflect risk factors for AKI in critical illness including the need for new nephrotoxic medication, sepsis and shock52. Whether targeting modifiable risk factors for early PO-AKI, for example, using perioperative haemodynamic interventions, will impact the incidence of AKI later in the postoperative period, >48 h after surgery, remains uncertain.Several patient-related, surgery-related, and anaesthetic-related risk factors for developing early PO-AKI during the first 48 h after surgery have been described53. However, existing validated risk scores for PO-AKI were developed using a variety of older (pre-KDIGO) definitions of AKI or were applied to select surgical settings46. More generalizable prognostic clinical tools are needed to accurately risk-stratify patients preoperatively, particularly to distinguish those patients at the highest levels of risk (e.g. >20–30%). In the absence of such tools, patients with CKD and those with diabetes can reasonably be considered to be at increased risk of PO-AKI. The KDIGO guideline for the diagnosis, evaluation and management of AKI suggests that age >60 years, emergency surgery, elevated American Society of Anesthesiologists physical status classification score and preoperative comorbid illnesses, including diabetes and any chronic disease of the heart, lung or liver, are risk factors for PO-AKI42.Use of a validated clinical risk score is a potentially useful approach to risk stratify patients for targeted interventions or to facilitate the conduct of randomized controlled trials (RCTs)Understanding the pathophysiology and epidemiology of PO-AKI is central to the identification and development of novel preventative therapies and treatments. This understanding may be improved by conducting prospective studies that include collection of biological samples for histological and biomarker examination as well as retrospective analyses that examine shared pathogenesis across a spectrum of perioperative complications. The incidence of PO-AKI in the day and ambulatory surgery settings is uncertain and should be investigated in epidemiological studies. Development of better risk prediction tools for PO-AKI could help to risk-stratify patients and set thresholds of predicted risk that merit adjustments in perioperative care.54 and is supported by evidence from numerous clinical studies57. Furthermore, within the 7-day window, different timing of AKI may be indicative of the nature of the kidney insult, that is, intra-operative events versus post-operative insults.Although the KDIGO definition of AKI is now widely employed in the perioperative literature, the exact implementation of this definition varies considerably in terms of timeframe of diagnosis. We recommend defining PO-AKI as occurring when existing KDIGO criteria for AKI are met within 7 days of an operative intervention Box . AKI occ51. Use of a KHA was previously recommended by ADQI in the context of quality improvement processes for monitoring patients at a high risk of AKI before surgery58. A KHA comprises a structured evaluation that includes previous history of AKI, current medications, cardiovascular health, haemodynamic status and markers of kidney function (i.e. serum creatinine) and kidney damage (i.e. urine dipstick). Use of a KHA in the perioperative period involves integrated assessment of patient-specific and procedure-specific risk factors, clinical context and resource setting.Notably, AKI could potentially be present before surgery , highlighting the importance of a risk-based kidney health assessment (KHA)Importantly, the KHA approach can be used to perform context-dependent evaluation both before and after surgery, when AKI is diagnosed and during follow-up Fig. . A perio59, which may spuriously increase or decrease measured values, as well as perioperative changes in creatinine production that alter serum creatinine levels independently of changes in GFR60. Urine output is also likely to be influenced by multiple physiological factors specific to the perioperative period, including intravascular volume status, relative or absolute hypotension and neurohormonal response to surgery. Data suggest that although intraoperative oliguria is associated with PO-AKI, the positive predictive value is low32. We therefore recommend that urine output is interpreted in the context of severity of oliguria, clinical setting and other corroborating evidence of PO-AKI. Thus, isolated perioperative oliguria meeting AKI stage 1 criteria in the context of transient hypotension should not be viewed as being the same as more severe or prolonged oligo-anuria unresponsive to haemodynamic interventions. The presence of new evidence of kidney tubular damage (e.g. on urine microscopy or from novel biomarkers) or evidence of evolving sepsis in the context of early oliguria would be suggestive of AKI; these factors can be identified and integrated within a timely postoperative KHA.Conventional diagnostic criteria for AKI might be influenced by multiple factors specific to the perioperative period, rendering their predictive utility for PO-AKI less certain. Serum creatinine concentration is subject to abrupt changes in total body waterBox 2 Definition of PO-AKIConsensus Statement 1aWe recommend defining postoperative acute kidney injury (PO-AKI) as occurring when existing Kidney Disease Improving Global Outcomes (KDIGO) criteria for AKI are met within 7 days of an operative intervention (ungraded).Consensus Statement 1b.We recommend performing a risk-based Kidney Health Assessment (KHA) preoperatively and postoperatively, with the specific methods and frequency of assessment determined by clinical judgement based on individual patient-specific and procedural-specific risk factors and the available resources Consensus Statement 1cWe recommend using the term postoperative acute kidney disease (PO-AKD) when KDIGO criteria for AKI are present after postoperative day 7 and objective evidence of injury initiation was present within the first 7 postoperative days (ungraded).54 or AKD61 without the postoperative prefix. With this proposed nomenclature  occurs in patients with PO-AKI who continue to meet KDIGO criteria for AKI >7 days after surgery. Patients whose rise in serum creatinine level begins before postoperative day 7 but whose serum creatinine level does not increase ≥50% above baseline until 7–90 days after surgery would also meet PO-AKD criteria based on a slow progressive deterioration in kidney function in the postoperative period Box . Howeverure Fig. , it sougLimited information exists regarding the prognostic implications of the nature and timing of the AKI diagnosis within 7 days after surgery, and these important aspects of the PO-AKI diagnostic criteria warrant better characterization. Furthermore, to what extent indices of kidney injury other than serum creatinine and urine output, such as urinary or plasma biomarkers, are important for prognostication in PO-AKI is unknown and should be the subject of further study. Whether or not important differences exist in the epidemiology and prognostic significance of PO-AKD that arises as persistent PO-AKI compared with PO-AKD that develops after a gradual rise in creatinine after day 7 is also unknown.The impact of routine, structured KHA on the detection of PO-AKI remains to be determined. An expanded perioperative urinalysis conducted as part of a detailed KHA might identify previously unknown pathology and could potentially lead to additional, potentially expensive and/or invasive investigations that could delay surgery. The presumed benefits and cost-effectiveness of a KHA therefore require confirmation. Finally, the optimal interpretation of intraoperative and postoperative oliguria remains unclear and should be the subject of further study.62. Although the avoidance of potential nephrotoxins is considered to be a cornerstone of AKI prevention, there is a relative paucity of data supporting this approach. However, routine use of gentamicin for surgical prophylaxis was associated with an increased risk of AKI following orthopaedic surgery63 and, in the paediatric setting, systematic screening for nephrotoxin use, with a focus on close monitoring in patients exposed to multiple nephrotoxins and nephrotoxin cessation, has been shown to reduce rates of AKI65.Surgery is sometimes performed in patients with established AKI; however, common perioperative interventions focus on prevention of PO-AKI Box . As neph66.At present, common practice is to discontinue use of ACE inhibitors and ARBs before surgery to avoid perioperative hypotension. This approach is assumed to reduce the risk of perioperative AKI based on physiological rationale. In line with this established practice, we recommend discontinuing ACE inhibitors and ARBs for a minimum of 24 h before surgery to minimize the risks of perioperative hypotension and/or postoperative AKI Box . This re24.NSAIDs are generally avoided in surgical patients at a high risk of AKI, but might be beneficial as opiate-sparing agents in patients who are at a low risk of AKI. Although NSAIDs are widely used in patients with normal kidney function, their impact on risk of AKI in the postoperative period is unclear67.To date, no preoperative pharmacological intervention has been shown to reduce the risk of PO-AKI in RCTs. However, attention should be paid to ensuring euvolaemia prior to surgery and addressing or avoiding preoperative fluid losses caused by excessive fasting, bowel preparation and/or acute illnessBox 3 Prevention of PO-AKI: preoperative strategiesConsensus Statement 3a.We recommend that when considering use of medications or interventions that are associated with an increased risk