Enhanced external counterpulsation (EECP) is provided by a noninvasive device positively affecting cardiovascular function via mechanisms called diastolic augmentation and systolic unloading. The renal aspects of EECP therapy have not been extensively investigated.To assess the effect of EECP on renal function and to determine the application in patients with kidney disease.MEDLINE, EMBASE, SCOPUS, and Cochrane CENTRAL databases were searched for all studies involving EECP treatments. The title and abstract of all searched literatures were screened, and those focusing on renal outcome or conducting in kidney disease patients were selected.Eight studies were included in the qualitative analysis. EECP increases stroke volume, mean arterial pressure, renal artery blood flow, renal plasma flow, glomerular filtration rate (GFR), plasma atrial natriuretic peptide, urine volume, and urinary sodium chloride excretion, but reduces the plasma concentration of renin and endothelin-1 in healthy subjects. A single session of EECP after radioactive contrast exposure could provide increased contrast clearance, and this reduces contrast-induced kidney injury in patients, irrespective of previous kidney function. Thirty-five-hour sessions of EECP treatment were illustrated to increase long-term estimated GFR in patients with chronic angina and heart failure. In cirrhotic patients, EECP fails to improve GFR and renal vascular resistance. EECP device could maintain blood pressure, decrease angina symptoms, and increase cardiac perfusion in hemodialysis patients.EECP treatment potentially increases renal perfusion and prevents kidney injury in several conditions. EECP possibly provides beneficial effects on hemodynamics and cardiac function in hemodialysis patients. Figure 1). The device consists of 2–3 inflatable pneumatic cuffs applied around the thighs, legs, and buttock that can inflate or deflate synchronously with electrocardiography (ECG) waveform. Similar to the mechanism of IABP, EECP enhances cardiac function in both the systolic and diastolic phases [Enhanced external counterpulsation (EECP) is a therapy provided by a noninvasive device, which was initially utilized to maintain hemodynamic stability in patients with cardiac dysfunction for more than half a century . Before c phases , 6. In tc phases , 6.EECP has been demonstrated in many cardiovascular diseases, at least in part, due to reported positive cardiovascular effects of EECP in humans such as increasing myocardial perfusion and systolic function, improving endothelial function via a change in nitric oxide and endothelin-1 from shear stress generated from the counterpulsation, promoting angiogenesis of collateral coronary vessels, enhancing peripheral effects, and improving neurohormonal factors, all of which are similar to those from regular physical exercise ,8,9. TheAs stated above, EECP might potentially provide beneficial effects on renal hemodynamics and renal function. There have been certain available studies regarding this interesting issue. The present systematic review was conducted to determine the effect of EECP on renal function, and application of EECP in patients with kidney diseases.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .As studies of EECP therapy in general, and those focusing on renal outcome or studying in kidney disease patients are scarce, we chose a less strict searching strategy to cover all possibly relevant studies. We searched the MEDLINE, EMBASE, SCOPUS, and Cochrane CENTRAL databases from inception until February 2023 using the following key search terms: “enhanced external counterpulsation” or “enhanced external counter-pulsation” or “enhanced external counterpulsation” or EECP. In addition, references in the selected studies were screened manually for additional records.The title and abstract of all retrieved studies were independently screened and selected by two authors (TT and WK) and only studies focusing on renal outcome or kidney disease patients were selected. All disagreements were resolved by discussion and consensus with the third author (KT). EECP application of any protocols and durations, short- to long-term, was included and stated in the study details. The literature without a full text in English was excluded. Studies with reported quantitative outcomes and comparison groups were included for meta-analysis.The following data were extracted from the included studies: first author's name, year of publication, country of study, study design and comparison, included patients, study sample size, protocol, duration of EECP therapy along with data collection time, outcomes, measurements, contraindication for EECP, reported data, and adverse effects from the therapy.The process of quality assessment was similar to that for data screening and selection. The quality of the included literature was evaluated with Methodological Index for Non-Randomized Studies (MINORS) if there was no comparison group. There were 8 items for non-comparative studies and each item was scored 0 if not reported; 1 when reported but inadequate; and 2 when reported and adequate. The global ideal score was 16 for non-comparative studies. However, there was no validated cut point for the score . For ranAs the included studies could not be combined into a meta-analysis due to their lack of comparison group and difference in measured outcomes, our systematic review is presented mainly in a narrative manner and no statistical analysis was utilized.Figure 2. From an initial set of 1,578 records, 5 of 13 eligible studies were further excluded due to lack of full text to yield the final 8 studies for qualitative analysis. There was no study for meta-analysis due to their lack of similar qualitative outcomes and comparison groups. The characteristics of the included studies are detailed in Table 1. The MINORS quality assessment of the studies is demonstrated in Table 2. For an included randomized controlled trial, the Cochrane Collaboration's tool for assessing risk of bias reveals low risk of selection bias, detection bias, attrition bias, and reporting bias due to a clear explanation on randomization and low number of loss follow-up. There was no declaration about the allocation concealment. Despite a non-sham control manner, the performance bias was considered as low risk because all outcomes were objective laboratory measurements.The flowchart of study selection is depicted in P < 0.05) upon application of EECP [P = 0.0001) along with a peak systolic flow velocity increase of 8% (P = 0.006) [Figure 3). The cardiac cycle lengths were not affected by EECP therapy. Of interest, these renal and systemic augmentation were fully reversible after the termination of EECP session [In healthy subjects, EECP therapy has an immediate effect on systemic hemodynamics, local renal hemodynamics, and neurohormonal factors , 19. Str of EECP  with a t of EECP . The enh of EECP . This bl= 0.006) . The aug session .P < 0.05), glomerular filtration rate , and a trend in decreasing renal vascular resistance (68.5 ± 17.2 to 55.2 ± 12.5 mmHg·min/l) [2O (P < 0.05). Plasma epinephrine and norepinephrine concentrations were relatively unchanged [In consort with the systemic hemodynamic changes, renal hemodynamic parameters in healthy subjects as measured by indicator clearance technique also exhibited increased renal plasma flow  . Moreovenchanged .Patients with cirrhosis are well known to be at risk of renal hypoperfusion from splanchnic blood redistribution, resulting in acute to chronic renal dysfunction, so-called hepatorenal syndrome. Werner et al.  demonstrnd h after iopromide radiocontrast exposure for diagnostic computed tomography significantly enhanced the contrast clearance as calculated from 2-point plasma iopromide concentration measurements without urine collection . Moreover, EECP therapy yielded a significant reduction in the incidence of ≥10% increase in serum cystatin C at the 24th h after radiocontrast exposure  [th and 48th h after radiocontrast exposure was low in the study  [Zhang et al.  demonstr5–0.653) . However2, P = 0.047). The incidence of contrast-induced kidney injury was significantly lower with EECP therapy  along with lower high-sensitivity C-reactive protein, possibly reflecting reduced renal inflammation from contrast media [Zeng et al.  exploredst media .2 to 76.27 mL/min/1.73 m2 after a median follow-up time of 16 months after the treatment  with a trend in decreasing N-terminal pro b-type natriuretic peptide (NT-proBNP). A significant increase in estimated GFR was identified mainly in a subgroup of 12 patients with an initial estimated GFR of <60 mL/min/1.73 m2 and those with initial NT-proBNP of >125 pg/mL, probably representing patients with cardiorenal syndrome [Ruangkanchanasetr et al.  examinedsyndrome .Previously mentioned studies on renal outcomes of EECP included patients with relatively preserved renal function or early stage of CKD. In hemodialysis patients, Wu et al.  studied However, there has been no study applying EECP treatment during the dialysis session. There was only one case series by Onuigbo , who sucThere is an ongoing randomized controlled trial conducted by the authors of this systematic review regarding application of EECP during hemodialysis. A preliminary result reveals the maintenance of cardiac output and DBP, which are normally declined due to the ultrafiltration process. The incidence of intradialytic hypotension is absent in the EECP group and the longer term effects on cardiovascular parameters are under investigation .Two patients in the study by Wu et al.  did not EECP has long been shown in patients with coronary artery disease to increase cardiac output, coronary blood flow, and myocardial perfusion via the diastolic augmentation and systolic unloading mechanisms ,28,29. TTo date, there have been a few studies investigating the renoprotective effect of EECP, especially those with treatment duration of more than a single EECP session. The most promising renoprotective effect of EECP is the prevention of contrast-induced kidney injury. A recent randomized controlled study by Zhang et al. , which i2, especially in those with initial estimated GFR of <60 mL/min/1.73 m2 and high initial NT-proBNP level. This possible amelioration of progressive cardiorenal syndrome by EECP is limited by the study design that had no control group. As such, the change in estimated GFR might be mediated by other causes such as the heart failure treatment [Regarding patients with cardiorenal syndrome, a study by Ruangkanchanasetr et al.  applyingreatment . In cirrreatment  failed tIn hemodialysis patients, Wu et al.  demonstrIn terms of adverse effects, EECP application in the general population is a relatively safe treatment modality after appropriate patient selection and exclusion of contraindications such as significant aortic regurgitation and abdominal aortic aneurysm. Possible adverse effects to be monitored include intracranial hemorrhage, hemorrhoid bleeding, headache, minor muscular pain, and cutaneous irritation. However, the adverse effects of EECP application in patients with kidney disease have been rarely reported and require further surveillance.Despite being the first study to comprehensively review the application of EECP in patients with kidney disease, the limitation of our study is a few included studies. This also impedes the author from conducting meta-analysis. Moreover, the varied quality assessment of the included studies necessitates more mechanistic and clinical studies with good study design, larger sample size, more various patient subgroups, adequate follow-up time, and hard endpoints before the final consensus on this topic could be drawn.In conclusion, EECP therapy is safe and potentially able to prevent renal injury in patients with cirrhosis, heart failure, radiocontrast exposure, and chronic angina. In hemodialysis patients, EECP possibly poses a positive cardiovascular effect. However, closed surveillance for possible additional adverse effects after application of EECP in this group of patients is required.


