Nondiabetic kidney disease (NDKD), which is prevalent among patients with diabetes mellitus (DM), is considerably different from diabetic kidney disease (DKD) in terms of the pathological features, treatment strategy and prognosis. Although renal biopsy is the current gold-standard diagnostic method, it cannot be routinely performed due to a range of risks. The aim of this study was to explore the predictors for differentiating NDKD from DKD to meet the urgent medical needs of patients who cannot afford kidney biopsy.This is a retrospective study conducted by reviewing the medical records of patients with type 2 DM who underwent percutaneous renal biopsy at the Affiliated Hospital of Guizhou Medical University between January 2017 and May 2021. The demographic data, clinical data, blood test results, and pathological examination results of the patients were obtained from their medical records. Multivariate regression analysis was performed to evaluate the predictive factors for NDKD.P < 0.001), an absence of diabetic retinopathy , a high RBC count , and a negative of urinary glucose excretion test result ..A total of 244 patients were analyzed. The median age at biopsy was 55  years. Patients diagnosed with true DKD, those diagnosed with NDKD and those diagnosed with NDKD superimposed DKD represented 48.36% (118/244), 45.9% (112/244) and 5.74% (14/244), respectively, of the patient population. Immunoglobulin A nephropathy was the most common type of lesion in those with NDKD  and NDKD superimposed DKD . Independent predictive indicators for diagnosing NDKD included a DM duration of less than 5 years (odds ratio [OR] = 4.476; 95% confidence interval [CI]: 2.257–8.877; A DM duration less than 5 years, an absence of retinopathy, a high RBC count and an absence of urinary glucose excretion were independent indicators for the diagnosis of NDKD, suggesting that patients with NDKD may require a different treatment regimen than those with DKD. The worldwide prevalence of diabetes mellitus (DM) has increased considerably in the past decades, mainly due to the increased prevalence of type 2 DM (T2DM) . AccordiHowever, DKD is not the only form of kidney disease in patients with DM. Patients diagnosed with chronic kidney disease (CKD) and DM can have true DKD , nondiabetic kidney disease (NDKD) , or a combination of both DKD and NDKD . It has In clinical practice, the diagnosis of DKD relies predominantly on clinical presentations, including persistent proteinuria, hypertension, and a progressive decline in renal function. DiagnosGiven that the diabetic environment can alter the progression of NDKD, the form of kidney disease in patients with DM can only be reliably distinguished by performing renal biopsy . HoweverOur study protocol was conducted in accordance with the Declaration of Helsinki guidelines. Given this is a retrospective cohort study, consent for conducting present study was not obtained, but written consent for renal biopsy was obtained from all of the included patients.2 or markers of kidney damage , or both, for a duration of at least 3 months, regardless of the underlying cause  for skewed data and as the mean ± standard deviation for normally distributed data. For categorical variables, the data are presented as the frequency (%). Analysis of variance or the Kruskal–Wallis test was used to compare continuous variables. The chi-square (χ2) test was used to assess the differences between the categorical variables. Univariate and multivariate logistic regression analyses were used to screen for the factors  g/24 h. The numbers of patients with hypertension, dyslipidemia, anemia and DR were 155 (63.52%), 213 (87.30%), 155 (63.52%), and 96 (39.34%), respectively. The patients were divided into three according to the type of renal pathology: the DKD , NDKD , and MIX  groups. The baseline clinical characteristics of the three groups are shown in Table A total of 244 patients were analyzed, 66.66% of whom were male. The median age of the patients at the time of the renal biopsy was 55  years, the median DM duration was 5 1, 10) years, the median HbA1c was 7% , the median eGFR was 42.45  mL/min/1.73 m, 10 yearIt is noteworthy that up to 87.29% of patients in the DKD group had class III or IV of diabetic nephropathy at the time of renal biopsy, while 100% of the patients in the MIX group had the same Table . SimilarP < 0.001), absence of hypertension , absence of DR , eGFR , RBC count , Hb level , negative of urinary glucose status , urinary RBC count , and reduced UPE level (< 3.5 g/24 h) . The demographic data, including gender, age, BMI, and variables that were observed to be statistically significant in the univariate analysis were used to further multivariate logistic regression analysis to identify the factors closely related to NDKD. The results demonstrated that four indicators were statistically significant in the diagnosis of NDKD: a diabetes duration of less than 5 years , an absence of DR , the RBC count , and a negative test result for urinary glucose    and Hb level  are associated with NDKD, but after adjusting gender, age and BMI, et, multivariate logistic regression analysis showed that RBC count  not Hb level has a predictive value for the diagnosis of NDKD. It makes sense, synthesizing erythropoietin (EPO) is one of the key duty of kidney [To our knowledge, the present study suggested that higher RBC count and negative of urinary glucose had predictive value for diagnosis of NDKD for the first time. Previous studies have explored the level of Hb not RBC count in patients with NDKD and DKD, and suggested that higher level of Hb is a predictive factor for NDKD , 29. In f kidney . Renal af kidney . A studyf kidney , which cf kidney , it is nAs for other parameters, there is controversy of the clinical value of UPE between patients with DKD and NDKD , 34. TheAlthough many studies have explored the differential diagnosis of DKD and NDKD, the results from these studies varied, thus differentiating NDKD from DKD effectively and based on scientific evidence remains under further exploration. A strength of this study is that we recommended to all of the patients to undergo renal biopsy unless there were contraindications for it, which minimizes the selection bias as much as possible. In addition, we included as many relevant parameters as possible. However, there are also some limitations to this study. This study is retrospective and was performed at a single center, so applying the results to populations in other countries and regions will produce results that need to be interpreted cautiously due to the restrictive patient population in this study; Moreover, it cannot be entirely ruled out that there were no unmeasured confounding factors that could have possibly influenced the observed association in this study.Diabetic nephropathy is regarded as a major cause of end-stage renal disease, and true DKD usually requires treatment by glycaemic control and dual sodium-dependent glucose transporters 2 (SGLT2) and rennin angiotensin system (RAS) inhibition. However, NDKD may require additional therapeutic approaches. The present study demonstrated that a DM duration of less than 5 years, the absence of retinopathy, an elevated of RBC count, and the absence of urinary glucose excretion are independent indicators associated with the diagnosis of NDKD, suggesting that patients with NDKD may require a different treatment regimen than those with DKD when kidney biopsy cannot be performed. Given this study was performed at a single center, the conclusions derived from this work should be further analyzed and validated at other centers in China.