of acute kidney injury (AKI), the relative benefits and/or necessity of the medications or interventions should be balanced with the risk of postoperative AKI (PO-AKI). This recommendation applies to outpatient medications, new medications and exposures including contrast media Consensus Statement 3b.We recommend discontinuing angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) for a minimum of 24 h before surgery, dependent on the specific medication, to minimize the risks of perioperative hypotension and/or PO-AKI Consensus Statement 3c.We recommend assessing the relative risks and benefits of use of NSAIDs (including COX2 inhibitors) in individual patients. In general, in patients at a high risk of PO-AKI, the risk of AKI associated with use of NSAIDs might outweigh the benefits with regard to opioid minimization, whereas in low-risk patients, the benefits of use of NSAIDs might outweigh the risks. The risk–benefit profile in patients at a moderate risk of AKI is particularly uncertain 68. Algorithms that use cardiac output monitoring to optimize cardiac stroke volume and/or increase global oxygen delivery, known as goal-directed therapy, have been the subject of multiple RCTs. A Cochrane review and meta-analysis suggested that goal-directed therapy can reduce the risk of PO-AKI70. Large observational studies have shown strong associations between intraoperative hypotension and PO-AKI with the risk of organ injury a function of the severity and duration of the hypotension73. Both absolute mean arterial blood pressure (MAP (e.g. 60–70 mmHg)) and relative MAP (e.g. <30% reduction from baseline) thresholds are associated with PO-AKI. The results of an interventional trial of use of a MAP target of >65 mmHg as part of a goal-directed strategy have added weight to these observational data, demonstrating fewer postoperative complications (including AKI) and shorter length of hospital stay with this approach74. Similar to other settings such as sepsis, a higher MAP target may be beneficial in reducing the incidence of AKI in patients with poorly controlled pre-existing hypertension75.Intravenous fluid and haemodynamic management in the intraoperative and early postoperative period has a major impact on the development of PO-AKI Box . The REL78 .We recommend not using restrictive or zero-balance perioperative fluid regimens in major elective surgery (except in specific circumstances). Such regimens are associated with increased risk of postoperative acute kidney injury (PO-AKI) Consensus Statement 4b.We recommend the use of goal-directed haemodynamic therapy in high-risk patients to optimize volume status, blood pressure and cardiac output and reduce the risk of PO-AKI Consensus Statement 4c.We recommend maintaining an intraoperative mean arterial blood pressure (MAP) >65 mmHg to reduce the risk of PO-AKI. The risk of AKI is a function of both the magnitude of hypotension and its duration. For selected patients, including those with pre-existing systemic hypertension, a higher MAP target should be considered Consensus Statement 4d.We recommend the perioperative use of balanced crystalloids rather than 0.9% saline to reduce the risk of PO-AKI Consensus Statement 4eIn the setting of elective surgery outside of the ICU, insufficient evidence exists to make a recommendation regarding the choice of crystalloids or colloids for volume expansion to modify the risk of PO-AKI (ungraded).84. These findings suggest that maintenance of fluid and haemodynamic status in the early postoperative phase is important to avoid organ injuries including AKI28. A trial that used stroke volume optimization and vasopressors to target a MAP within 10% of the preoperative baseline value during and for 4 h after surgery showed that this strategy was associated with a reduction in postoperative organ injury, but was not powered to assess kidney-specific outcomes85. Further research is warranted to evaluate postoperative monitoring strategies and ideal clinical settings to reduce the risk of PO-AKI in moderate- to high-risk patients.The early postoperative period has not been extensively studied in relation to kidney outcomes. However, therapeutic strategies used in the hours after surgery could likely modify the risk of AKI Box . Clinica86. Failure to restart chronic ACE inhibitor or ARB therapy that was suspended preoperatively is associated with increased 30-day mortality88. However, the optimal time point to restart these medications has not been clearly established. Given their effects on the kidney, a reasonable approach would be to conduct a focused KHA before restarting ACE inhibitors or ARBs.As postoperative hyperglycaemia is strongly associated with AKI, avoidance of perioperative hyperglycaemia (>180 mg/dl) is recommended89.Enhanced recovery after surgery pathways are typically multimodal, multidisciplinary interventions that have been promoted to facilitate recovery from surgery. Use of NSAIDs to minimize opiate analgesia requirements is a common component of such pathways, along with avoidance of markedly positive fluid balance. Accordingly, in some settings such as colorectal surgery, implementation of an enhanced recovery after surgery pathway could be associated with a higher, rather than lower, incidence of AKIBox 5 Prevention of PO-AKI: postoperative strategiesConsensus Statement 5a.We recommend maintenance of adequate organ perfusion, including attention to euvolaemia and treatment of hypotension in the early postoperative period, to reduce the risk of postoperative acute kidney injury (PO-AKI). Monitoring and targeting haemodynamic parameters may require enhanced levels of monitoring and/or care depending on the clinical context and patient risk profile Consensus Statement 5b.We recommend treatment of postoperative hyperglycaemia (target glucose <180 mg/dl [<10 mmol/l]) to reduce the risk of PO-AKI Consensus Statement 5c.We recommend considering restarting angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) therapy within the first 48 h postoperatively in patients who are haemodynamically stable without evidence of PO-AKI. This decision should be individualized and context-specific Consensus Statement 5d.We recommend that enhanced recovery after surgery (ERAS) pathways  are individualized based on the baseline risk of PO-AKI. The impact of ERAS pathways on the incidence of PO-AKI is unclear owing to the potential for both beneficial and harmful effects As a general principal, treatment of AKI should be initiated as early as possible, including in patients with suspected AKI or who are at a substantially increased risk of AKI. Management of PO-AKI involves specific approaches, for example, avoidance of nephrotoxins, as well as general management principles that are common to all postoperative complications, such as haemodynamic optimization. Similarly, treatment of PO-AKI shares many features with the treatment of AKI in other settings Table . Treatme95. Nephrology or urology consultation should be sought if intrinsic causes of AKI or obstruction are suspected based on the KHA results. The frequency and duration of monitoring should be individualized based on patient risk, exposure and clinical course .We recommend that the underlying causes of postoperative acute kidney injury (PO-AKI) should be determined and treated whenever possible Consensus Statement 6b.In patients with PO-AKI, we recommend that criteria for implementation of kidney replacement therapy (KRT) should be based on directly anticipated or observed life-threatening AKI-related complications Consensus Statement 6c.We suggest that AKI phenotype is taken into account when deciding what type of treatment to implement 97. However, observational studies have shown that the severity of AKI correlates with short-term101 and longer-term102 adverse events, suggesting that interventions that reduce the severity of AKI could improve these outcomes.The treatment and prevention bundle outlined in the 2012 KDIGO clinical practice guideline for AKI consists of supportive measures, including volume management, maintenance of adequate blood pressure and judicious avoidance of nephrotoxins. Two RCTs demonstrated that implementation of this bundle in high-risk patients identified by biomarkers after surgery significantly reduced the occurrence of PO-AKI, but were not powered to demonstrate differences in longer-term patient outcomesKidney replacement therapy (KRT) may be required in patients with severe AKI who develop medically refractory fluid and/or metabolic complications. In the absence of conventional indications, the best timing for initiating KRT in PO-AKI is uncertain. Potential rationales for initiating KRT during PO-AKI are avoidance of fluid accumulation, maintenance of acid-base and electrolyte control, provision of space to accommodate nutritional and medication needs, and abrogation of the interaction between the kidney and other organs. Theoretically, early initiation of KRT could facilitate better fluid, electrolyte and acid–base homeostasis. However, KRT is associated with increased risk of complications, healthcare costs and clinical workload.104 have stimulated debate regarding the best timing for KRT in patients with established AKI and differences in study design, patient populations, definitions of early and late RRT initiation, and inclusion criteria make head-to-head comparisons difficult.The 2012 KDIGO guidelines recommend starting KRT “when life threatening changes in fluid, electrolyte, and acid-base balance exist” and considering the “broader clinical context, the presence of conditions that can be modified with [KRT], and trends of laboratory tests — rather than single BUN and