Hypertension is a prevalent chronic disease associated with an increased risk of cardiovascular (CV) premature death, and its severe form manifests as resistant hypertension (RH). The accurate prevalence of resistant hypertension is difficult to determine due to the discrepancy in data from various populations, but according to recent publications, it ranges from 6% to 18% in hypertensive patients. However, a comprehensive understanding of the pathogenesis and treatment of RH is essential. This review emphasizes the importance of identifying the causes of treatment resistance in antihypertensive therapy and highlights the utilization of appropriate diagnostic methods. We discussed innovative therapies such as autonomic neuromodulation techniques like renal denervation (RDN) and carotid baroreceptor stimulation, along with invasive interventions such as arteriovenous anastomosis as potential approaches to support patients with inadequate medical treatment and enhance outcomes in RH. Hypertension is one of the leading chronic diseases, ranking high among risk factors for cardiovascular (CV) death in patients. About one billion patients struggle with it, which poses quite a challenge to modern medicine ,2,3. A pIt is important that in patients with inadequate BP control, either ambulatory blood pressure monitoring (ABPM) or home blood pressure monitoring (HBPM) should be checked in patients with confirmed normal compliance. According to many studies, the estimated range of RH prevalence is not conclusive due to varieties in populations and calculation methods ,14,15,16Due to the analysis of data from the Scandinavian clinical program, initial higher BP, male gender, diabetes, left ventricular hypertrophy, increased body mass index (BMI), and higher alcohol consumption are the risk factors of RH development . ATRH isThe efficiency of antihypertensive treatment is variable, which is confirmed by the differences in the results of studies on arterial hypertension. This effectiveness is lower in patients with comorbidities such as diabetes or chronic kidney disease . Most paEpidemiological studies on the occurrence of apparent and true RH are necessary to more precisely define the frequency of these types of hypertension and better assess this problem in the general population. The RH guidelines focus on the white coat effect, compliance, and pharmacological adherence as components that need to be considered when diagnosing RH  to excluThe overarching goal of hypertension treatment is to lower BP values so as to reduce the risk of CV events ,21. The Target BP values vary depending on age and concomitant diseases . AccordiThe basis for diagnosing RH is a properly performed BP measurement . HoweverA standard BP cuff is 12–13 cm wide and 35 cm long . A propeOne of the causes of a misdiagnosis of RH is incorrect BP measurement ,24. CommDespite the availability of various arm cuff sizes, BP measurement in obese individuals with a large arm circumference can be problematic ,29. In aFor a correct diagnosis of RH, it is necessary to measure BP out of office to exclude the white coat effect . AccordiOther reasons for a misdiagnosis of RH include poor adherence to medical recommendations  . FurtherTo estimate the OSA risk, various questionnaires are used. The commonly used ones include the Berlin questionnaire, Epworth Sleepiness Scale, and the STOP-Bang questionnaire . To confThe most common endocrine cause of RH is hyperaldosteronism, with the prevalence of primary aldosteronism from 5 to 15% in the hypertension population . PrimaryThe early detection of secondary hypertension enables prompt intervention to mitigate its effects and prevent permanent changes in the body. That is particularly crucial in younger patients, as interventions later in life are less likely to be cured due to the permanent vascular and organ damage caused by chronic hypertension in these individuals.Inadequate drug adherence is a significant problem in the treatment of AH. It is a common cause of poor BP control, as well as pseudo-RH (≤50% of patients). It usually occurs as irregular medication intake and early treatment discontinuation. The consequence of nonadherence is an increased risk of CV events . There aA post hoc analysis by Kario et al.  aimed toSimilar results regarding the percentage of nonadherence to medication were provided by a meta-analysis by Bourque et al. , where nCoinciding with the aforementioned are the results of a meta-analysis by Lee et al. , where aAdherence to AHM recommendations is also important in terms of the occurrence of CVD events. Feng et al.  proved tThe reasons for such high nonadherence include the high cost of treatment, the complicated dosing of AHM, the occurrence of numerous side effects, or therapeutic inertia, among others .Articles summarizing studies evaluating treatment adherence are presented in There are several ways to improve drug adherence. These include simplifying therapy, i.e., preferring long-acting drugs, avoiding the complex dosing regimen of AHM, and using single-pill combinations as often as possible. In addition, it is important to avoid the side effects of the drug and to make therapy as compatible as possible with the patient’s financial capabilities. It is also essential to try to look for ways to improve adherence that are individually tailored to the patient, to motivate and encourage them to take care of their health .Recent studies suggest that telemetry may be important in improving drug adherence . High hoIt is very important to start the whole treatment process first with nonpharmacological methods that will, of course, only gently support pharmacological therapy. It is well known that a healthy lifestyle is a very important factor affecting quality of life and health, and in this case, BP values . PatientA blocker of the RAA system, with special emphasis on angiotensin-converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs);A long-acting calcium channel blocker (CCB), most commonly amlodipine;A long-acting thiazide diuretic, i.e., indapamide or chlorthalidone.Pharmacological treatment consists of a group of ≥3 hypotensive drugs. The classic regimen consists of the use of:We also have other drugs like aldosterone synthase inhibitors or dual endothelin antagonists to choose from .These three large groups of drugs are complementary in their mechanisms affecting the lowering of BP values by which, through the choice of doses and frequency of administration, they should give due therapeutic effect and reduce the risk of death ,8. The mThe PATHWAY-2 (Prevention and Treatment of Hypertension With Algorithm-Based Therapy) trial showed a significant benefit when patients were supplemented with another drug, spironolactone, a mineralocorticoid receptor antagonist, in addition to the above-mentioned drugs. It is worth adding that due to side effects such as gynecomastia, impotence, and menstrual disorders, not all patients will tolerate mineralocorticoid receptor antagonists (MRAs) well. Due to the small number of studies on patients with renal impairment, we can use this drug for those with eGFR ≥45 mL/min and serum potassium levels ≤4.5 mmol/L . An alteA group worth paying special attention to because of the increased risk of dehydration and orthostatic hypotension are the elderly. As they age, their physiological mechanisms weaken, and both thirst and the accumulation of primary urine in the kidneys decrease. Their total body water decreases, which consequently leads to lower fluid stores. In addition to physiological causes, we can add risk factors such as cognitive impairment, dysphagia, and deliberately reduced fluid intake, for example, due to fear of incontinence. Furthermore, frequent use of diuretics leads to an even further intensification of the emptying of fluid stores, further aggravating dehydration . OrthostRenal denervation (RDN) is a minimally invasive, investigational procedure that has emerged as a potential therapeutic option for patients with RH. Its mechanism of action includes interrupting the renal sympathetic activity by destroying sympathetic nerves localized close to the renal arteries. Activation of the efferent renal sympathetic nerves leads to renin secretion, sodium absorption as well as increased renal vascular resistance. Consequently, it contributes to the pathogenesis of hypertension. The result of RDN is a reduction in sympathetic activity and, therefore, a decrease in renin activity and an increase in renal blood flow . RH is aEarly trials on RDN are focused on severe hypertension, with SBP over 160 mmHg ,53,54. IMore recent studies have focused on patients with lower BP values ,56,57. TThe aim of baroreflex activation therapy (BAT) devices is to artificially strain the carotid baroreceptor and, as a consequence, induce a decrease in BP. Carotid baroreceptor stimulation (CBS) was initially associated with a high number of adverse incomes, mainly related to periprocedural complications due to the implantation of electrodes around both carotid arteries. However, the procedure has developed and shown promising effects in recent years.The first-generation CBS system called Rheos consists of two electrodes implanted bilaterally in the region of the carotid sinus and conducted between the heads of the sternocleidomastoid