We propose a consensus definition of “an exaggerated orthostatic pressor response” in subjects in whom systolic blood pressure increases ≥20 mmHg when going from the supine to standing posture. This definition can be extended for seated to standing measurements. We reserve the term “orthostatic hypertension” if this pressor response leads to an upright systolic blood pressure ≥140 mmHg. We believe this consensus definition will help in the study of the pathophysiology, clinical impact, and potential treatment of these entities, and the identification of patients that are at greater cardiovascular risk. Standing imposes a major hemodynamic burden on the cardiovascular system. In healthy persons, neurohumoral reflex mechanisms, particularly the arterial baroreflex, maintain blood pressure with standing. However, the counterregulatory response overshoots in some people such that blood pressure increases with standing. This orthostatic pressor response may result in hypertensive blood pressure levels only when standing or further exacerbate arterial hypertension. The condition is often referred to as orthostatic hypertension and appears to be associated with increased cardiovascular risk , 2. Yet,exaggerated orthostatic pressor response is a sustained increase in systolic blood pressure by at least 20 mmHg when changing from the supine to the standing position regardless of absolute blood pressure while standing.An Orthostatic hypertension is defined as an exaggerated orthostatic pressor response associated with systolic blood pressure of at least 140 mmHg while standing.Our goal was to arrive at a pragmatic definition that could be utilized in the setting of mechanism-oriented studies, epidemiological surveys, and clinical trials. We are aware that the suggested definitions are based on clinical reasoning and normative data rather than cardiovascular risk estimates and that the definitions as well as blood pressure cutoff values may have to be refined in the future. While orthostatic hypertension may be associated with cardiovascular risk as outlined in the sections below, more data is required to allow for a risk-based definition.In the Malmö Preventive Project, blood pressure measurements after 10 min in the supine position and after one-minute standing were measured in 33,346 persons aged 45.7 ± 7.4 years [An increase in diastolic blood pressure on standing can be normal. Moreover, in individuals with excessive orthostatic heart rate increases, cardiac stroke volume may decrease such that pulse pressure is narrowed. The resulting spurious diastolic blood pressure increase is difficult to interpret. The panel, therefore, suggests that systolic blood pressure may be more practical when diagnosing orthostatic hypertension.A recent publication by an expert group suggested that a systolic blood pressure increase while standing by at least 20 mmHg or above 140 mmHg in otherwise normotensive persons could serve as criteria for orthostatic hypertension . The limWith standing, approximately 500–1000 ml blood is pooled below the diaphragm and hydrostatic pressure shift fluids from the intravascular to the interstitial compartment. In healthy persons, compensatory autonomic reflex mechanisms withdraw cardiac parasympathetic drive and raise cardiac and vascular sympathetic activity thereby leading to an increase in heart rate, a drop in systolic blood pressure <20 mmHg, and a small increase in diastolic blood pressure . Small-sThe epidemiology of orthostatic hypertension and the data regarding the cardiovascular risk associated with orthostatic hypotension have been reviewed recently . BrieflyWhen diagnosing an exaggerated orthostatic pressor response or orthostatic hypertension, blood pressure measurements with a brachial cuff suffice. Pulse rate should also be recorded. Preferably, measurements should be conducted in a quiet surrounding to limit confounding of orthostatic blood pressure responses by external stimuli. Supine blood pressure and heart rate should be measured after five minutes of rest in the supine position. Then, patients should stand up actively. Upright blood pressure and heart rate should be measured after 1, 3, and 5 min, at least, to confirm the persistent character of the blood pressure change. We suggest that the measurements after 3 and 5 min be averaged to determine standing systolic blood pressure. For screening purposes, a single measurement after 3 min may be sufficient.Beat-to-beat blood pressure and heart rate recordings can be applied in selected patients but are not widely available .We recommend that repeated orthostatic testing should be conducted on a different day to confirm the diagnosis.Because supine and upright blood pressure measurements may be difficult to conduct in all patients, seated to upright measurements may be considered for screening purposes. Since hemodynamic changes when standing up from the seated standing position are less compared with standing up from the supine position, the orthostatic pressor response may be less pronounced such that the sensitivity in detecting orthostatic hypertension may decrease. Similar limitations have been observed when using seated to standing measurements when diagnosing orthostatic hypotension . TherefoHead-up tilt testing may be considered for persons unable to undergo an active standing test.There is no evidence that individuals with an exaggerated orthostatic pressor response and blood pressure within the normal range require antihypertensive therapy. It may be prudent to follow up these patients’ blood pressure given the increased risk for progression to arterial hypertension. In patients with isolated orthostatic hypertension in whom blood pressure is only in the hypertensive range while standing, ambulatory blood pressure monitoring may have utility in gauging the overall blood pressure load during the day and detecting abnormalities in diurnal blood pressure patterns, such as extreme blood pressure dipping during the night or masked morning hypertension , 14. Pat