creatinine thresholds alone — when making the decision to start [KRT]”. Contradictory trial results105. Importantly, STARRT-AKI included a large cohort of patients with PO-AKI (n = 965) and showed no evidence of a benefit of early commencement of KRT in this subgroup . Thus, decisions regarding timing of KRT initiation remain a complex issue that should be individualized based on the clinical state of the patient, in particular imminently anticipated or observed life-threatening complications of AKI.Following our consensus conference, the results of the STARRT-AKI study provided high-quality evidence that early commencement of KRT based on AKI criteria in the absence of conventional indications does not improve survival, may needlessly expose some patients to KRT and results in a higher risk of KRT dependence at 90 days compared with a conservative strategy for starting KRT106. Until these strategies are better embedded in clinical practice, the potential benefits of individualized therapy for AKI subtypes cannot be clearly established.AKI is a heterogeneous condition consisting of distinct endotypes and phenotypes based on its aetiology, prognosis and molecular pathways. Different subtypes might require and benefit from different therapeutic strategies. However, current AKI definitions based on serum creatinine and urine output provide no information on AKI subtypes. Studies using various damage and functional AKI biomarkers suggest that these new tools might overcome the limitations of current AKI definitions to improve AKI phenotypingWe recommend further trials to assess the optimal choice and volume of fluids for intravascular expansion and vasopressor therapy in patients with PO-AKI as well as the use of protocol-based management of haemodynamic and oxygenation parameters to prevent worsening of PO-AKI. Investigation of individual components of the KDIGO guideline to identify those that are the most effective in treating PO-AKI is important given that these were largely assembled based on expert opinion. Better understanding of AKI phenotypes is required to enable further trials to assess the response to implementation of the KDIGO bundle and other treatment options according to different AKI endotypes and phenotypes.115 39.Patients with PO-AKI or AKD are at an increased risk of recurrent episodes of AKI and progressive deterioration of kidney function, including CKD and dialysis dependency108. Among 1,200 patients who underwent non-cardiac and non-vascular surgery, of whom 6.7% met the RIFLE criteria for AKI (2), the risk of non-cardiovascular complications (such as pneumonia and stroke) was similar in those with and without AKI over the first 2 weeks after surgery, but thereafter, patients with AKI experienced a significantly higher rate of cardiovascular events, including acute coronary syndrome, acute heart failure and arrhythmias107. Similarly, AKI after abdominal surgery has been associated with an increased risk of in-hospital complications, including perioperative acute myocardial infarction, pneumonia and sepsis (P < 0.001)29.Other postoperative complications, including infection, prolonged mechanical ventilation, tracheostomy and cardiovascular events are also more common in patients who develop AKI after surgery than in those who do not117. Furthermore, the risks of 30-day re-admission and discharge to a nursing facility or rehabilitation centre are increased31. PO-AKI has also been associated with important, but difficult to quantify adverse outcomes, including reduced quality of life and perceived well-being118 as well as with increased risk of malignancy, possibly due to interactions with the immune system119.Unsurprisingly, the development of PO-AKI is associated with substantial increases in hospital costs and resource utilization, as well as longer stays in the ICU and in hospital120 and limited evidence is available to support any specific monitoring paradigm. However, adverse outcomes are increased in patients with prolonged, more severe AKI, and in those whose kidney function does not recover to baseline levels, suggesting that use of these factors to inform the initial frequency and nature of follow-up would be a reasonable approach122.Given the increased risk of short- and long-term adverse outcomes following PO-AKI, appropriate monitoring and follow up of this cohort is a potential approach to improving patient outcomes. Although follow-up schemas for all-cause AKI have been proposed, the optimal timing of follow-up after a postoperative episode of AKI or AKD is unknown123. When available, further work-up and monitoring of kidney function with other diagnostics may be appropriate. Changes in management based on this monitoring should be communicated with the patient as well as with other treating physicians.Monitoring pathways should be developed in conjunction with nephrologists, but may be directly provided by perioperative physicians, primary care physicians or other specialists Fig. . As a mi58. A multi-disciplinary follow-up approach  combined with patient education is recommended but has not been formally studied. Undoubtedly, drug selection, dosing and monitoring are important and should be guided by personalized clinical decision making and regular assessment of kidney function. Both the use of nephrotoxic drugs and the inappropriate exclusion of potentially beneficial medications  need to be avoided125. In patients with AKD, in particular, a substantial risk exists of potential therapeutic failure caused by underdosing or avoidance of effective cardiovascular drugs. Close collaboration between the relevant clinical teams is paramount and future research efforts should clarify the ideal timing and method for providing post-AKI and AKD care .The incidence of kidney and non-kidney adverse events is increased following postoperative acute kidney injury (PO-AKI) or postoperative acute kidney disease (PO-AKD). In particular, patients with surgery-associated AKI or AKD are at an increased risk of new or worsening chronic kidney disease (CKD), which is associated with increased long-term mortality Consensus Statement 7b.The prevention of new or worsening CKD following an episode PO-AKI or PO-AKD is a public health priority. We recommend that all patients undergo at least one kidney health assessment once the acute phase of AKI or AKD is complete. This approach will require optimal communication and AKI education between health-care professionals and the patient or caregiver A need exists for epidemiological research to describe the frequency, nature and severity of PO-AKI and PO-AKD progression to CKD. The effectiveness and cost-effectiveness of supplementing a simple KHA with enhanced kidney health care in the prevention of CKD progression after surgery-associated AKI or AKD should be evaluated in appropriately selected patients. Finally, research is needed to explore a possible common aetiology between surgery-associated AKI or AKD and other chronic organ injuries as the longer-term effects of AKI do not only involve the kidney.PO-AKI is a sentinel postoperative event that is strongly associated with both short-term surgical complications and long-term adverse outcomes. In this Consensus Statement, we provide an overview of PO-AKI and management. However, PO-AKI is a heterogeneous syndrome with a variable clinical course. In many areas the strength of evidence is poor and accordingly our recommendations are weak. We provide research recommendations for these areas to enable greater clarity to be achieved in the future. Overall, we believe that an integrated approach to PO-AKI management requires serial evaluations of AKI risk, diagnosis, progress and outcomes throughout the perioperative period, which can be constructed as a series of focused KHAs.Supplementary Information


The cortical bone trajectory (CBT) has been introduced with the aim of better screw hold, however, screw-rod constructs with this trajectory might provide less rigidity in lateral bending (LB) and axial rotation (AR) compared to the constructs with the traditional trajectory (TT). Therefore, the addition of a horizontal cross-connector could be beneficial in counteracting this possible inferiority. The aim of this study was to compare the primary rigidity of TT with CBT screw-rod constructs and to quantify the effect of cross-connector-augmentation in both. Spines of four human cadavers (T9 –L5) were cropped into 15 functional spine units (FSU). Eight FSUs were instrumented with TT and seven FSUs with CBT pedicle screws. The segments were tested in six loading directions in three configurations: uninstrumented, instrumented with and without cross-connector. The motion between the cranial and caudal vertebra was recorded. The range of motion (ROM) between the CBT and the TT group did not differ significantly in either configuration. Cross-connector -augmentation did reduce the ROM in AR , LB  and flexion-extension FE  for the TT group and in AR  for the CBT-group. The primary rigidity of TT and CBT single level screw-rod constructs did not show significant difference. The minimal reduction of ROM due to cross-connector-augmentation seems clinically not relevant. Based on the findings of these study there is no increased necessity to use a cross-connector in a CBT-construct. Dorsal pedicle screw instrumentation of the spine is an effective method to attain primary stability required for bony fusion. More rigid instrumentations lead to higher fusion rates  and consIntuitively, this raises the question whether these narrower constructs provide less shear stability which could be disadvantageous in construct stiffness, particularly in axial rotation (AR), lateral