muscle to a pulse generator. Rheos has been improved to a second-generation device, which is Barostim neo™ . It diff® device was used. After 3 months of baroreceptor stimulation, a reduction in SBP of 21 mm Hg and DBP of 12 mmHg was achieved. The reduction in BP was even greater after 12 and 24 months of treatment. The Rheos Pivotal Trial [The first study assessing BAT with a long-term implantable device was the DEBuT-HT trial  . CVRx Rhal Trial , a doublal Trial . At 3 moClinical trials on BAT are still in progress. The MobiusHD™ is a device undergoing investigation that is not based on electrical stimulation . This stArteriovenous anastomosis is another of the invasive treatments for hypertension . It has A meta-analysis by Scholz et al.  evaluateTo properly diagnose RH, BP measurement should be performed meticulously following the recommendations of current guidelines. It is also necessary to exclude factors that may contribute to the misdiagnosis of RH, so-called pseudo-RH.In the diagnosis of RHT, it is crucial to consider the presence of secondary causes that contribute to its development. Early detection of secondary HT, particularly in younger patients, holds the potential to prevent irreversible structural and functional changes in organs, thus mitigating the risk of organ failure.The pharmacological treatment of RH is complex. The main groups of drugs are blockers of the RAA system, long-acting calcium channel blockers, and long-acting thiazide diuretics, which are combined in polytherapy. In addition to pharmacological treatments, all patients at every stage of therapy should take care of the principles of a healthy lifestyle, i.e., limit salt intake, increase physical activity, and reduce body fat. Furthermore, the use of autonomic neuromodulation therapies in RH treatment has become very promising recently. Clinical trials on device-based therapies are still ongoing.


Anemia is common in chronic kidney disease (CKD) and is associated with outcomes. In addition, serum soluble Fas (sFas) levels are related to anemia and erythropoietin (EPO) resistance.Firstly, to compare clinical data and serum levels of sFas, EPO, and pro-inflammatory markers between patients with non-dialytic CKD (NDD-CKD) and healthy subjects. Subsequently, to compare and evaluate the relationship of serum EPO, sFas levels with anemia, and outcomes in patients with NDD-CKD over a long follow-up period.We performed a retrospective study in 58 NDD-CKD patients compared with 20 healthy subjects on complete blood count, kidney function, serum EPO, sFas, and inflammatory markers  at baseline. We then compared the same baseline data between patients with NDD-CKD who evolved to anemia and those who did not have anemia over the follow-up. We also evaluated the frequency of outcomes in patients with CKD with higher sFas levels. Finally, we performed a multivariate analysis of factors associated with CKD anemia.There were lower eGFR and Hb but higher serum inflammatory markers, sFas levels, sFas/eGFR, and EPO/Hb ratios in patients with NDD-CKD. Comparatively, on the other hand, NDD-CKD patients with anemia had lower eGFR but were older, had more diabetes, and had higher sFas/ eGFR, EPO/Hb ratios, and serum levels of IL-6 and sFas than NDD-CKD without anemia for an extended period. In addition, there was an association in a multivariate analysis of diabetes, age, and sFas levels with kidney anemia. Furthermore, there were higher frequencies of outcomes in increased serum sFas levels.As an elective risk factor, serum sFas levels, in addition to age and diabetes, were independently associated with kidney anemia for an extended period. Thus, more studies are necessary to analyze the proper relationship of sFas with kidney anemia and its outcomes and therapy in CKD. Anemia is a common complication of chronic kidney disease (CKD) . It is aEPO is a glycoprotein composed of 166 aa, and its production occurs mainly in the kidneys. EPO acts as a peptide hormone on its specific receptor (EPOR), distributed in the most diverse tissues , 5. HoweManagement of CKD-related anemia consists of erythropoiesis-stimulating agents (ESA) and iron replacement –12. EvenThe soluble form of the Fas receptor (sFas) is produced as an alternative splicing product of CD95/ Fas mRNA or can be cleaved by metalloproteases that lack the transmembrane domain , 18. ThuIn addition, studies reported that serum sFas levels are associated with autoimmunity and hyporesponsiveness to EPO and are predictors of the need for ESA therapy in patients with CKD , 17. ThuWe performed a retrospective study of NDD-CKD patients at the outpatient clinic of the Nephrology Service of the Federal University of São Paulo (UNIFESP) between late 2007 and 2019. In addition, we were able to evaluate data from our database that included patients with NDD-CKD from other publications. , 19.The current study was handled according to the guidelines of the Declaration of Helsinki of 1975, revised in 2013. The Institutional Committee on Human Research, Review and Ethics Board of the Federal University of São Paulo/UNIFESP, SP, Brazil approved the study .No individual personal data was revealed or included in our study. All participating volunteers had access to the free and informed consent term. After reading, they signed it, authorizing and approving their participation. Volunteers who agreed to participate in the research could request their withdrawal from the study at any time without interference or discrimination in the treatment they were undergoing. Informed consent included using clinical data and results from anonymously extracted and personally de-identified sera. The researchers stored the patients’ data, and it is under total ethical secrecy.2 and no marker of kidney damage, such as proteinuria. Therefore, we formed the NDD-CKD and non-CKD groups and evaluated and compared some variables at baseline. So, we performed correlations of the variables in the study of NDD-CKD patients. After, we created two new other subgroups and realized comparisons of parameters at baseline between CKD patients with and without long-term anemia. The analysis of long-term anemia occurred by Hb concentration at the end of follow-up up to 12 years, at the last Hb assessment before initiating kidney replacement therapy, or in evaluating Hb concentration before death among all patients with NDD-CKD. Afterward, we performed multivariate analyzes between NDD-CKD patients using long-term anemia as a dependent variable and variables at baseline as predictor variables.First, all the clinical data of the patients were obtained through the chart medical records. Then, we analyzed the clinical data and serum levels of pro-inflammatory markers, EPO, and sFas, in patients with CKD undergoing conservative treatment. Our data bank contains all the enrolled CKD patients with serum measurements of EPO, sFas, osteo-metabolic and inflammatory markers collected between December 2007 to July 2008. Next, the data from patients with CKD were compared with 20 healthy volunteer individuals. All healthy volunteers had no CKD, diabetes, or hypertension. Thus, the healthy subjects had an eGFR above 60 ml/min/1.73 mFor all analyses, 126 patients with NDD-CKD filled out the following inclusion criteria: age >18 years and CKD diagnosis for at least three months with CKD stages according to the eGFR between stages 1 to 5, ranging from 11.2 to 91.1 ml/min/1.73. All CKD patients have had proteinuria for at least three months, and blood tests at baselines, such as complete blood count, iron status, kidney function, and serum levels of molecules of interest measured in the study. The exclusion criteria were a change of center and nephrological medical service, referral for kidney transplantation with a living or deceased donor within six months of enrolling in the outpatient clinic, iron depletion, presence of hematological diseases, active bleeding, abnormality in mean corpuscular volume and mean corpuscular hemoglobin, and need for ESA within 30 days after admission to the Nephrology outpatient clinic. In addition, we also excluded patients with a diagnosis of neoplasia, chronic viral diseases such as human immunodeficiency virus, hepatitis B and C infection, and those who started dialysis as a kidney replacement therapy or died within three months of enrollment in the NDD-CKD outpatient clinic. Thus, we excluded 68 patients from the study .We collected clinical and demographic data at baseline from patient charts. They included age, sex, active smoking, CKD etiology, body mass index (BMI), complete blood count , iron status, serum albumin, and medications in use, such as a renin-angiotensin system blocker, iron replacement, and use of ESAs. In addition, serum levels of pro-inflammatory markers, EPO, intact parathyroid hormone (iPTH), and sFas, were analyzed from our database of the same patients and with extracted blood sample in the exact period that the clinical tests were analyzed. We also computed the need for ESA, need for red blood cell transfusion, need for dialysis, and mortality as outcomes for up to 12 years of follow-up since admission to the Nephrology outpatient clinic. So the last follow-up and anemia data analysis were for up to 12 years. Or we also got data, such as Hb concentration, before initiating kidney replacement therapy or at the previous assessment before death among all patients with NDD-CKD. We used the sFas/eGFR ratio in correlations and comparisons because that is the molecule evaluated as a uremic retention solute related to clinical outcome.Anemia was defined as Hb<13.0 g/dl for men and postmenopausal women or Hb<12.0 g/dl for premenopausal women , 23. ESA®, Belo Horizonte, Brazil). We considered proteinuria values greater than 10mg/dl by dipstick urine analysis . Therefore, proteinuria with a value greater than 0.1g/l or when the albumin-to-creatinine ≥30mg/g over three months determined kidney damage Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: No**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: These are my comments to the author:In Material, Methods section:- It is a retrospective study for non-dialysis CKD patients presented to outpatient clinics, you should give some details about stages of CKD you are followed- You followed only 97 patients?