Sodium is reabsorbed all along the renal tubules. The positive impacts of sodium-glucose cotransporter-2 inhibitors (SGLT2i), angiotensin receptor neprilysin inhibitor (ARNI) and mineralocorticoid receptor antagonists (MRA) on hard renal and/or cardiac endpoints calls for the role of diuretics in nephroprotection and cardioprotection in patients with diabetes mellitus to be reviewed. Here, we review: (a) the mechanisms of action of the available natriuretics; (b) the physiological adaptations to chronic loop diuretic usage that lead to increased sodium reabsorption in the proximal and distal convoluted tubules; (c) the physiology of sodium retention in patients with diabetes mellitus; and (d) the mechanisms of aldosterone breakthrough. We show the rationale for combined diuretics to target not only the loop of Henle, but also the proximal and distal convoluted tubules. Indeed, higher residual proteinuria in patients treated with renin-angiotensin-aldosterone system (RAAS) blockers portends poorer renal and cardiovascular outcomes. Diuretics are known to optimize the reduction of proteinuria, in addition to RAAS blockers, but may favor aldosterone breakthrough in the absence of MRA. The aim of our study is to support a combined diuretics strategy to improve the management of patients with diabetes mellitus and chronic kidney disease or heart failure. Diabetes mellitus (DM) is often complicated by diabetic kidney disease (DKD), which is the most frequent etiology of end-stage renal disease . DM alsoEven more than tight glycemic control per se, the choice of glucose-lowering drugs may influence clinical outcomes . Indeed,ARNI decrease both hospitalization for HF and cardiovascular mortality, compared to angiotensin receptor blockade alone, irrespective of DM status. This effect was observed in patients featuring HF with a reduced  and maybSalt restriction and diuretics may foster aldosterone breakthrough in patients treated with ACEI or ARB . This coIn our study, we reviewed the rationale for combining different classes of diuretics , Table 1Sodium retention is a key player in numerous pathological conditions, most of which are frequently diagnosed in patients with DM. Increased sodium retention and hypervolemia strongly contribute to hypertension in patients with DM . IncreasMore recently, magnetic resonance imaging analysis confirmed that patients with T2D exhibit increased skin and muscle sodium content, which is related to organ damage . In addiIn physiology and pharmacology, escape to a monotherapy acting on a single pathological pathway is an expected outcome. The doxazosin arm of ALLHAT study was terminated early because of a 25% higher rate of combined cardiovascular events, including congestive HF. Indeed, alpha-blockers monotherapy induce sodium retention and adrenergic and renin-angiotensin-aldosterone system activations, contributing to increased risk of congestive HF. ALLHAT study also showed that chlorthalidone was superior to ACEI for the prevention of major cardiovascular disease events,  suggestiRAAS blocking agents were mainly used in association with diuretics in cardiovascular outcome trials. PROGRESS trial showed that only patients receiving both ACEI and diuretic were protected against recurrent stroke .Carbonic anhydrase inhibitors (CAI) indirectly inhibit Na/H exchanger type 3 (NHE-3) at the apical membrane of proximal convoluted tubule and have a modest effect on distal tubule, where they inhibit pendrin (a HCO3/Cl exchanger) . The ove2, SGLT2i have a marginal effect on glycemia, but still exert clinically significant antihypertensive activity: this modest inhibition of sodium reabsorption in proximal convoluted tubule is still significant in these patients. While GFR decreases, more glucose is filtered in every single remaining nephron and osmotic-driven sodium secretion is increased in the tubule [SGLT2i act by inhibiting Na-Glucose cotransport in proximal convoluted tubule. After starting SGLT2i, a clinically significant natriuresis is exhibited with rapid body weight loss, mimicking thiazide-induced volume depletion, and with dose-dependent decrease in blood pressure . When GFe tubule . SGLT2i e tubule . The cone tubule .At an early stage of diabetes (mainly type 1), proximal convoluted tubule hyperplasia with increased expression of SGLT2 contributes to increased sodium reabsorption. Although assessing the precise diuretic effect of SGLT2i is methodologically challenging,  SGLT2i dThe abolition of glucose-reabsorption-induced hyperfiltration causes an initial transient decrease in GFR, subsequently offset by increased sodium reabsorption along the loop of Henle . This suIn DAPA-HF, dapagliflozin decreased the risk of worsening HF or cardiovascular death, irrespective of DM status , indepenHowever, beyond decreased intra-glomerular pressure, the benefit of SGLT2 inhibitors is most likely explained by pleiotropic mechanisms, including blood pressure reduction as well as favorable effects on vascular function, reduction in tubular workload and hypoxia and metabolic effects .LD are secreted into tubular lumen through the organic anion secretory system of the proximal convoluted tubule. They inhibit the NKCC2 symporter of the ascending limb of the loop of Henle, where up to 20–25% of the filtered sodium is reabsorbed. NKCC1 receptor’s inhibition may contribute to the vasodilatory effect on vascular muscles. Enhanced secretion of renal prostaglandins further contributes to vasodilation, especially on afferent arteriole, resulting in increased glomerular sodium filtration.In spite of powerful acute natriuretic activity, numerous mechanisms can contribute to treatment escape to LD, irrespective of the administration regimen.(i)The short half-life of LD underlies the acute braking phenomenon,  with a d(ii)After several days, sodium depletion and rising prostacyclins activate RAAS. LD inhibit NKCC2 on macula densa, resulting in the inhibition of the tubuloglomerular feedback, ,43 preve(iii)The long-term effect of LD is further chronically limited by the compensatory hypertrophy of early distal convoluted tubule. Thus, LD synergy with thiazides is expected. Thiazides limit the enhanced distal reabsorption of sodium and the prolong LD effect with their longer half-life, as a perfect example of sequential nephron blockade. However, the association of LD and thiazides exposes patients to a risk of hypokaiemia, which can be prevented by a triple combination with a potassium-sparing diuretic, especially since the activation of thiazide-sensitive NCC during LD treatment is partially aldosterone-induced ,43.tubular lumen through the organic anion secretory system and inhibit NCC, a Na/Cl cotransport on distal convoluted tubule, where up to 7% of the filtered sodium is reabsorbed. Nevertheless, blood pressure reduction persists even after volume depletion is balanced by compensatory mechanisms, which suggests a long-term effect on vascular resistances, probably by opening a calcium-activated potassium channel. Other mechanisms remain controversial: calcium antagonist activity, calcium desensibilization, or reverse whole-body regulation [Thiazide diuretics are secreted into the gulation .On the B-intercalated cells of the collecting duct, pendrin activation contributes to thiazide resistance , in addiIn sodium-sensitive hypertension, thiazides can decrease blood pressure enough to decrease intraglomerular pressure and thus the amount of filtered sodium, thereby allowing a new sodium balance . HoweverMRA are characterized by a slower onset of natriuresis, when