bending (LB) and lateral shear (LS). Vice-versa CBT screws are aligned more sagittal which could be beneficial in flexion-extension (FE) . SupportThe addition of horizontal cross-connectors (CC) to TT screw-rod constructs has been demonstrated to increase rigidity of dorsal instrumentations in AR and LB –12. WithThese considerations lead to three hypotheses: 1) CBT-constructs are less stable in AR, LB and LS, 2) CBT constructs are more stable in FE and 3) CC-augmentation is more effective in CBT-constructs compared to TT-constructs. Therefore, the aim of this study was to compare the primary rigidity of single level CBT-constructs to TT-constructs and to quantify the effect of CC-augmentation on both trajectories.Four fresh frozen cadavers of human donors  were used for this study . EthicalBiomechanical testing was performed on a biaxial linear-torsion static testing system . The machine consists of a traverse to generate compression and tension along the z-axis and a torsion motor to generate torque around the z-axis. This machine was supplemented with a setup consisting of an x-y-table and holding arms that allow for specimen fixation in a vertical orientation for axial compression-decompression (AC) and axial rotation (AR) and in a horizontal orientation for lateral bending (LB) and anteroposterior shear (AS) as well as flexion-extension (FE) and lateral shear (LS) . FE, LB After five preconditioning cycles, the movement of the cranial and caudal vertebral body was recorded over one cycle with a motion capturing system ). The segments were frequently sprayed with phosphate buffered saline (PBS) to prevent tissue dehydration. The loading protocol was repeated for all six loading directions. Every FSU was tested under three conditions—1) uninstrumented, 2) instrumented and 3) instrumented with horizontal CC, whereby the test sequence of 2) and 3) was reversed for half of the samples to prevent potential bias. Uninstrumented testing was performed with implanted pedicle screws, but without vertical rods. For instrumented testing, pedicle screws were interlinked with vertical rods  on either sides and set screws were tied with 9 Nm torque according to the standard surgical technique. One CC  was mounted horizontally in the center of the two rods and locked with 5.5 Nm torque according to the standard surgical technique . A camerScrew-length, screw-diameter and bone density of the two groups were normally distributed and thus were compared using the two-sample t-test. Motion tracking data was used to calculate the ROM over the whole loading amplitude. For translational directions , the relative translational movement of the markers was assessed. For rotational movements , the projected angular movement in the motion plane was evaluated. Shapiro-Wilk parametric hypothesis tests of composite normality showed not all residuals of the ROM and reduction values to be normally distributed. Thus, the Mann-Whitney U test was used to evaluate the difference between the two trajectory groups and the Wilcoxon signed-rank test for paired data was used to compare the effect of the instrumentations on the ROM . The sigThe specimens of the two groups did not differ significantly in CT-values with 125 ± 17.8 HU (mean ± SD) for TT and 113 ± 28.8 HU for CBT (p = 0.37). The ROM in the uninstrumented configuration of the TT and CBT group did not differ significantly (p>0.05) , Table 2No significant correlation was observed between the rod-rod distance and the ROM (p>0.05) . HoweverPedicle screws following the cortical bone trajectory have been demonstrated to be a valid alternative to TT pedicle screws in multiple aspects . There aWhile CC-augmentation has been observed to increase rigidity of TT-constructs in AR and LB –12, no sThe presented results provide further evidence that CBT constructs are associated with equivalent primary stability compared to TT constructs. This information is crucial for the clinical practice as it prevents unnecessary cross-connector-augmentation, which is related to additional implant cost and surgical time and has been associated with soft tissue irritation and delayed infection  and evenThere are some limitations to this study. The study investigates the primary, passive rigidity in the six major axes in well defined, isolated load applications. However, in reality an infinite number of complex combined loading situations occurs . The effWe conclude that the rigidity of lumbar and lower thoracic CBT-constructs is not significantly different to that of TT-constructs. Furthermore, our findings indicate that cross-connector-augmentation does not increase the rigidity of CBT or TT instrumentations in a clinically relevant way.S1 File(ZIP)Click here for additional data file.


Fishes usually stop eating food when they are sick, and treating diseased fish with oral drugs is a serious hurdle in the aquaculture industry. Tylosin tartrate is a potent bacterial-killing agent useful against frequently occurring bacterial fish infections. We tested the effectiveness against pathogenic bacteria and the human safety of the drug for possible application to cultured olive flounder, one of the most important culture species in far eastern Asian countries. Tylosin tartrate was very effective in killing the pathogenic bacteria grown in artificial culture media, and it was also demonstrated that the drug reached body concentrations in olive flounder, high enough to kill the pathogen. In addition, we also determined how long to wait until the fish clears the injected drug out and it is possible for human consumption. These results will pave a new method for disease treatment useful for olive flounder farming.n = 240) and intravascular  injections. Serum concentrations of tylosin were determined using a validated liquid chromatography-tandem mass spectrometry method. According to the non-compartmental analysis, the bioavailability of TT was 87%. After the IV injection, the terminal half-life, total body clearance, volume of distribution, and mean residence time of TT were 21.07 h, 0.07 L/kg/h, 2.15 L/kg, and 16.39 h, respectively. Rapid absorption (Tmax 0.25 h), prolonged action , and linear dose–response relationship  were monitored following 10 and 20 mg/kg IM injection. The withdrawal time of TT from muscle  was 9.84 days, rounded up to 10 days (220 degree days). Large Cmax/MIC90, AUC0-inf/MIC90, and T > MIC90 values were obtained for Streptococcus isolates and these PK/PD indices satisfied the criteria required for efficacy evaluation. This study lays a foundation for the optimal use of TT and provides valuable information for establishing therapeutic regimens.The objective of this study was to demonstrate the pharmacokinetic–pharmacodynamic profile, bioavailability, and withdrawal time of tylosin tartrate (TT) administered to olive flounder via intramuscular (IM, 10 or 20 mg/kg,  Paralichthys olivaceus) typically remain calm during injection, so it can be treated without causing excessive stress. A few single and complex antibiotics that could be administered via injection, such as ampicillin, amoxicillin, ceftiofur, and the florfenicol-amoxicillin combination, have been successfully implemented to treat P. olivaceus in the Korean aquaculture industry [Antibiotics are typically administered to farmed fish via the oral route. However, the critical disadvantage of oral administration is the difficulty in administering the drug to a sick fish with a poor appetite . Despiteindustry .Streptomyces fradiae [Tylosin tartrate (TT) is a macrolide antibiotic used in veterinary medicine and is extracted from the soil microbiome called  fradiae . It is a fradiae . TT is k fradiae . To spec fradiae ,7,8.P. olivaceus infected with Streptococcus parauberis [P. olivaceus infected with S. parauberis intramuscularly (IM) yielded excellent results: a 90% relative survival rate and no hematological or histopathological side effects. These pharmacological characteristics of TT are beneficial in treating infections caused by Gram-positive bacteria, such as S. parauberis, in P. olivaceus.A previous research study examined the efficacy and side effects of TT used to treat rauberis . The admP. olivaceus.TT distributes extensively in body fluids and tissues because of its high lipid solubility and 40% plasma protein binding ,11. The Moreover, most antimicrobial use in the global aquaculture industry is not related to classification of the target bacteria or susceptibility to the range of available antimicrobials , and the2HPO4), sodium dihydrogen phosphate (NaH2PO4), formic acid, and tricaine methanesulfonate (MS-222) from Sigma . The HLB Oasis column  purchased from Waters  was used for solid-phase extraction (SPE). Brain and heart infusion agar and Muller-Hinton broth were obtained from Difco  and lysed horse blood was obtained from Oxoid . Parenteral TT injection formulation  was purchased from Eeglevet Veterinary Medicine Co., LTD.  and diluted with phosphate-buffered physiological saline.Analytical standard tylosin tartrate (≥98%) was obtained from Dr. Ehrenstorfer GmbH . All primary analytical reagents for HPLC were purchased from Merck . All chemicals were of ACS grade purchased from the following sources: sodium phosphate dibasic  × 3.0 m (W) × 1.0 m (H) in size and fed 1% of their body weight with commercial pellet feeds  per day. Water quality was checked at 09:00 am every day and maintained at approximately 30 PSU, 7–8 mg/L of dissolved oxygen, pH 8.1, and temperature of 22 ± 0.5 °C. All fish were fasted before the experiment to avoid potential adverse effects caused by residual food in their gastrointestinal tract. They were weighed and administered doses based on their body