- You should specify the date of sample collections and then the follow up period- You have used healthy control group; you should give some details about how you chose them- During follow up period which is 12 years, what about outcomes, how many patients, is any patient started dialysis, is there any patients died, any patients transplanted, also you mentioned the exclusion criteria, is it on initial screening or during follow up period?- Retrospective study means you depend on patients file review, do you do routine laboratory tests including pro-inflammatory cytokines and sFAS???Result section:- In table 1 the number on control group is 20, however, in the flow chart figure, the number is 21m which one is correct?- The mean eGFR for NDD-CKD was 35.7ml/m±2.5, however the eGFR after 12 years follow up for non-anemic NDD-CKD patients in table 2 was 54.9±5.26, is it worth for CKD patients over the long period of follow up?? It should be lower than at the initial presentation?- It is better to compare between groups at initial presentation and after follow up period- There are few language errors like in row 8 after table 3 (seven (17.9%) out of 39, the correct out of-Discussion part:- The discussion section looks more as an introduction part with no more details regarding comparing your study with other previous studies- There are some few language errors in some words for correction pleaseReviewer #2: Comments1- There are few grammar mistakes, confused sentences, and typos throughout the manuscript. It should be revised carefully.2- The introduction should be one way or another to be extended to include dissertation about erythropoietin (EPO).3- References should be updated in both the introduction and the discussion (lack of 2021 &2022 references).4- Results are well organized, but correlations were not discussed in the discussion part of the manuscript.5- The discussion is poor and lack explanations of many subtracted points, in addition it should be extended; more other previous studies in the same area of the present study should be used to show that the present results match or do not with that of other previous studies.6- The conclusion was limited only to diabetes mellitus, age, and higher serum sFas levels although the study includes several other variables such as Hb, serum albumin IL-6, IFN-γ, Serum EPO and iPTH which were not discussed well.7- There are some weak points in the present study: First, the sample sizes of the patients, and the healthy volunteers who participated in the study were small as proven in the article by the authors and this is againt editorial policies and submission guidelines of PLOS ONE. Also, the researchers did not carry out any intervention in the group of patients and they focused on patients with NDDCKD and serum levels of inflammatory markers (which were not referred to in the discussion) and sFas that are associated with anemia and hyporesponsiveness to EPO, with most of the patients already reported.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentComments on PLOS ONE article.docxSubmitted filename: Click here for additional data file. 1 May 2023To the Academic Editor:1. Respond point-by-point to the reviewers' comments. #1Comments, we appreciate the comments of all reviewers, and we have modified all suggested points in detail.2. Check the manuscript for grammatical and syntax errors. #2Comments, we greatly appreciate this comment and reinforce the correction of grammar and syntax errors. 3. Discussion section needs to be rewritten. #3Comments, we’d like to thank you for the comment. We emphasize that several parts of the discussion were modified, aiming to adapt the language, clarify information and further enrich the content. Corrections related to journal requirementsAll the notes made by the journal were made by the authors, which included: the revision of the formatting, notes added to the topic ‘’ethical issues’’, and the clarification related to the Data Availability statement. _________________________________________________________________Comments to the authors:Reviewer #1: In Material, Methods section:1- It is a retrospective study for non-dialysis CKD patients presented to outpatient clinics, you should give some details about stages of CKD you are followed1#Comments,Correspondent author: We thank you a lot for this important observation. Yes, We followed up with 58 patients diagnosed with CKD undergoing conservative treatment between stages 1 to 5 according to the eGFR, which ranged from 11.2 to 91.1 ml/min/1.73, and proteinuria after exclusion criteria. Here eight individuals in stage 1 or 2 who had been healthy volunteers with eGFR >60 but had a marker of kidney damage - proteinuria in late 2007/early 2008 in urine analysis in samples collected and analyzed with an interval of more than three months. So, they are currently our CKD outpatients since the diagnosis. As suggested, we wrote the patients' stages and eGFR in the Materials and Methods section.2- You followed only 97 patients?2#Comments,Correspondent author: Thank you again for this comment. In fact, in our database, there were 126 outpatients admitted between December 2007 and July 2008 with CKD under conservative treatment, presenting an evaluation of blood count, iron status, kidney function, and urine I. In parallel, they were enrolled for research in our database, with measurement of serum levels of sFas, EPO, osteo-metabolic, and inflammatory markers. We had not reported these patients before because 29 patients changed centers or were followed up for kidney transplants within six months of registration in our database. As amended, described in the main text and Figure 1 after we've looked it over carefully. Thus, we could follow up with 58 patients with CKD after exclusion criteria over 12 years.3- You should specify the date of sample collections and then the follow up period.3#Comments,Correspondent author: We thank you for this information, and we have edited all method sections to describe better the date of sample collections and the follow-up period.We also computed the need for ESA, need for red blood cell transfusion, need for dialysis, and mortality as outcomes over 12 years of follow-up since admission to the Nephrology outpatient clinic. So the last follow-up and anemia data analysis were for up to 12 years. Thus, we also got data before initiating kidney replacement therapy or at the previous assessment before death among all patients with NDD-CKD.4- You have used healthy control group; you should give some details about how you chose them.4#Comments,Correspondent author: Thank you for this encouraging comment. We have included more details about the healthy volunteers in the second paragraph in Patients and Data collection.5- During follow up period which is 12 years, what about outcomes, how many patients, is any patient started dialysis, is there any patients died, any patients transplanted, also you mentioned the exclusion criteria, is it on initial screening or during follow up period?5#Comments,Correspondent author: Thank you for this critical observation. We have reported the outcomes in Methods and Results. So, we have computed the need for ESA, need for red blood cell transfusion, need for dialysis, and mortality as outcomes for up to 12 years of follow-up since admission to the Nephrology outpatient clinic. During the 12 years of follow-up, 11 patients required a dialysis program. Unfortunately, there were five non-survivor patients over 12 years of follow-up. 6- Retrospective study means you depend on patients file review, do you do routine laboratory tests including pro-inflammatory cytokines and sFAS???6#Comments,Correspondent author: Thank you for this relevant point. In the current study, we have included patients with iron status, complete blood count, kidney function, and urine analysis and collected that patient data from charts. In addition, we have a database for research with serum analysis of sFas, inflammatory markers, and bone profiles parallel to the charts' clinical data. Prof. Miguel Angelo has been studying serum levels of sFas, and inflammatory markers related to anemia in CKD since his Master's degree. Thus, he stores the database safely and ethically. Therefore, after 12 years of follow-up and observing outcomes, we decided to