compared to other diuretics. MRA act on basolateral side of aldosterone-sensitive cells of the collecting duct, and compete with aldosterone on its cytosolic receptor, thereby preventing its nuclear translocation. MRA thus lead to the proteasome-mediated degradation of ENaC . In prinSpironolactone and eplerenone have a dose-dependent diuretic and potassium-sparing effect. These MRA showed reduced mortality in patients with heart failure with a reduced ejection fraction, irrespective of DM status ,18. EpleFinerenone, a non-steroidal MRA, reduced the risk of CKD progression and cardiovascular events in patients with T2D in addition to conventional RAAS blockade ,20. The Treatment with MRA on top of ACEI or ARB can be limited by hyperkalemia, more frequent in type 2 diabetes. Associations with LD, thiazides, or potassium binders may be required to prevent RAAS blockers discontinuation. SGLT2i may be useful to limit the rise of serum potassium, since SGLT2i have a kaliuretic effect by increasing flow in distal tubules . FinerenENaC inhibitors  directly block sodium channels in the principal cells of the collecting duct . In the The natriuretic effects of ENaC inhibitors and MRA are roughly equivalent: 2–4% of filtered sodium, or even more if sodium reabsorption is inhibited upstream (by loop or thiazide diuretics), or in the case of hypermineralocorticism-induced hypervolemia. ENaC inhibitors could be more efficient in edema resulting from nephrotic proteinuria with low RAAS activation. In those cases, sodium reabsorption is a consequence of the plasmin-induced increase in ENaC activity, rather than the aldosterone-induced increase in ENaC abundance. This mechanism might prove especially useful in patients with DKD and nephrotic syndrome. Conversely, ENaC inhibitors may be less effective with cirrhotic ascites with strong RAAS activation.Beneficial effects of natriuretic peptides  on the cSacubitril potentiates natriuretic peptides’ physiological increase by inhibiting neprilysin (NEP), the enzyme responsible for the inactivation of natriuretic peptides. In patients with DM, neprilysin seems to be upregulated, and response to natriuretic peptides is blunted . Since Np < 0.001), [2/year; p < 0·0001) [Natriuretic peptides mainly increase natriuresis in proximal cortical tubule, but also in distal tubule with downregulation of ENaC, following an inhibited aldosterone and renin release. Natriuretic peptides also inhibit angiotensin II-induced release of vasopressin and angiotensin II effect on V2 receptors, increasing diuresis . Natriur 0.001),  although 0.001), . This ma 0·0001) . Overall 0·0001) .Sacubitril-valsartan may contribute to improved glycemic control in patients with T2D, as it fosters the beneficial effect of natriuretic peptides on metabolism ,64. MoreIn the collecting duct, vasopressin binds to its V2 receptors, thereby promoting the trafficking of aquaporins-2 (water channels) to the apical membrane. This mechanism allows water reabsorption following an osmotic gradient . TolvaptThe effect of tolvaptan on albuminuria is controversial and has been exclusively investigated in patients with polycystic kidney disease . TreatmeTolvaptan may help to alleviate fluid retention in patients with DM and nephrotic syndrome, but this benefit needs to be confirmed by further studies . In factAldosterone levels may increase during RAAS blockade with ACEI or ARB in patients with DM. The incidence of aldosterone breakthrough is up to 53% after one year of RAAS blockade, with a liberate definition  . Its occSeveral negative outcomes could be expected after aldosterone breakthrough , includiThe prescription of diuretics should be guided by the best available evidence, therefore following approved indications and existing guidelines. Moreover, the right diuretic combination should be wisely tailored according to each patient’s characteristics, comorbidities, and risk factors. It should be kept in mind that the benefit of most diuretics has been demonstrated in addition to RAAS blockers (ACEI or ARB), which are the cornerstone of nephroprotection in the setting of proteinuric kidney disease. Our suggested approach is depicted in In most patients with T2D and established cardiovascular and/or kidney disease, SGLT2i are of utmost importance, and may be started on top of metformin regardless of glycemic control. This suggestion dovetails the recommendations of American and European learned societies . On anotDiuretics acting on the collecting tubule may be particularly useful in patients at risk of aldosterone breakthrough. In particular, finerenone might be a relevant addition to conventional RAAS blockade (with ACEI or ARB) and SGLT2i in patients with diabetic kidney disease . OtherwiLD remain the handiest treatment in the case of residual or acute volume overload, due to their rapid onset of action and powerful natriuretic activity. If resistance to LD occurs, combination with thiazide diuretics can achieve efficient sequential nephron blockade. Nevertheless, because of the harmful counter-regulations arising from their use, LD should be promptly down-titrated to the minimal effective dose after the emergency phase.Each diuretic comes with its own safety profile and array of adverse drug reactions (ADR), as summarized in the table. Combining low dose diuretics might alleviate the burden of each specific ADR compared to the prescription of full dose monotherapies, and improve drug acceptability. However, some ADR, shared by several diuretics, are subject to concerning additive or synergistic interactions.Among those ADR, the risk of prerenal acute kidney injury following excessive sodium depletion is high, and common to all diuretics. Therefore, heightened caution is warranted when introducing and uptitrating diuretics, especially when combined. Extracellular volume status should be meticulously assessed before each diuretic escalation. In the case of normovolemia or hypovolemia, therapeutic escalation should be ideally postponed or associated with transient (or sustained) down-titration of LD. Likewise, patients should be educated to self-monitor blood pressure and weight, if necessary daily. Patients should also be taught to transiently discontinue their diuretic treatments in risky situations, such as diarrhea or significant decrease in dietary intakes. These simple measures may allow early warning in the case of excessive volume depletion, and may prevent the occurrence of prerenal failure.Dyskalemia is another frequent concern in patients treated with diuretics. LD and thiazide diuretics may cause hypokalemia, while diuretics acting on the collecting duct increase the risk of hyperkalemia. Patients with DM are, per se, prone to hyperkalemia, especially when also treated with ACEI or ARB ,81. In tWe reviewed and discussed