weight. This study followed the protocol approved by the Institutional Animal Care and Use Committee  from the National Institute of Fisheries Science (NIFS). No fish died during the adaptation and sampling period. The absence of tylosin in the muscle and serum was confirmed in five fish before commencement of the experiments.Healthy n = 90, each group), 90 (IV dose of 10 mg/kg), and 60 (IM dose of 10 mg/kg) fish in each.The fish were divided into three groups: pharmacokinetic, bioavailability, and tissue depletion group, with 180  in the pharmacokinetic study. The chosen dose was 10 mg/kg TT because a previous pilot study showed that a single IM injection of TT was effective in combating bacterial infections, such as S. parauberis [Single IM injections of TT were administered into the thick and muscular area under the dorsal fin at 10 and 20 mg/kg to fish maintained at 22 ± 0.5 °C  were obtained from the caudal vein using a 1-mL syringe and transferred to a serum separator tube (SST). The blood samples were centrifuged at 3000× Tissue depletion studies of the withdrawal time were performed in fish maintained as described above. Fish were single-IM injected with 10 mg/kg TT. Muscle samples were obtained 1, 2, 3, 4, 5, and 7 days following the end of the last administration. Muscles were sampled and stored at −80 °C pending analysis for the residue depletion test.g for 10 min for extraction. The clear supernatant was transferred to a 15-mL conical polypropylene tube and the extract was evaporated in a stream of nitrogen at 50 °C. Na2HPO4-NaH2PO4  was added to the residue. The mixture was loaded onto methanol-water pre-activated (5 mL pre-rinsing) HLB cartridge columns, flushed with 5 mL of 5% aqueous methanol, and eluted with 5 mL of methanol. The eluted extract was evaporated in a stream of nitrogen at 50 °C. The residue was reconstituted with 1 mL of HPLC-grade methanol:water (1:1) and filtered using 0.2-µm syringe filters; 10 µL of the residue were analyzed using the HPLC-MS/MS system.The serum or muscle TT concentration was measured using a modified high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) described in the Korean Food Standards Codex . To simp18 reverse-phase 1.8-µm Agilent Zorbax RRHD SB column (2.1 × 50 mm), which can be maintained at a temperature of 40 °C. During the mobile phase, isocratic elution was performed using the mixture of acetonitrile:water (7:3) containing 0.1% formic acid at a flow rate of 0.25 mL/min. an MS/MS detector was used using the following parameter settings: capillary voltage at 4000 V; nebulizer gas (N2); nebulizer gas flow at 11 L/min; nebulizer pressure at 45 psi; gas temperature at 350 °C; and delta EMV+ at 500 V. TT was evaluated in multiple-reaction monitoring mode for positive charges. Quantification and qualification ions were m/z 916.5 to174.1 and from m/z 916.5 to 101.1, respectively.The HPLC-MS/MS system consists of an Agilent 1260 Infinity series LC  combined with an Agilent 6430 Triple Quad detector. Chromatography separation was performed using a C2 > 0.99. Limit values of detection (LOD) and quantification (LOQ) were determined from signal-to-noise (S/N) ratios of 3:1 and 10:1, respectively. Recovery rates for the extraction procedure from olive flounder tissues were determined by spiking known concentrations of standard solution at three levels ) into serum or muscle samples. In addition, to avoid the influence of variable instrument sensitivity over time, the linearity of the standard curves for spiked TT standards was checked at the beginning and end to ensure absence of significant fluctuations.The analytical method was validated according to the criteria of the validation procedure . Linearimax) and time to peak serum concentration (Tmax) were determined directly from the experimental data. The area under the concentration–time curve (AUC) was determined by the trapezoidal rule. Bioavailability (F) was calculated as F (IM) = 100 × (AUCIM x doseIV)/(AUCIV × doseIM).The pharmacokinetic parameters for serum concentration were analyzed based on the non-compartmental analysis model using the PKSolver, an add-in program for Microsoft Excel . The pea5 colony-forming units (CFU)/mL and cultured at 28 °C for 24 h. The MIC was determined as the lowest antibiotic concentration at which bacterial growth was visibly inhibited. The quality control was performed using Escherichia coli ATCC 25922/NCIMB11210; Aeromonas salmonicida subsp. ATCC 33658/NCIMB1102; and Staphylococcus aureus ATCC 25923 for the precision and accuracy of the test.In vitro antibacterial activity tests were performed in accordance with the CLSI guideline explained for MIC tests . Bacterimax)/MIC, area under the concentration–time curve (AUC)/MIC, and the duration of time (T > MIC) over MIC, were determined using in vitro MIC data and in vivo PK parameters obtained following IM injection of TT at 10 and 20 mg/kg to olive flounder [Surrogate markers for antibacterial efficacy, including the peak serum concentration  was performed in accordance with the muscle drug concentration by applying a method developed by the Committee for Veterinary Medicinal Products . The mat2 = 0.999) range of 0.5–50 ng/mL. The inter-day and intra-day precision (coefficient of variance) were below 12.8% for three concentrations: 0.5, 5, and 50 ng/mL. The accuracy (recovery rate) ranged from 83.2% to 103.0%, satisfying the criteria of the Bioanalytical Method Validation Guidance for Industry [An HPLC-MS/MS analysis was used to validate a 0.5 ng/mL limit of quantification used. The retention time of tylosin in the serum was found to be about 0.6 min . The linIndustry . DetailsF of tylosin based on the noncompartmental analysis model. After the IV injection, the terminal half-life (t1/2λz), total body clearance (Cl), volume of distribution (Vz), and the mean residence time (MRT) of TT were 21.07 h, 0.07 L/kg/h, 2.15 L/kg, and 16.39 h, respectively. After the IM injection, Cmax was 10.76 and 16.60 µg/mL, and it took 0.25 h (Tmax) after the administration to reach Cmax, at doses of 10 and 20 mg/kg, respectively. The extent of TT absorption indicated the linear dose–response relationship with AUC following 10 mg/kg IM injection (123.55 µg/mL*h) proportionally increased the 246.05 µg/mL*h at 20 mg/kg. The t1/2λz and MRT of 10 mg/kg IM injection were 33.96 and 43.66 h, respectively. For the 20 mg/kg IM injection, t1/2λz and MRT were 26.04 and 33.09 h, respectively. The TT bioavailability after IM injection was 86.98 to 87.32%. Detailed pharmacokinetic parameters are given in All fish tolerated TT administered by IM or IV injection well, and no adverse effects were noted. Streptococcus isolates are shown in S. iniae strains showed an MIC range between 0.125 and 0.5 μg/mL. Twenty S. parauberis strains showed an MIC range between 0.5 and 2 μg/mL.The MICs of 43 clinical Streptococcus isolates (MIC50 and MIC90) are shown in max/MIC, AUC/MIC, and T > MIC ratios. According to S. iniae and S. parauberis were calculated with large Cmax/MIC and AUC/MIC values. Moreover, T > MIC was maintained for at least about 2 days, with serum concentrations exceeding the MIC for both strains.The MICs of TT that restrained 50 and 90% of the clinical The residue depletion of tylosin from muscle was monitored after TT single injection at 10 mg/kg, and the results as a function of time are shown in P. olivaceus. Our study provides meaningful pharmacokinetic results of injectable TT in olive flounder, which, in relation to the absorption of TT, is quite rapid from the IM site, showing a long t1/2λz and MRT is beneficial for efficacy, and a linear dose–response relationship with AUC. In addition, by providing a degree day through muscle residue analysis, it was demonstrated that TT is an appropriate drug for treating fish for food.To the best of the authors’ knowledge, this is the first study describing the pharmacokinetic profile of TT in fish including F of other routes of administration, such as oral and IM injection. The dose of IV injection was set at 10 mg/kg to calculate factors, such as F or Vz.The dose of IM injection in the pharmacokinetic study was set at 10 mg/kg based on a previous pilot study. Although the pharmacokinetic parameter of IV administration to cultured fish is not important in the application to real practice, it is necessary to calculate the 1/2λz after IV injection was 21.07 h, indicating that the rate of drug elimination is relatively slow in P. olivaceus, possibly due to differences between species. Fish have a lower metabolic rate than mammals (t1/2 = 4.52 h) [1/2 = 2.04 h) [P. olivaceus. Vz represents the volume of distribution to body tissues and fluids, and this was high in P. olivaceus (2.15 L/kg). The high Vz of TT observed in the present study suggests a high volume of distribution to body tissues of P. olivaceus, supported by the results of a previous study in which Vz higher than 1 L/kg defined the high volume of distribution of the drug [P. olivaceus.The t 4.52 h)  and bird 2.04 h) , which ethe drug . The extthe drug . Overallmax of 10.76 µg/mL at 0.25 h (Tmax), after being rapidly absorbed into the serum. Although the information about pharmacokinetics of TT after IM injection in other fish may not be applicable to this study, the rapid rate of absorption was similar to the values reported (Tmax within 1 h) in ducks, cattle, buffaloes, and sheep [F of TT was calculated to be 87.35% in P. olivaceus after IM injection at 10 mg/kg, supporting the rapid and nearly complete absorption following IM administration. The comparative pharmacokinetic parameters for the administered dose all showed a fast absorption rate , and when administered at 20 mg/kg rather than 10 