publish these data with approval by the ethics committee of UNIFESP.Result section:7- In table 1 the number on control group is 20, however, in the flow chart figure, the number is 21m which one is correct?7#Comments,Correspondent author: Thank you for this critical observation.We have made a mistake in typing. The correct number is 20 healthy individuals. We corrected it in the figure and the main text.8- The mean eGFR for NDD-CKD was 35.7ml/m±2.5, however the eGFR after 12 years follow up for non-anemic NDD-CKD patients in table 2 was 54.9±5.26, is it worth for CKD patients over the long period of follow up?? It should be lower than at the initial presentation?8#Comments,Correspondent author: Thank you for this critical observation.We appreciate your intelligent and solid question. We redid all the statistical analysis and looked over the typing again.In fact, we retitled Table 2 for clarity. Thus, in this table and the results in the main body of the text, we analyzed two more subgroups of 58 patients with CKD for the long-term anemia outcome. Thus, we were able to compare patients with CKD and anemia in the long term and patients with CKD without anemia for a long time. However, baseline eGFR, as expected, was higher in the group without long-term anemia .Consequently, we used baseline variables in these comparisons and then performed binary logistic regression with long-term anemia as the dependent variable.However, these analyses supported our hypothesis, as these two subgroups did not have statistically significant serum EPO levels, although patients with long-standing anemia had higher EPO values. These same patients had higher levels of sFas, sFas/eGFR ratio, and EPO/Hb, indicating that the lower relative production is the main factor of anemia but that some uremic solutes retained in renal dysfunction contribute to anemia in these patients.9- It is better to compare between groups at initial presentation and after follow up period 9#CommentsCorrespondent author: Again, thanks for your critical opinion. We believe there was a mistake from us, and for better understanding, we changed the title of Table 2 to “Comparison of factors at baseline between chronic kidney disease patients who had anemia and those who did not have anemia at the end of follow-up”. We compared the eGFR values at baseline of patients with CKD-NDD who did not have long-term anemia (54.9±5.26) with those who were long-time anemic (27.7±.72 ), both at the end of the follow-up.10- There are few language errors like in row 8 after table 3 (seven (17.9%) out of 39, the correct out of10#Comments,Correspondent author: Thanks a lot. As suggested, we have made changes for better understanding.Discussion part:11- The discussion section looks more as an introduction part with no more details regarding comparing your study with other previous studies11#Comments,Correspondent author: Again, thank you for your opinion. We sought to enrich the Discussion section by comparing our findings with recent archives in the literature.12- There are some few language errors in some words for correction please12#Comments,Correspondent author: Thank you. As suggested we try to fix language errors in the Discussion sectionReviewer #21- There are few grammar mistakes, confused sentences, and typos throughout the manuscript. It should be revised carefully.1.2#Comments, Correspondent author: Thank you for this comment. As suggested, we carefully proofread the text, removing confusing phrases.2- The introduction should be one way or another to be extended to include dissertation about erythropoietin (EPO). 2.2#CommentsCorrespondent author: Again, thanks for your critical opinion. As suggested, we have included more concepts about erythropoietin (EPO) in the introduction.3- References should be updated in both the introduction and the discussion (lack of 2021 &2022 references).3.2#Comments,Correspondent author: Many thanks, for your opinion.As suggested, we have included new references in the Introduction and Discussion section after careful reading and review in line with our writing.4- Results are well organized, but correlations were not discussed in the discussion part of the manuscript.4.2#Comments, Correspondent author: Thank you again for this comment.As suggested, we reported the relationship of sFas (the focus of our study) with eGFR and some results of the correlation between sFas and both erythropoietin hyporesponsiveness (EPO/Hb ratio) and inflammation (IL-6) in the Discussion Sector.5- The discussion is poor and lack explanations of many subtracted points, in addition it should be extended; more other previous studies in the same area of the present study should be used to show that the present results match or do not with that of other previous studies.5.2#Comments,Correspondent author: Thank you for this critical observation. As suggested, the discussion has been expanded and matched with other research.6- The conclusion was limited only to diabetes mellitus, age, and higher serum sFas levels although the study includes several other variables such as Hb, serum albumin IL-6, IFN-γ, Serum EPO and iPTH which were not discussed well.6.2#Comments,Correspondent author: Again, thank you for this critical observation. As suggested, we discuss more Hb, IL-6 serum albumin, IFN-γ, serum EPO and iPTH in the Discussion Sector. Thus, as suggested, we have included a few more inflammatory markers in the Discussion and Conclusion.7- There are some weak points in the present study: First, the sample sizes of the patients, and the healthy volunteers who participated in the study were small as proven in the article by the authors and this is againt editorial policies and submission guidelines of PLOS ONE. Also, the researchers did not carry out any intervention in the group of patients and they focused on patients with NDDCKD and serum levels of inflammatory markers (which were not referred to in the discussion) and sFas that are associated with anemia and hyporesponsiveness to EPO, with most of the patients already reported.7.2#Comments,Correspondent author: We appreciate this feedback, critical observation, and opinion.Indeed, as we reported in the manuscript, our study had many limitations. Our sample size at enrollment was larger by our database, which is stored safely and ethically. However, there were many reasons for exclusion, such as the preferential request for a referral for a kidney transplant in the first six months of outpatient follow-up, even non-survival or need for dialysis, or use of ESA in the first three months of outpatient follow-up. In addition, there were severe comorbidities such as HIV, chronic hepatitis, neoplasms, or even hematological diseases that directly interfered with the anemia of these patients. Likewise, our control group was initially formed by 30 volunteers. All of them were employees from our University environment (not described in the manuscript). Of these, seven were excluded due to incomplete data. Unfortunately, two still died in the pandemic, and one asked to no longer participate in the study.AttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 25 May 2023A retrospective view of the relationship of soluble Fas with anemia and outcomes in chronic kidney diseasePONE-D-23-00605R1Dear Dr. Andrade,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Lakshmi KannanAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 22 Jun 2023PONE-D-23-00605R1 A retrospective view of the relationship of soluble Fas with anemia and outcomes in chronic kidney disease Dear Dr. Andrade:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Lakshmi Kannan Academic EditorPLOS ONE


In the adequate perfusion group, infarct proportion was closely associated with SBPdropmax ; 1.22 ± 0.48, (< 0.01)), SBPincmax ), SBPcv (0.61 ± 0.15 (< 0.01)), SBPsd (0.66 ± 0.08 (< 0.01)), and SBPmean (0.71 ± 0.37 (0.053) before recanalization. The associations remained significant only in SBPdropmax, SBPincmax, and SBPmean after recanalization. SBPincmax, SBPcv and SBPsd showed significant associations with modified Rankin Scale score at 3 months in the pre-recanalization period. In the poor perfusion group, none of the SBP indices was associated with any stroke outcomes regardless of recanalization status. BP may show differential associations with stroke outcomes by the recanalization and baseline perfusion status.We hypothesized that the association between BP and endovascular treatment (EVT) outcomes would differ by baseline perfusion and recanalization status. We identified 388 ICA or M1 occlusion patients who underwent EVT ≤ 24 h from onset with successful recanalization (TICI ≥ 2b). BP was measured at 5-min intervals from arrival and during the procedure. Systolic BPs (SBP) were summarized as dropmax , incmax , mean, coefficient of variation (cv), and standard deviation. Adequate baseline perfusion was defined as hypoperfusion intensity ratio (HIR) ≤ 0.5; infarct proportion as the volume ratio of final infarcts within the T In this subset of patients, periprocedural medical treatment might be effective in achieving good functional outcomes2. As there are individual differences in the viability of salvageable ischemic tissue and ischemic progression after LVO3, modulating systemic blood pressure (BP) peri-procedurally may be a