the available literature regarding the role of diuretics for cardioprotection and nephroprotection in patients with DM . Despite our best efforts, some studies may have escaped our scrutiny. Nonetheless, the antiproteinuric effect of each diuretic monotherapy encourages the benefit of their synergistic associations to be considered. Combined diuretics may help to overcome the escape to monotherapies. While adequately individualized associations may also decrease the risk of adverse drug reactions , caution should be taken when introducing and titrating each different class to avoid massive natriuresis, excessive hypovolemia, and prerenal failure.Achieving dry body weight is pivotal in patients with DM, since sodium retention occurs in diabetes at an early stage and a fortiori after the development of DKD and/or HF. Hypervolemia has detrimental effects on cardiovascular outcomes, and worsens DKD through uncontrolled proteinuria. Thiazides and LD were for a long time the main natriuretic drugs used to control this hypervolemia. However, sodium retention in patients with DM mainly occurs in proximal and distal convoluted tubules, where thiazides and LD increase sodium retention, and favor a deleterious aldosterone breakthrough. Therefore, diuretics acting on proximal tubules (ARNI and SGLT2i) and aldosterone blockade may be required in patients with DM. Additional studies are needed to assess whether proteinuria reduction translates to nephroprotection in diuretics other than finerenone or SGLT2i , while c


The co-administration of loop diuretics with thiazide diuretics is a therapeutic strategy in patients with hypertension and volume overload. The aim of this study was to assess the efficacy and safety of treatment with bumetanide plus chlorthalidone in patients with chronic kidney disease (CKD) stage 4–5 KDIGO.A double-blind randomized study was conducted. Patients were randomized into two groups: bumetanide plus chlorthalidone group (intervention) and the bumetanide plus placebo group (control) to evaluate differences in TBW, ECW and ECW/TBW between baseline and 30 Days of follow-up. Volume overload was defined as ‘bioelectrical impedance analysis as fluid volume above the 90th percentile of a presumed healthy reference population. The study’s registration number was NCT03923933.2 (2.2–29) were included. There was decreased volume overload in the liters of total body water (TBW) on Day 7  and Day 30 ; and in liters of extracellular water (ECW) on Day 7  and Day 30 . There was also a decrease in systolic blood pressure on Day 7  and Day 30  and in diastolic blood pressure on Day 7  and Day 30 .Thirty-two patients with a mean age of 57.2 ± 9.34 years and a median estimated glomerular filtration rate (eGFR) of 16.7 ml/min/1.73 mIn CKD stage 4–5 KDIGO without renal replacement therapy, bumetanide in combination with chlorthalidone is more effective in treating volume overload and hypertension than bumetanide with placebo. Approximately 85% of patients with advanced chronic kidney disease (glomerular filtration rate < 30 ml/min) suffer from hypertension mostly as a result of volume increase . Given tA single-center, randomized, double-blind (nephrologist and patient), parallel, 1:1 allocation ratio, bumetanide plus placebo (control group) trial was conducted to compare bumetanide plus chlorthalidone (intervention group) in patients with advanced chronic kidney disease (KDIGO stages 4 and 5) with hypertension, volume overload and chronic loop diuretic use. We defined chronic loop diuretic use as patients with at least three consecutive months of furosemide (at least 40 mg daily) or bumetanide (at least 1 mg daily) use. Bumetanide , 11 and Randomized groups were assigned to receive initial treatment with either 3 mg bumetanide + 50 mg chlorthalidone or 3 mg bumetanide + placebo. Bumetanide was administered on the following schedule: 2 mg at 10 am and 1 mg at 4 pm. This bumetanide dose schedule was defined according to the experience in the use of bumetanide from our department and based on pharmacokinetic studies . Chlorth2, 16 × 30 cm2, or 16 × 36 cm2).The inclusion criteria were uncontrolled hypertension (systolic blood pressure (SBP) > 140 mmHg and/or diastolic blood pressure (DBP) > 90 mmHg) despite antihypertensive treatment, volume overload , chronic loop diuretic use (at least three months), chronic kidney disease stages 4 or 5 diagnosed at least three months previously, age between 18 and 75 years, and a signed informed consent form. The exclusion criteria were contraindications for chlorthalidone use , pregnancy, breastfeeding, cognitive deterioration, non-steroidal anti-inflammatory drug (NSAID) use, acute heart failure, chronic liver failure, respiratory insufficiency, and any cancer. Eligible patients were recruited from the nephrology clinic of our hospital and scheduled for the initial consultation of the treatment period, which consisted of basal blood testing to screen for treatment contraindications, as well as the estimated initial glomerular filtration rate by CKD-EPI; the etiology of chronic kidney disease was attributed to hypertension or diabetes in the case of having these long-standing diseases, without clinical evidence of another contributing factor. One patient with a biopsy showing lupus nephropathy participated in the clinical trial, and those patients without comorbidities and without a biopsy showing the etiology of kidney disease were classified as of unknown cause. Bioelectrical impedance analysis was also performed at this time using a seca mBCA 514 medical Body Composition Analyzer . Blood pressure measurements were performed by trained physicians blinded to the treatment group under the 2018 ESC/ESH guidelines for the management of arterial hypertension using a mercury sphygmomanometer of adequate size , extracellular water (ECW) and ECW/TBW between baseline and Days 7 and 30 of follow-up. Electrical bioimpedance vector analyses were performed to evaluate these primary outcomes. Secondary outcomes included differences in baseline SBP, DBP, mean arterial pressure (MAP), fraction of sodium excreted (FENA), and brain natriuretic peptide (BNP) measurements compared with Days 7 and 30 of follow-up.The following effect outcomes were recorded: impaired kidney function (defined as an increase in creatinine serum ≥ 0.3 mg/dL from baseline); creatinine doubling (compared with baseline creatinine); hyponatremia ; hypokalemia (serum potassium ≤ 3.5 mEq/L); hyperuricemia (serum uric acid ≥ 8 mEq/L); cardiovascular events ; and mortality for all causes or cardiovascular causes.n = 32 for this simple comparison between groups. Measurements were compared at the various time points. Randomization was performed by the nephrology research director using a simple 1:1 model and online software . Parametric and nonparametric statistical tests were performed depending on the distribution of variables for the evaluation of each of the primary and secondary outcomes. The mean and standard deviation are shown for variables with a normal distribution, and the median and range are shown for variables without a normal distribution. Effect size was compared with Cohen's D test between groups for body water parameters ; blood pressure ; and of the changes of FENA and BNP. A p value < 0.05 was considered statistically significant.A double-blind (nephrologist and patient) method was utilized, whereby the patient and researcher were unaware of allocations to the bumetanide plus placebo and treatment groups. As part of the statistical plan, an Excel database was created in which the content of the data collection sheets was captured. The information was later exported to the STATA 14 statistical program . A statistical power analysis was performed to estimate sample size based on data from the study of chlorthalidone effects on blood pressure, pulse and weight in patients with low kidney function . With anFifty-one patients were assessed for eligibility, and 34 were randomized (17 in each group). Finally, sixteen participants from each group were included in the analysis. Two participants (one from each group) were excluded from the analysis because they did not complete the follow-up. The recruitment period lasted 3 months , and 17 patients (53.1%) were in KDIGO stage 4. No significant differences in baseline characteristics were observed between groups , the mean time from CKD diagnosis was 24 months 3–120), 68.8% were diabetic, the median diuretic use was 12 months (3–60), and furosemide was the most common loop diuretic (93.8%). The median glomerular filtration rate was 16.7 ml/min/1.73 m20, 68.8%p = 0.003] and Day 30 [intervention: -5.3  vs. control: -0.07  p = 0.016]; in liters of ECW on Day 7 , and Day 30 ; and in the ECW/TBW ratio on Day 7  and Day 30   vs. control: -0.59 , p = 0.073] and Day 30 ; in the DBP on Day 7 ; and in the MAP on Day 7  and on Day 30 , see Fig. p = 0.009); but not in patients with bumetanide and placebo .There was a decrease in SBP on Day 7 [intervention: -18  vs. control: -7.5 , p = 0.104 and p = 0.605, respectively)  or at 30 days (p = 0.371). Brain natriuretic peptide levels were also evaluated. At the start of follow-up, the bumetanide plus placebo group showed an increase in BNP on Day 7 to 33.6  and on Day 30 to 213.5  without statistical significance (p = 0.175). The intervention group showed a decrease of -39.3 on Day 7  and -0.64 on Day 30  without a significant difference (p = 0.221). Similarly, intergroup analysis did not reveal any significant differences  to 0.59 , while the bumetanide plus placebo group showed a change from baseline to day 30 of -0.34 , both groups without intragroup statistical significance (d greater than 0.5) were observed for changes in blood pressure  and changes in body water .Effect size analysis is shown in Table p = 0.013). Nevertheless, none of the patients exhibited a two-fold increase in their serum creatinine levels at the end of follow-up, nor did they require renal replacement therapy. Other adverse effects are listed in Table p = 0.001) and twenty-eight days (p < 0.000). In this same group, a significant decrease in the glomerular filtration rate was observed at seven (p = 0.004) and twenty-eight days (p = 0.003). Regarding urea levels, both groups showed significant increases when comparing baseline with Days 7 and 30. The comparison of serum levels of creatinine and urea and the glomerular filtration rate estimated by CKD-EPI between the control and intervention groups did not show significant differences. During the follow-up time of the clinical trial patient, none started renal replacement therapy and none presented clinical data of volume depletion.The most common adverse event observed in this study was increased creatinine levels Cr > 0.3 mg/dL), which were present in eleven cases with a range of 2.63 mg/dL 68.75%), compared with four cases in the bumetanide plus placebo group with a range of 1.86 mg/dL (25%) ; however, when they were randomized, furosemide was changed to bumetanide, a more potent loop diuretic with a dose equivalent to 160 mg furosemide QD . This prThiazide diuretic use, particularly chlorthalidone, seems to provide additional benefits; chlorthalidone has a potency at least threefold that of hydrochlorothiazide, as well as a longer half-life . A recenActual estimates indicate that nearly 40% of patients diagnosed with advanced chronic kidney disease decide not to start renal replacement therapy, choosing conservative treatment instead . Our resOn the other hand, BNP has been correlated with volume overload in patients with chronic kidney disease measured by bioimpedance , 34; howWe observed the same phenomenon in plasma urea levels and glomerular filtration rate (GFR), both associated with a nonsignificant deterioration in patients on bumetanide plus chlorthalidone treatment compared with bumetanide plus placebo. The proportion of patients in the bumetanide plus chlorthalidone group who experienced an increase in plasma creatinine levels could be conceptually defined as having acute kidney injury . NeverthOne of the main adverse effects reported in the literature associated with thiazide use is hydroelectrolytic imbalance, hyperuricemia or an increased diabetes risk –42. Our This study has some others limitations. First, the follow-up in our study was short; therefore, further prospective trials are needed to assess the long-term safety and efficacy of this regimen. Second, there was patient noncompliance with dietary recommendations, even though all patients were evaluated and counseled in terms of renal nutrition, with an emphasis on sodium dietary restriction, as it is known that sodium excretion is directly proportional to kidney function and sodium dietary intake . Third, In conclusion, our study demonstrated the efficacy of chlorthalidone and bumetanide on volume overload and blood pressure reduction in advanced chronic kidney disease patients. Our results suggest that at the one-month follow-up, bumetanide plus chlorthalidone therapy is effective in patients with advanced chronic kidney disease. Volume overload reduction was adequately tolerated in this population; however, further studies with larger samples and longer follow-up periods are warranted. Based on this trial and those conducted previously on thiazide use for advanced chronic kidney disease, we propose that current guidelines should be reviewed to perhaps change the notion that thiazides should not be used in advanced CKD patients, as it has been demonstrated that this class of diuretics can be helpful in the treatment of this population.