mg/kg, the Cmax was absorbed 1.5 times higher. The AUC was shown to be concentration dependent (123.55 vs. 246.05 at 10 and 20 mg/kg) according to the administered dose.After IM injection at 10 mg/kg, TT reached a Cnd sheep ,17,32. Tnd sheep , which pThe calculated withdrawal times by applying an official MRL of 0.1 µg/g  were 10 It is well recognized that macrolides antibiotics as a class exert their activity in parallel with the time length over which tissue concentrations are maintained above the effective level, e.g., MIC . They armax/MIC, T > MIC, and AUC/MIC values. A Cmax/MIC ratio greater than 10 is considered the indicator of activity for concentration-dependent antibiotics [max/MIC90 values for the two test strains  obtained for S. parauberis lasted for 44 h after single IM injection at 10 mg/kg, and S. iniae lasted about 20 more hours (Streptococcus.The clinical efficacy of antibiotics against their target pathogens can be predicted by PK/PD indices, including Cibiotics . TT, witflounder . The timre hours . These PP. olivaceus and suggested an estimate of its withdrawal time after single IM administration. In addition, clinical parameters were established by examining the PK/PD relationships using MICs of clinical Streptococcus isolates. These data form an important foundation for optimal use of TT as a treatment for systemic infection in P. olivaceus and this provides valuable information for establishing scientific and effective treatment regimens.This study has shown the beneficial pharmacokinetic profile, including the bioavailability, of an injectable TT formulation in


We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n = 46), atypical parkinsonism patients  and non-neurological controls (n = 39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters  and age improved the discrimination of PD vs. AP. Our results show that the discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson’s disease (PD,  However, many of these symptoms have not developed fully in early disease stages, explaining why the rate of misdiagnosis could be up to 20%, even in the hands of movement disorders experts7. Therefore, especially in early disease stages, reliable biomarkers are needed for accurate differentiation between PD and AP. Such a timely distinction is important, e.g., for patient counselling since the forms of AP usually have a faster disease progression than PD, with little or no clinical response to levodopa medication. Being able to reliably separate the different parkinsonian syndromes at the earliest possible stage is also critically important for research purposes, allowing the correct patients to be recruited into trials.There is currently no reliable objective test to discriminate Parkinson’s disease (PD) during lifetime from the various forms of atypical parkinsonism (AP), which include multiple system atrophy (MSA), progressive supranuclear palsy (PSP), dementia with Lewy bodies (DLB), corticobasal syndrome (CBS), and vascular parkinsonism. Discrimination of these disorders based on the clinical presentation alone can often be puzzling, especially early in the disease course when symptoms overlap across the different parkinsonism conditions. The clinical diagnosis is based on the most recent specific criteria defined for each disease and includes clinical features, imaging, rate of disease progression, and response to dopaminergic medication9. However, this assay could not discriminate PD from MSA or DLB. Quantification of neurofilament light chain (NfL) in either CSF or blood may discriminate PD from AP12, but additional biomarkers may help to increase the specificity to discriminate PD from AP.Cerebrospinal fluid (CSF) is a rich source for the identification of potential fluid biomarkers for neurodegenerative disorders due to its close proximity to the brain. The CSF composition may directly reflect pathological changes in the brain. Although several studies have identified potential biomarkers for parkinsonian syndromes, none have yet been implemented in clinical practice. So far, quantification of α-synuclein (α-syn) by real-time quaking-induced conversion (RT-QuIC) proved very useful to discriminate parkinsonian disorders with an underlying α-synucleinopathy, such as PD, DLB, and MSA, compared to other types of proteinopathies, such as the tauopathies PSP and CBSThe aim of this study was to identify proteins, that could assist in the discrimination of PD from AP in relatively early stages of the disease, and assess their diagnostic value in our cohorts. Such biomarkers may alert clinicians for a timely diagnosis of AP which is more rare than that of PD. The identification of proteins in our study was based on tryptic peptide biomarkers, that are produced after enzymatic digestion of CSF proteins with trypsin and enable mass spectrometric analyses. We used non-targeted (shotgun) proteomics for the discovery of protein biomarkers and targeted  mass spectrometry (MS) for validation of our findings. We performed our discovery and validation experiments using patients from a unique longitudinal cohort followed up for up to 12 years. Importantly, all participants had an uncertain diagnosis at the time of inclusion, thereby replicating the clinical challenge faced by clinicians to provide a correct diagnosis, i.e., at a phase in the disease process when many clinical symptoms are overlapping and where diagnostic biomarkers could be very useful.p-value < 0.05) between PD and MSA.Using shotgun proteomics, 5,543 tryptic peptides were identified in the PD, MSA, and non-neurological controls groups. Of these 5,543 peptides, 191 peptides had significantly different levels  in our study. Total protein concentration was higher in the AP group (mean = 579 mg/L) compared to PD (mean = 533 mg/L) and controls , due to high total protein levels in the PSP group (see Table p < 0.05). Therefore, age was included as covariate for group comparisons. Clinical assessment of disease severity ) positively correlated with 6/32 peptides in the AP group, with correlation coefficients ranging from 0.4 to 0.6 (p < 0.05). No significant correlation of (other) clinical parameters with the levels of any of the tryptic peptides in the PD or AP group was observed.For group comparisons, we considered MSA and PSP as one group (AP) because of the relatively low number of PSP cases (p = 0.023) in the PD group, but not in other groups. The diagnostic accuracy of the 14 peptides to discriminate PD from AP, i.e., the AUC of the ROC, was moderately high and ranged from 0.60 to 0.76  the 14 tryptic peptides which were differentially expressed in PD vs. AP, 2) the 14 tryptic peptides and previously identified biochemical markers such as NfL, α-syn, amyloid β42, total tau, phosphorylated tau, and RT-QuIC analysis of misfolded α-syn; (3) the 14 tryptic peptides, the above-mentioned biochemical markers and clinical assessments, such as UPDRS, ICARS, MMSE scores; (4) a combination of the previously identified biochemical markers (as in model 2) with clinical assessments (as in model 3). An overview of the biochemical markers and clinical parameters that were included, next to tryptic peptides, in the models for the discrimination of PD and AP is provided in Supplementary Table  the 14 tIn this study, we used untargeted MS to identify tryptic peptides in CSF as potential biomarkers that could discriminate parkinsonian disorders, and performed an independent validation of our findings by targeted MS. For this purpose, we purposely included only patients with clear signs of parkinsonism but an uncertain diagnosis at the time of inclusion and CSF collection, but in whom a silver standard diagnosis was made 3−12 years later based on the rate of progression, response to treatment and possible development of red flags. This approach served to replicate the challenge that clinicians face in everyday clinical practice when a clinical diagnosis has to be established in movement disorders patients with only partially developed clinical syndrome. Under such circumstances, having reliable diagnostic biomarkers would be very helpful.Both untargeted and targeted MS methods proved to be reliable and robust methods to identify tryptic peptide biomarkers and provided a relative quantification of the levels of these peptides in CSF. We developed a protocol for the evaluation of SRM analysis of tryptic peptides in CSF. The newly developed assay procedure was very robust since it proved to be very stable during several measurement days (CV < 10%), it was reproducible across different sample preparation days, and was resistant to multiple freeze/thaw cycles. Therefore, this SRM assay may be useful for other CSF biomarker studies as well.The SRM assay confirmed our findings for many tryptic peptides from the discovery experiment, illustrating the robustness of the shotgun proteomics for biomarker identification. For 14 tryptic peptides, we found lower CSF levels in AP compared to non-neurological controls and PD, both in the discovery and validation experiments, and they individually discriminated PD from AP with a diagnostic value up to 76%. Multivariate analysis by random forest modelling did not increase the discriminative value between PD and AP when only peptides were included in the model. The lower discriminative value generated by random forest modelling compared to individual tryptic peptides (53% vs. 76%) could be explained by the low number of variables (14 peptides) included in the analysis, and on top of that, the model was developed in 70% of our cohort and validated in remaining 30%. However, by including more variables, such as other CSF protein biomarkers and/or clinical assessments, the