treatment target to maintain the tenacity of the ischemic penumbra before recanalization and to minimize hemorrhagic complications after EVT.Revascularization therapy can bring a straightforward functional recovery in patients with acute ischemic stroke due to large vessel occlusion (LVO) with a number needed to treat (NNT) of 4. However, there remains a substantial proportion (54%) of patients in whom functional recovery is insufficient despite endovascular treatment (EVT)7. However, studies analyzing the relationship between BP level and stroke outcomes have a major limitation, as these studies do not exclude the possibility that higher BP can occur as a consequence of severe ischemia8. A recent randomized controlled trial (BP-TARGET) failed to show a benefit in functional outcomes and hemorrhagic complications between low BP and standard management arms9. Multiple observational studies have shown varying results11. Because of the lack of any robust data, many guidelines either do not comment on absolute BP targets in the management of patients after successful reperfusion12, or they recommend the same BP targets suggested for post-intravenous thrombolysis care, i.e., ≤ 185/110 mm Hg13.During the peri-EVT period, high or low BP measurements are usually negatively associated with the treatment outcome14. We, therefore, hypothesize that the baseline brain perfusion status likely influences the relationship between systemic BP and stroke outcome in patients with LVO and that such a relationship is present only before successful recanalization is achieved. To this end, we analyzed 388 anterior circulation LVO patients with post-EVT modified thrombolysis in cerebral infarction (mTICI) ≥ 2b who underwent intensive BP monitoring after arriving at the emergency department.Biologically, leptomeningeal collateral (LMC) perfusion after LVO depends on the pressure gradient across these collateral channels; thus, augmentation of blood flow through LMCs using systemic BP modification may only work before recanalization is achieved. Moreover, elevated systemic BP may only be capable of increasing cerebral blood flow (CBF) when local cerebral autoregulation capacity is depleted, a condition that is likely influenced by the severity of baseline perfusion defectInformed consent from patients or the next of kin was obtained for the prospective stroke registry under the approval of the Seoul Nation University Bundang Hospital institutional review board . All experimental protocols and analysis plans were approved by the Seoul Nation University Bundang Hospital institutional review board . All human research was conducted according to the Declaration of Helsinki.15. Among the patients who underwent EVT, 192 were excluded due to (1) posterior circulation (n = 90), (2) poor-quality baseline images (n = 16), (3) lack of perfusion studies before EVT (n = 41), and (4) unsuccessful recanalization with mTICI less than 2a (n = 45).This was an observational, single-center, retrospective analysis of prospectively acquired data. Between January 2016 and March 2020, 580 endovascular recanalization treatments for acute ischemic stroke patients were performed in the Cerebrovascular Center of the Seoul National University Bundang Hospital, which participates in a prospective multicenter stroke registry16. In our center, general anesthesia is not recommended during EVT, and conscious sedation is selectively administered to some uncooperative patients as determined by the treating team. Successful recanalization was defined as post-EVT mTICI 2b or 3. The time of recanalization was defined as the earliest time when successful recanalization was observed.Acute stroke management was performed as per institutional protocol based on the current guidelines at the time of practice and at the discretion of the individual physicians13. The institutional protocol recommends as per the acute care guideline to maintain SBP < 185 mm Hg; the uses of BP-lowering intravenous medications were decided by an interventionalist or attending physician after considering neurological deficit, BP level, and degree of recanalization. All BP measurements were recorded in the institutional registry database along with the time of the measurements. The use of BP-lowering medication and their time were retrospectively collected.When patients arrive at our stroke center with stroke-like symptoms, BP recordings at 5-min intervals using a portable patient monitor  were recorded, starting from hospital arrival. During the EVT, noninvasive BP was recorded using a monitoring device  until the end of the procedure as part of usual care. After EVT, all patients were transferred to a stroke unit where BP is measured as per the guidelines, i.e., at least every hour until 24 h after the procedure15. Data on the 3-month mRS score were prospectively collected during a regular clinic visit or through a structured telephone interview conducted by a trained research nurse.Baseline clinical information and follow-up functional recovery data were retrieved from the prospective stroke registryFollow-up images were routinely acquired 3–5 days after the procedure or at the time of neurologic deterioration to evaluate hemorrhagic complications and final infarct volume. Magnetic resonance imaging is the recommended imaging modality, but CT scans were accepted based on clinical circumstances.max ≥ 6 s. The baseline Tmax ≥ 6 s map from CT or MR perfusion images was reconstructed using Olea Sphere 3.0 . The perfusion map was exported to Analyze 14  and co-registered to the follow-up diffusion-weighted images (DWI). Artifacts found on the Tmax map were manually removed. Only lesions with high signal intensity on the DWI and an apparent diffusion coefficient less than 600 μm2/s within the region of Tmax ≥ 6 s were counted as the final infarction. Infarct proportion was quantitated by a research technician with supervision by a vascular neurologist.Infarct proportion was measured as a ratio of the volume of final infarction over the volume of baseline Tdropmax, the maximum decrease between two consecutive SBP measurements; SBPincmax, the maximum increase between two consecutive SBP measurements; SBPmean, the average of SBP measurements; SBPsd, the standard deviation of SBP measurements; and SBPcv, the coefficient of variation of SBP measurements.We summarized systolic BP (SBP) measurements with the following indices: SBPmax–SBPmin), the successive variation (SBPsv), and the average real variability (SBParv) were analyzed against the infarct proportion and presented as supplemental data.Additionally, the range . Alberta Stroke Programme Early Computed Tomography Score (ASPECTS) was evaluated on the first images, using computed tomography (CT) in 235 (60.6%) and diffusion-weighted image (DWI) in 153 (39.4%). ASPECTS were comparable between CT and DWI . Hemorrhagic transformation was evaluated on follow-up imaging using the Heidelberg Bleeding Classification (HBC)18. We defined significant hemorrhage as HBC class 2 or HBC class 3, i.e., PH2 hemorrhage or intracerebral hemorrhage outside the infarcted brain tissue or intracranial-extracerebral hemorrhage.The hypoperfusion intensity ratio (HIR) was defined as the volume of tissue with Tt-test for interval variables. Due to the right-skewed distribution of final infarct proportion, multivariable gamma regression models were used to test the effect of BP indices on this variable. Infarct proportion was designated as the primary outcome variable. Binary logistic regression models were used when the outcome was significant hemorrhage, while ordinal logistic regression models were used when the outcome was the overall distribution of mRS scores at three months. Multivariable models were adjusted for covariates with clinical significance or a bivariate P value < 0.10, including age, the time last known well to arrival, baseline NIHSS score, ASPECTS, occlusion location, hypertension, procedural sedation, and intravenous BP-lowering medication. Significance levels were set at P < 0.05, with all statistical tests being two-tailed. The P-values of multiplicative interaction terms were considered to have marginal significance when P-value was between ≥ 0.05 and < 0.1019. All tests were considered exploratory and hypothesis-generating; therefore, no adjustment was made for multiple tests20. All statistical analyses were performed using R, version 4.1.0 .Baseline characteristics were summarized and compared using the chi-squared test for categorical variables and an independent A total of 388 anterior circulation LVO patients ≤ 24 h from their last known well (LKW) with baseline perfusion images were selected for the current analysis. The average age of the study population was 70.6 ± 12.7 years, and 39.7% of participants were female. The median baseline National Institutes of Health Stroke Scale (NIHSS) score was 14 , and the median time from LKW to arrival was 3.3 h . Forty-nine percent (n = 188) of cases involved M1 occlusion at baseline, and intravenous thrombolysis was given in 31% (n = 122) of cases. The mean SBP at the initial presentation was 153 ± 52 mm Hg. No patient received general anesthesia during the EVT procedure, and 29% (n = 113) had conscious sedation. Patients with adequate baseline perfusion, i.e., baseline HIR < 0.5, were