Blood pressure management is crucial in the treatment of patients with aneurysmal subarachnoid hemorrhage (aSAH). Possible association between the blood pressure increase and the risk of delayed cerebral ischemia (DCI) and different systemic complications after aSAH is still a matter of debate. This study aims to elucidate the influence of blood pressure levels on the outcome of aSAH. All consecutive aSAH patients (n = 690) treated between 01/2003 and 06/2016 were included. The mean value of the mean arterial pressure (MAP) during 14 days after ictus was calculated for each individual. According to the institutional standards of vasospasm management, the mean 14 days MAP ≥ 95 mmHg was referred as increased (IMAP) and the patients with and without vasospasm were analyzed separately. Study endpoints were the occurrence of DCI on computed tomography scans, development of cardiac and nephrological complications, and poor outcome 6 months after aSAH (mRS > 2). Associations were tested in univariable/multivariable binary logistic regression analysis. IMAP was documented in 474 (68.7%) cases and was more common in individuals with poor neurological conditions at admission (p < 0.001), severe amount of intracranial blood (p = 0.001) and premorbid hypertension (p < 0.001). IMAP was independently associated with the occurrence of DCI  and poor functional outcome  in patients with vasospasm, but not in counterparts without vasospasm (p = 0.113/p = 0.086). IMAP had no influence on cardiac or nephrological complications. In aSAH individuals with cerebral vasospasm, sustained increase of blood pressure exceeding the therapeutic targets is strongly associated with the risk of DCI and poor outcome. Therefore, such an intrinsic increase of blood pressure might reflect the autoregulatory mechanisms against the impending cerebral ischemia in patients with cerebral vasospasm.Trial registration number: German clinical trial registry . Multiple factors including cerebral vasospasm, microthrombosis, activation of inflammatory pathways, and spreading cortical depolarization contribute to development of infarction in former unaffected brain areas2. Preventive measurements include administration of nimodipine and endovascular intervention in case of vasospasm4.Aneurysmal subarachnoid hemorrhage (aSAH) is a subform of hemorrhagic stroke with high morbidity and mortality. Besides the initial bleeding and possible rebleeding event, patients with aSAH are endangered by multiple subsequent complications, especially during the first weeks after aneurysm rupture. One major contributor to poor outcome is delayed cerebral ischemia (DCI)6. However, the only randomized controlled trial so far showed that an extreme increase of the mean arterial pressure (MAP) does not result in better outcome, but is related to higher risk of cardiac complications8. According to the recent aSAH guidelines9, blood pressure management remains the cornerstone element of aSAH treatment including the avoidance of hypotension, and the maintenance of the MAP over certain cut-off values widely varying throughout the neurovascular centers.Induced hypertension had a long-standing role in the prevention of DCI and associated infarction11. Under certain circumstances, MAP might exceed the therapeutic targets and necessitate the treatment with anti-hypertensive agents. In this context, the value of hypertensive and non-hypertensive blood pressure values for the course and outcome of aSAH remains unclear.Along with this vasospasm-targeted MAP-based blood pressure management, patients’ premorbid conditions, initial severity of bleeding, later complications, and different medical interventions during hospitalization may also contribute to blood pressure alterations after aSAHThis study aims to elucidate the clinical impact of sustained blood pressure increase exceeding the aimed therapeutic frame on the risk of DCI associated infarction, different systemic complications and poor outcome after aSAH.All adult patients treated for aSAH between January 2003 and June 2016 at our neurovascular center were eligible for this study. The exclusion criteria were: (a) hospital admission later than 48 h after ictus; (b) hospital stay at our clinic for less than 72 h; (c) no aneurysm treatment; (d) mycotic aneurysm as a bleeding source; (e) no data on MAP values.The approval of the institutional ethics committee  for this study was obtained and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The study was registered in the German clinical trial registry . All patients or their relatives gave written informed consent within the treatment contract before inclusion into the database.All patients with a suspected aSAH received radiographic imaging  or computed tomography (CT) angiography of the head) for identification of the bleeding source. Commonly, aneurysm treatment was performed within 24 h after hospital admission by endovascular coiling or microsurgical clipping. Acute hydrocephalus was treated by insertion of an external ventricular drain allowing the measurement of the intracranial pressure (ICP). Raised ICP (> 20 mmHg) was treated conservatively by drainage of cerebrospinal fluid, head elevation, osmotherapy, deep sedation, and relaxation. Patients with increased ICP refractory to conservative management were referred to decompressive craniectomy. Cerebral perfusion pressure (CPP) was maintained over 60 mmHg. Posthemorrhagic hydrocephalus was treated with ventriculoperitoneal shunt placement. Follow-up CT scans of the head were performed in the first 24 h after aneurysm treatment, and if clinically indicated .All patients were administered nimodipine orally for 21 days after ictus. Fluid management included the maintenance of euvolemia. In addition, transcranial doppler (TCD) ultrasound was performed at least once a day for the first 14 days after ictus. Patients showing clinical signs of vasospasm in form of (a) TCD velocities higher than 120 m/s and compromised consciousness; (b) new neurological deficits or a decline in the Glasgow coma scale over 2 points not related to other reasons  were scheduled for immediate DSA for the verification and invasive endovascular treatment. As first line therapy, pharmacologic angioplasty was performed using intraarterial nimodipine. Mechanical angioplasty  