random forest algorithm was capable to provide a better discrimination between disease groups, increasing the accuracy to 86%. Interestingly, very comparable AUC values (0.86−0.88) were obtained for the models 2, 3, and 4, suggesting that a combination of the tryptic peptides identified in the current study with established protein biomarkers  has similar additional diagnostic value as clinical data in combination with these established markers. These models including CSF tryptic peptides and clinical assessments offers a great advantage to help clinicians to identify a correct diagnosis of parkinsonian disorders, but need to be tested in independent cohorts.13. Two other tryptic peptides/proteins found in our study, SLIT and NTRK-like protein 115, and Amyloid-beta precursor protein16, also play a role in synaptogenesis. Dysfunctional synapses contribute to neurodegeneration17, and dysregulation of these proteins may add to such dysfunction in AP syndromes. A meta-analysis on imaging studies showed that presynaptic dopaminergic function is 34% lower in PSP as compared to PD and MSA18. Moreover, in a study published after this meta-analysis, evidence-based on DAT SPECT data was obtained supporting a faster decline of presynaptic function in MSA compared to PD as well19. Our findings added several potential molecular biomarkers to this imaging-based evidence of synaptic dysfunction. Studies using immunohistochemistry on brain tissues, animal and in vitro studies may be useful in confirmation of altered expression of the proteins in AP and their localization.Aside from a potential role in differential diagnosis, several of the 14 identified tryptic peptides, which are derived from 13 different proteins 27. Functional studies demonstrated the presence of LINGO-1 in dopaminergic neurons and oligodendrocytes29. A meta-analysis identified LINGO-1 polymorphisms related to decreased risk of PD, but not of MSA30. In MSA, accumulation of misfolded α-syn occurs in oligodendrocytes, which are the cells responsible for myelin maintenance. Myelin dysfunction in MSA precedes α-syn accumulation and neuronal loss31, therefore myelin dysfunction might be an important early mechanism of neurodegeneration in MSA. In previous studies of our group, we found increased levels of MBP in the CSF of MSA patients compared to PD patients33. Although the specific mechanism underlying the lower LINGO-1 levels in MSA compared to PD remains unclear, abnormal levels of the peptide LTVFPDGTLEVR may be an indication of early disturbances in oligodendrocyte myelin production in MSA, consistent with the increased CSF MBP levels in MSA.Lower CSF levels of the peptide LTVFPDGTLEVR  in AP compared to PD could be related to demyelination in MSA as compared to PD. LINGO-1 is a transmembrane protein that negatively regulates oligodendrocyte differentiation and axon myelination35. Therefore, disruptions in secretogranin-2 levels might be related to altered levels of dopamine release in the synaptic cleft. Recently, one study showed co-localization of secretogranin-2 with aggregated α-syn and phosphorylated tau in brain tissue of a PD animal model, suggesting an involvement of these proteins in synaptic trafficking36. A previous proteomics study identified lower CSF levels of secretogranin-2 in PD compared to controls24, consistent with our results. The secretogranin-2 might be useful as an early biomarker to demonstrate dopamine disturbances in parkinsonian syndromes.The peptide VLEYLNQEK (secretogranin-2), was the only peptide in our study, which discriminated PD from both controls and AP. Secretogranin-2 is a protein that is cleaved into peptides and secreted in vesicles, releasing the neuropeptide named secretoneurin, a peptide that stimulates dopamine release in striatal neurons and basal gangliaThe remaining three identified tryptic peptides were derived from proteins involved in the regulation of cellular communication , extracellular structural function (Extracellular matrix protein 1), and protein glycosylation , with no known relation with neurodegeneration or previous link with parkinsonism.37. In this study, none of the features could discriminate PD from controls, whereas four proteins or protein fragments  discriminated either MSA or PSP from PD/controls. Four peptides of secretogranin 1 were identified in our discovery experiment, and at lower levels in MSA compared to PD, confirming these previous findings. However, the peptides belonging to this protein did not qualify for our SRM assay, and therefore we could not confirm it in our validation experiment. In yet another study, using Orbitrap MS, 5,043 protein-derived tryptic peptides were identified in CSF in a discovery cohort of PD, AP , and controls38. The number of peptides is quite comparable to our findings . In their discovery and validation experiments, up to 90 peptides were detected at significantly lower levels in AP compared to controls (p < 0.05), but there were no differences for PD vs. AP or PD vs. controls, as we observed in our study.Several previous studies aimed to discriminate PD from AP by using CSF proteomic profiling. In one study 2,000 (poly)peptides in CSF of PD, AP , and controls were analyzed using the method of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)39, and no differences were found over time. Furthermore, all PD and AP samples in our study were retrieved in the same period, and therefore, we do not expect that storage time is a major factor that may have affected the results of the differential levels in these patients. A second limitation may be related to the final diagnoses of the patients, which were based on clinical assessments and not on neuropathological examinations. However, given the very long follow-up of the patients in our cohorts (up to 12 years), and the independent assessment by two experienced movement disorder specialists, we believe that the rate of misclassification has been reduced to a minimum. Importantly, the long follow-up time allowed us to consider the rate of progression, response to therapy, and development of any red flags into the diagnostic process. We also included brain imaging findings in the diagnostic process. Based on these clinical parameters, a reliable ‘silver standard’ diagnosis can be made in most patients. Third, for 18 tryptic peptides, selected from our discovery experiment for validation, the results did not match in both experiments, which reinforces the need of robust independent validation studies before conclusions can be reached, which applied to the remaining 14 tryptic peptides. Fourth, apart from MSA, we only had a limited number of other cases with other causes of AP, such as PSP, CBD, or DLB, in our validation study, due to the relative low representation of these patients, for which also CSF was available, from our longitudinal cohort. Since AP comprises a heterogeneous group of disorders, including both synucleinopathies and tauopathies, the small number of AP cases other than MSA also limits the translation of our findings to potential disease mechanisms in e.g., PSP, and will probably mainly reflect changes in MSA.Few limitations may apply to our study. First, the long storage time of CSF samples may have affected our results. A previous study investigated the stability of CSF proteins up to 12 years storage on −80 °COne of the strongest aspects of our study is the use of two independent cohorts of patients for discovery and validation. In addition to that, we also performed our validation using a different MS technique (SRM) than in the discovery, and we confirmed the consistency of 14 tryptic peptides to discriminate PD from AP. A second strong point is the unique longitudinal study, in which patients were initially included with clear parkinsonian symptoms, but with an uncertain diagnosis at baseline, i.e., at a time in the diagnostic process where fluid biomarkers are needed most. As such, our cohort offers excellent opportunities for fluid biomarker discovery and validation, as we demonstrate here. Besides providing new insights for potential biomarkers to help clinicians to discriminate parkinsonian disorders, this may also provide new insights into differences in the underlying pathophysiological processes for PD as compared to AP.In summary, proteomics is a powerful tool to identify peptides in CSF for discrimination of parkinsonian disorders. Our newly developed SRM assay proved to be very robust and offered a reliable relative quantification of tryptic peptides in CSF. Our validation experiment confirmed the potential of 14 CSF peptides to discriminate PD from AP, already at an early disease stage when there is still a high level of uncertainty about the underlying aetiology of the specific movement disorder. The discriminative value of these tryptic peptides could be enlarged by the combination with existing biochemical markers or clinical assessments. Finally, our study may provide new insights into the underlying pathophysiological processes of each disorder.40, who all had clear clinical signs of parkinsonism, but with an yet unclear diagnosis at the time of inclusion, and who had been recruited from our movement disorders outpatient clinic between January 2003 and December 2006 at the Radboud University Medical Centre . In total, 25 CSF samples were included in the discovery experiment . For validation of our initial findings of the discovery phase we used 110 CSF samples from PD (n = 46), MSA (n = 17), PSP (n = 8), and non-neurological controls (n = 39).For both the discovery and validation experiments, we included participants from a longitudinal study40. After three and 12 years of inclusion, the diagnosis of all