more likely to be female with atherosclerotic stroke and to have a lower baseline NIHSS score and longer last known well (LKW) to arrival interval. The adequate perfusion group had a relatively low infarction proportion but showed comparable significant hemorrhage and functional recovery to the poor baseline perfusion group. The five selected BP indices were not different according to the baseline perfusion subgroup  of BP measurements was 47 ± 16.5 between arrival and 24 h after groin puncture, and the measurement interval was a median of 33 min . The median interval of BP monitoring during the EVT procedure was 5.2 min . The median counts of BP measurements were 9  in the pre-recanalization period and 33  in the post-recanalization period  and ≥ 13 values in the post-recanalization modeling (n = 387) to reliably calculate the SBP indices . Similarly, a positive correlation with infarct proportion was noted for SBPincmax, SBPcv, and SBPsd. In the post-recanalization period, SBPdropmax, SBPincmax, and SBPmean showed significant association with greater infarct proportion, but the significance was lost with SBPcv and SBPsd. Likewise, a higher mRS score at three months, representing worse functional recovery, was associated with SBPincmax, SBPcv, and SBPsd in the pre-recanalization period and with SBPincmax in the post-recanalization period. There was no correlation of significant hemorrhage with any of the SBP indices in the pre- and post-recanalization periods in the adequate perfusion subgroup.All five SBP indices analyzed in patients with adequate baseline perfusion, i.e., HIR < 0.5, showed significant association with infarct proportion in the pre-recanalization period  showed marginal significance in the conditional coefficient in the pre-recanalization period  and significance in the post-recanalization period  by the level of HIR. However, the associations between the global variability (SBPcv) or the overall level (SBPmean) and infarct proportion were not modified by the level of HIR in the peri-EVT periods .We further plotted the predicted infarct proportion from SBPnge Fig. . The raprange, SBPsv, and SBParv, and infarct proportions  widespread occlusion of capillaries and irreversible infarctions are already established23, or (2) these patients may have severely depressed LMC perfusion, unable even to cause any change in CBF from the fluctuation of systemic BP25. Patients with adequate baseline cerebral perfusion, with fully recruited LMCs alongside depleted autoregulatory function, may accommodate changes in systemic BP by changing CBF.As regional perfusion pressure decreases after LVO, the pial artery dilates to provide maximal blood flow within its capacity26. On the other hand, hemorrhagic transformation after EVT may be caused by ischemic injury to the cerebral vascular beds and parenchymal inflammation, resulting in increased permeability of the arteriolar or capillary wall and the extravasation of blood components into the brain tissue27. Thus, it may be inferred that hemorrhagic complications after recanalization treatment may be attributed not to BP changes but to recanalization itself and the microvascular ischemic injury that accumulated before recanalization.SBP levels and changes had no association with follow-up hemorrhage regardless of baseline perfusion status or recanalization period in our study. The reasoning that BP contributes to intracerebral hemorrhage is based on the idea that hydrostatic pressure causes the rupture of fragile arterioles or microaneurysmsdropmax and SBPincmax, or from the overall level, i.e., SBPmean, on the infarct proportion persist after recanalization. In contrast, the effect of global variability of BP, i.e., SBPsd and SBPcv, on ischemic injury became negligible after recanalization.Successful recanalization in LVO patients induces changes in cerebral hemodynamics. As forward flow is restored, the role of collateral flow through the LMC channel is reduced, and physiological levels of CBF are reinstated. Thus, the relationship between systemic BP and regional CBF may differ by the reperfusion status. Our study documented that the contributions from rapid fluctuation, i.e., SBP10. Mean BP, which reflects the steady and stable BP over a certain period, is probably the most widely used index, but it does not capture minute-by-minute fluctuations. It cannot be used to establish a causal relationship with an event during a measurement period5. Global BP variability has been summarized using the standard deviation, coefficient of variation, and variation independent of the mean, which together successfully demonstrate the long-term variation for stroke survivors28. However, their role in acute management is relatively limited in that the variability values vary depending on the BP measurement density, and the BP measurement interval is not incorporated in calculating them. The authors developed BPdropmax and BPincmax, which are intuitive and easy to calculate and are applicable to acute stroke management29. These indices may quantify rapid fluctuations over a very short period. In the current study, these SBP indices behaved differently according to baseline perfusion and recanalization status. Thus, each of these BP indices may also have a specific role in understanding the effect of systemic BP on outcomes in patients with acute ischemic stroke.Theoretically, systolic and diastolic BP measurements are generated with every cardiac contraction; BP can be measured repeatedly and changes according to various internal and external conditions. It is crucial to choose the most appropriate BP summary index for clinical or research purposes30. It can be speculated that ischemic injury rapidly develops in a case with poor LMCs after LVO, and the only effective treatment strategy is to expedite recanalization. Augmentation of CBF with therapeutically elevated BP would work only when the local cerebral autoregulation capacity is depleted, and adequate LMC channels are present. In contrast, in the presence of moderate LMCs, tissue viability mainly depends on systemic perfusion, and clinicians should be sensitive to even minute changes in blood pressure. At the same time, in patients with excellent or good collaterals, one can expect a higher tolerance to modest BP fluctuations. This conjecture needs to be validated in the future.Early infarct growth may be a function of time from onset, ischemic severity, i.e., baseline LMC perfusion, and tissue vulnerabilityBP levels and changes during the peri-EVT periods were found to aggravate the ischemic injury, especially in the pre-recanalization periods of the adequate baseline perfusion subgroup. However, their magnitudes of coefficient estimates were small, which may explain weak associations from BP indices with functional recovery three months after stroke.In this study, we acknowledge that our current analysis does not capture the entire extent of blood pressure variability and its potential impact on patient outcomes. While our approach focused on the SBPdropmax, we recognize that incorporating additional methods, such as the delta (SBPmax–SBPmin), the average variability between measurements, or moving averages, could provide a more complete understanding of variability (see Supplemental Table As the precise association between blood pressure profiles and stroke outcomes remains elusive, we acknowledge in this study that blood pressure medications play a role in the management of acute stroke patients. Despite the fact that our analysis did not prove the effects of blood pressure medications on variability (see Supplemental Table 20. Third, pre-and post-recanalization SBPs are inherently not independent due to repeated measurements. Fourth, we investigated SBP indices for increasing clinical practicality and reducing the BP data dimension. Fifth, we did not incorporate procedural information which could influence treatment outcomes. Sixth, anterior circulation LVO patients without perfusion images were excluded from analyses (see Supplemental Table A few points need further clarification. First, this is a retrospective study from a single center with all of the shortcomings associated with the design, resulting in a possible selection bias. However, we collected all the BP data according to an institutional protocol and selected the study subjects through prespecified steps to minimize selection bias. Second, we analyzed various outcome measures and multiple SBP indices, and multiple comparisons were inevitable in our analysis scheme. Inflation of type I error was possible, but we accepted a conventional level of significance considering the exploratory nature of our studyWe documented that the contribution of the SBP profile to stroke outcomes may depend on the baseline perfusion and the recanalization status in anterior circulation LVO patients with successful recanalization. SBP during the pre-recanalization period is equally important and is a determinant of outcomes, and the contribution may be affected by the baseline perfusion status. Our study suggests that a more sophisticated BP management strategy during the acute stroke period is required and needs to reflect various conditions, such as baseline perfusion, recanalization, and cerebral autoregulation capacity.Supplementary Tables.