participants was critically revised again and a silver standard clinical diagnosis was established by two independent movement disorder specialists. To establish this diagnosis, the clinical experts used the most recent clinical criteria at that time42, combined with the now available long-term response to therapy, the rate of disease progression, and the possible development of red flags, which may alert clinicians to an alternative diagnosis. Patient characteristics are presented in Table 44. For details on the assays used for quantification of these protein biomarkers, see At the time of inclusion, patients underwent a structured standardized neurologic examination by movement disorders specialists. Lumbar puncture, was performed within 6 weeks after the initial visit. The design of this study, methodology, and patient inclusion have been extensively described45, Unified Parkinson’s Disease Rating Scale (UPDRS)47, Mini-Mental State Examination (MMSE)48, and International Cooperative Ataxia Rating Scale (ICARS)49.Clinical assessments at baseline and after 3 and 12 years of follow-up included the Hoehn and Yahr scoresFor comparison, we selected a group of non-neurological control patients who had underwent a lumbar puncture because of a suspected central neurological disorder. All selected control cases were free of neurological disease, as determined after careful examinations. Moreover, their CSF composition, such as leukocyte and erythrocyte count, glucose, blood pigments, lactate, and (if assessed) oligoclonal immunoglobulin G bands were all within the reference ranges for their age group.g, aliquoted, and stored in polypropylene tubes at −80 °C until use. All patients with PD or AP provided written informed consent and the study was approved by the local Medical Ethics Committee . The use of CSF leftovers from the control patients who had been seen as part of daily care in research projects was approved by the local Medical Ethics Committee.All CSF samples included in this study were collected in polypropylene tubes, centrifuged at 800 × 50.Total protein concentration in CSF was determined by using the 2D Quant kit , according to the manufacturer’s protocol, and 400 µg total protein was used as input for profiling. All samples were loaded on an affinity removal column for the depletion of the 14 most abundant proteins . After tryptic digestion, CSF samples were fractionated in 20 fractions using high pH reversed-phase C18 LC and each fraction was subsequently analyzed by nanoflow liquid chromatography  connected online to an ultra-high resolution quadrupole time-of-flight tandem mass spectrometer  as described previously51 with pre-defined Qq-ToF parameter settings against the RefSeq (release 55) human protein sequence database. We set cysteine carbamidomethylation as a fixed modification, whereas N-terminal acetylation, methionine oxidation, and deamidation of glutamine and/or asparagine were set as variable modifications. For further statistical analysis, only peptides with intensity above the detection limit in at least 75% of the samples in one of the groups  were used.Raw MS data were analyzed by MaxQuant software version 1.5p-value below 0.05 determined by Mann−Whitney U test comparing PD vs. MSA; (2) ratio of intensity (PD:MSA) of at least 1.5; (3) intensity values above MS detection limit in at least 75% of samples in both PD and MSA groups; (4) peptide length of maximal 20 amino acids; (5) uniqueness (assignment to only one protein); (6) information available in Uniprot52 or PeptideAtlas53; (7) exclusion of peptides with susceptibility to post-translational or chemical modifications, such as methionine and cysteine oxidation, a potential deamidation site, or N-terminal cyclization.For the selection of tryptic peptides for the SRM assay, additional criteria were used: (1) 13C615N2) or arginine (13C615N4) residue was added to each sample to allow peptide identification and relative quantification. Samples were cleaned by passing them over a 0.22 µm filter and stored at −80 °C until MS analysis.The CSF samples for SRM and MS analysis were processed in randomized order using 50 µL of CSF from each patient as input. Prior to protein digestion, samples were subjected to overnight freeze-drying to concentrate the sample. On the next day, the sample was reconstituted with 4.3 µL of 8 M urea solution in 10 mM Tris-HCL, and diluted with 4.3 µL 10 mM Tris-HCL to reach a final urea concentration of 4 M. Protein reduction was performed by incubation with 0.5 µL of 10 mM dithiothreitol for 30 min at room temperature, followed by alkylation by incubation with 0.5 µL of 50 mM 2-chloroacetamide for 30 min at room temperature, kept in the dark. Samples were then incubated with 1 µL of 0.5 μg/μl Lysyl endopeptidase C for 3 h at room temperature, resulting in a volume of 10.6 µL, which was subsequently diluted four times with 31.6 µL of 50 mM ammonium bicarbonate and incubated with 1 µL of trypsin (1 μg trypsin/50 μg protein) for 4 h at 37 °C. Digestion reaction was stopped by adding 4.8 µL of 10% trifluoroacetic acid. A cocktail of synthesized isotope-labelled “heavy” peptides  on the C-termini of the target peptides at either a lysine (Samples (2 µL) were subjected to LC-MS analysis in randomized order on the Acquity MClass UPLC Xevo TQ-S (Waters), coupled with an ionKey/MS system using a Waters peptide BEH C18, 130 Å, 1.7 μm, 150 μm × 100 mm ionKey column for chromatographic separation using a 30 min linear gradient of acetonitrile ranging from 3 to 35% with 0.1% formic acid at a flow rate of 2 μl/min.To optimize SRM settings in the SRM method development step, we used a pooled trypsin digested CSF sample spiked in with a cocktail of heavy labelled peptides , and specifically, the cone voltage and collision energy were optimized for each peptide fragment. For each peptide, we started with a selection of at least 10 peptide fragments per precursor (transitions). For the final multiplex SRM assay, at least 2 transitions with the highest signal intensity and lack of interference were selected for each peptide target. For each peptide fragment, retention time windows of 1 min were used, allowing both endogenous and heavy labelled peptides to have at least 8 data points per chromatographic peak with an average dynamically dwell time of 250 ms.R2) below 0.7 were excluded. The calibration curve was used to determine the best heavy labelled peptide concentration for the clinical samples and a new peptide cocktail was prepared; (2) intra-assay variation < 20% for 1 pooled digested CSF sample injected five times on the same day; (3) inter-assay variation < 20% for 1 digested CSF sample measured on 10 different days; (4) inter-assay sample preparation < 20% for 5 identical aliquots of pooled CSF samples, all digested and measured on the same day; (5) sample stability on the autosampler which was set at 10 °C by injecting 1 sample repetitively from the same plate every 4 h for 24 h; (6) freeze/thaw effect < 20% for 1 pooled and digested CSF sample subjected to up to 5 freeze/thaw cycles; (7) freeze/thaw effect < 20% for a pooled CSF sample subjected to 3 freeze/thaw cycles prior to the digestion procedure. CV was calculated between technical replicates and a CV of 20% was regarded as acceptable. Results of these 7 criteria for the SRM method validation for all included tryptic peptides are shown in Supplementary Table Our newly developed SRM method was validated using a pooled digested CSF sample mixed with a cocktail of heavy labelled peptides and the following criteria were investigated: (1) linearity to provide a calibration curve, by using a dilution series of the cocktail of heavy labelled peptides  spiked into pooled digested CSF in three replicates. Peptide fragments with a linear regression coefficient  for automated measurement.54. For data analysis, the relative quantification was determined by calculation of the ratio between endogenous and heavy labelled peptides. We normalized each ratio of endogenous : heavy labelled peptides for total CSF protein concentration as these markers were identified in the proteomics profiling where also a normalization on total protein content was applied.Skyline software version 20.1  was used to process raw data from SRM assay to confirm peak detection, correct integration, and calculation of the peak area55. Receiver operating characteristic (ROC) was used to determine the diagnostic accuracy by calculating the area under the curve (AUC).Analyses were performed in R software version 3.5.3 (Austria), IBM SPSS Statistics 25 , or GraphPad Prism 5 . Groups were compared by using two-sided Student’s T-test or Mann−Whitney U test in the case of two groups depending on the data distribution (parametric or non-parametric), and two-sided analysis of variance (ANOVA) with Bonferroni’s multiple correction as a post hoc test or Kruskal−Wallis one-way analysis of variance with Dunn’s as post hoc test when more than two groups were analyzed. Rank analysis of covariance was used for group comparisons taking age as a covariate, including Bonferroni’s multiple correction as a post hoc test. Correlation of peptides with age and clinical parameters, such as disease duration, UPDRS, ICARS, MMSE scores, was performed using Spearman’s rank correlation coefficient. Random forest was applied for multivariate analysis to generate decision trees to improve group discrimination. The models generated by random forest were developed in 70% of our cohort and validated in 30%. For random forest analysis, an imputation method  was used to fill in missing valuesFurther information on research design is available in the Supplementary InformationReporting Summary