Prevalence of asthma is increasing steadily among general population in developing countries over past two decades. One of the causative agents of broncho-constriction in asthma is thromboxane A2 receptor (TBXA2R). However few studies of TBXA2R polymorphism were performed so far. The present study aimed to assess potential association of TBXA2R rs34377097 polymorphism causing missense substitution of Arginine to Leucine (R60L) among 482 patients diagnosed with pollen-induced asthma and 122 control participants from West Bengal, India. Also we performed in-silico analysis of mutated TBXA2R protein (R60L) using homology modeling.1), FEV1/Forced vital capacity (FVC) and Peak expiratory flow rate (PEFR) were assessed using spirometry. Patients’ sensitivity was measured by skin prick test (SPT) against 16 pollen allergens. Polymerase chain reaction-based Restriction fragment length polymorphism was done for genotyping. Structural model of wild type and homology model of polymorphic TBXA2R was generated using AlphaFold2 and MODELLER respectively. Electrostatic surface potential was calculated using APBS plugin in PyMol.Clinical parameters like Forced expiratory volume in 1 second (FEVA. indica (87.68%) followed by C. nusifera (83.29%) and C. pulcherima (74.94%). Significant difference existed for pollen sensitivity in adolescent and young adult (P=0.01) and between young and old adult (P=0.0003). Significant negative correlation was found between FEV1/FVC ratio and intensity of SPT reactions (P<0.0001). Significant association of FEV1, FEV1/FVC and PEFR was observed with pollen-induced asthma. Furthermore, risk allele T was found to be clinically correlated with lower FEV1/FVC ratio (P=0.015) in patients. Our data showed R60L polymorphism, which was conserved across mammals, significantly reduced positive electrostatic charge of polymorphic protein in cytoplasmic domain thus altered downstream pathway and induced asthma response.Genotype frequencies differed significantly between the study groups (P=0.03). There was no significant deviation from Hardy-Weinberg equilibrium in control population (χ2=1.56). Asthmatic patients have significantly higher frequency of rs34377097TT genotype than control subjects (P=0.03). SPT of patients showed maximum sensitivity in The present in-silico study is the first one to report association of TBXA2R rs34377097 polymorphism in an Indian population. It may be used as prognostic marker of clinical response to asthma in West Bengal and possible target of therapeutics in future. Asthma is correctly termed the 21st century epidemic and modern age disease and represents a substantial public health burden in many countries including India . The preSo, in the present study, attempts have been made to investigate the association of TBXA2R rs34377097 polymorphism with pollen induced bronchial asthma among population of West Bengal, India. rs34377097 is the only non-synonymous polymorphism within TBXA2R gene reported till date . In thisThe Clinical Research Ethics Committee of Allergy and Asthma Research Center, West Bengal, India approved the present study protocols vide CREC-AARC Ref: 62/21. Written informed consent was taken from each patient and control subjects of this study and preserved for future reference.A total of 632 individuals  were selected for this case-control study during March to December, 2021. Pregnant and lactating women (n=5), patients suffering from illness other than asthma (n=11) and who did not provide the written informed consent (n=12) were excluded from this study. Finally, 482 patients and 122 control subjects were included in this present study. Details regarding epidemiology viz. age, gender, habitat, age of onset of symptoms, life-style, family history , clinical features, aggravating factors and non-specific stimuli such as cold, exercise and other irritant factors, etc. were recorded in a self-prepared questionnaire. 482 participants diagnosed of having aero-allergen induced bronchial asthma and reported to be suffering from different asthmatic manifestations like airway hyper responsiveness, recurring episodes of airway obstruction, wheezing, dyspnoea and cough either alone or in different combination were considered as cases. Both patients and controls were classified according to their age like children (age 5–12 years), adolescents (age>12–19 years), young adults (age >19–40 years) and old adults (>40-70 years). Peripheral blood samples were collected from all the participants and centrifuged for separation of serum and kept in −20°C refrigerator for further analysis.1), Forced vital capacity (FVC), FEV1/FVC ratio and Peak expiratory flow rate (PEFR). 20% or more reduction of FEV1 and PEFR value than predicted is considered as asthmatic  guidelines 2017 . The folsthmatic . FEV1/FVsthmatic .Azadirachta indica, Cocos nucifera, Caesalpinia pulcherrima, Brassica nigra, Pinus sylvestris, Carica papaya, Zea mays, Triticum aestivum, Lantana camara, Poa pratensis, Chenopodium album, Peltophorum pterocarpum, Areca catechu, Parthenium hysterophorus, Lolium perenne and Plantago lanceolata. Histamine phosphate and normal saline were used as positive and negative controls respectively. Wheel diameter ≥3mm was considered as positive response to that particular allergen.Asthmatic patients those were positive in Spirometry test were undergone skin prick test (SPT) using 16 common aero allergen extracts  viz. Extraction of genomic DNA was performed from blood samples of 482 patients and 122 control subjects using QIAamp DNA blood mini kit . TBXA2R rs34377097 polymorphism was identified using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) technique . Significance level was taken as P<0.05.Continuous data are described by mean ± standard error (SE) and categorical data by number with percentages. Patients and controls were assessed by independent t test for continuous variables or Pearson chi-square test for categorical variables. Genotypic and allele distributionin patient and control group was evaluated using contingency chi-square or Fisher’s exact test, where applicable. Risk analysis was performed by odds ratio (OR) under additive, recessive and dominant models. Hardy-Weinberg equilibrium (HWE) was checked for the genotypes by goodness-of-fit chi-square test with one degree of freedom (df). SPT sensitivity for pollens among different age groups was analyzed using One way ANOVA followed by Tukey’s Multiple Comparison test. The association between FEV1, FEV1/FVC ratio and PEFR, a significant deviation was observed from the predicted value both in male and female patients in all age groups as compared to control , followed by Cocos nucifera (83.30%), Caesalpinia pulcherrima (74.95%), Brassica nigra (72.23%), etc. Sensitivity of patients to different pollen allergens categorized according to age is depicted in The highest prevalence of sensitization was obtained with The association of FEV1/FVC ratio with atopy is depicted in The genotype frequencies differ significantly between the study groups   than T allele bearing controls; while that in G allele bearing patient and control individuals remained non-significant  (1/FVC ratio differ significantly between G and T bearing asthma patients (P=0.04) while no significant difference is observed in controls (P=0.91)  denoted that the R60 amino acid is conserved across the species . As per our knowledge is concerned, our study which is based on both structural and functional aspects, is the first one to report the association of TBXA2R rs34377097 polymorphism in an Indian population. It is also the first extensive in silico analysis that perform both polymorphism study and modeling approach to assess TBXA2R rs34377097 polymorphism clinically. Another study conducted in Japanese population reported lack of association of rs34377097 polymorphism with bronchial asthma . The pre4 synthase enzyme and inactivates it or this regulation may involve another regulatory protein which is yet to be discovered. Inactivation of LTC4 synthase leads to reduction of Leukotriene C4 (LTC4) biosynthesis in platelets. However, in the case of risk allele rs34377097 T-bearing individuals, the non-synonymous mutation (R60L) in TBXA2R protein inhibits the interaction between GNA11 and TBXA2R due to the change in the positive charge potentiality at the cytoplasmic domain. This is in line with Hirata et al. 1996  . IP3enhaal. 1996 , who demThe present study provided several lines of evidence to support that TBXA2R rs34377097 polymorphism act as a potent risk factor for asthma in West Bengal population, India. More studies are warranted in diverse ethnic groups to validate the above findings. The present in-silico study will be useful for the advancement of gene based therapy for better management and treatment of asthma in near future.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: SCV003761534 (ClinVar).The studies involving human participants were reviewed and approved by Clinical Research Ethics Committee, Allergy and Asthma Research Center, West Bengal, India (CREC-AARC Ref: 62/21). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Collection of samples was done by IG, SS and NS. IG and AL contributed to data compilation and wrote first draft of the manuscript. PB did the structural modeling. SM diagnosed the disease and provided the samples. Statistical analysis was done by IS, HB and AL. SP conceived the study design, supervised the work, critically revised the manuscript and made the final draft. All authors contributed to the article and approved the submitted version.