was performed only as a rescue option in large proximal intracranial arteries. In case of recurrence of new deficits or increased TCD values, endovascular therapy was repeated up to twice daily until the resolution of vasospasm signs. Post angiographic TCD and/or clinical recovery was reevaluated to determine the success of treatment.Until obliteration of the aneurysm, systolic blood pressure was kept < 150 mmHg using antihypertensive drugs, if needed. After aneurysm treatment, MAP was retained at ≥ 70 mmHg for the first 14 days after ictus in all aSAH individuals. In patients with confirmed clinical and angiographic vasospasm, the MAP was raised to ≥ 90 mmHg using norepinephrine administration via a central line, if necessary. In cases where the designated MAP target could not be reached with norepinephrine alone, the intensive care physician used additional vasopressors depending on the individual patient's situation. MAP target was kept for the duration of VS. In patients with an unvoluntary systolic blood pressure (SBP) over 220 mmHg, blood pressure was lowered up using antihypertensive medication without compromising the designated MAP. Blood pressure was monitored continuously via arterial line and was documented every 2 h in the patients’ chart.The variables of interest, including radiographic, clinical, laboratory and demographic characteristics of the patients, were collected from the electronic patients’ records and the institutional prospective aneurysm database. The radiographic data were separately reviewed by the senior author (RJ) blinded at this time for any clinical information.12 and was dichotomized into good (WFNS 1–3) and poor grade (WFNS 4–5) for statistical analysis. The original Fisher scale was used to assess the radiological severity of bleeding13, with further dichotomization into high (Fisher 3–4) and low (Fisher 1–2) grades. Occurrence of new cerebral infarction(s) was judged upon the follow-up CT imaging up to 6 weeks after aSAH. New hypodensities visualizable within 48 h after early aneurysm occlusion were referred as early infarcts, whereas later infarct events were allocated as DCI associated infarction.Initial clinical severity of aSAH was classified using the World Federation of Neurological Surgeons (WFNS) scaleA mean value of MAP and SBP for the first 14 days after ictus were calculated using the daily mean MAP/SBP values collected from the ICU documentation system. For further statistical evaluation, the 14-days mean MAP values were dichotomized according to the institutional standards of blood pressure management after aneurysm treatment: within  and over  the maximal therapeutic target. No patient showed the 14-days mean MAP value < 70 mmHg. The total dosage of used norepinephrine (in milligram [mg]) was extracted for each patient in the cohort.Depending on the presence of clinical signs and radiographic proof of cerebral vasospasm  the patients in the cohort were referred to vasospasm (VS) and no vasospasm (NO VS) groups ;impaired renal function defined as any decrease of the mean glomerular filtration rate under 60 ml/min/1.73 macute coronary syndrome (ACS) based on the changes in the cardiac enzymes and/or electrocardiography (ECG); 12-channel ECG and laboratory work-up was performed if changes in continuous ECG monitoring  or clinical symptoms of ACS occurred.14.Finally, all chest x-rays during the first 14 days were screened to identify new pulmonary congestion not present on the admission X-ray utilizing the Shochat score (cut-off: 2 points)Along with preexisting conditions recorded from the medical charts, the occurrence of following systemic complications during the hospital treatment was also collected:15. An mRS > 2 was defined as poor outcome.The outcome was assessed at a 6-month follow-up and documented using the modified Ranking scale (mRS)The correlation of the 14-days mean MAP values with the functional outcome at 6 months and DCI associated infarction development were set as the primary endpoint. Secondary endpoint included the impact of the 14-days mean MAP values on the occurrence of systemic complications during aSAH.All statistical analyses were performed using SPSS Version 27 for Mac (IBM Corp). The significance level was set to p < 0.05. Missing data were addressed using multiple imputations.Univariate analysis was performed to address the correlation of the 14-days mean MAP with the patients’ baseline characteristics and initial severity of aSAH as well as secondary endpoints. Chi-Square test was used for dichotomous/dichotomized variables; for samples with a size smaller than 5, the Fisher exact test was used. Continuous variables were tested with the Students t-test for normally distributed data and the Mann–Whitney-U test for non-normal distributed data. Multivariable binary logistic regression analysis was performed for the primary study endpoints to prove independent correlations. For the primary endpoints, the results were adjusted for initial clinical and radiological aSAH severity, the need for ICP treatment, presence of premorbid arterial hypertension and patients’ age. In addition, the analyses were performed for aSAH individuals in the VS and NO VS groups separately.Ethik-Kommission, Medizinische Fakultät der Universität Duisburg-Essen, Registration number: 15-6331-BO.A total of 690 patients were included in the final analysis  was independently associated with poor outcome in the VS group  was independently associated with the occurrence of DCI associated infarction in the VS, but not in the NO VS group  vs. 23.2 mg (interquartile range:1.12–100.51 mg), but not in the VS group , ACS (p > 0.99/p = 0.380) and impaired kidney function  does not translate into better outcome but more cardiac complications. Of note, the control group in this trial was kept over a MAP of 80 mmHg16.The influence of increased blood pressure and respectively MAP on the outcome following aSAH has been discussed extensivelyIn our study, IMAP beyond the therapeutic target independently correlated with unfavorable clinical and radiographic outcome of aSAH in individuals with cerebral vasospasm. At the same time, blood pressure values did not show impact on the study endpoints in patients that did not develop vasospasm.18. The time point of this blood pressure increase after aSAH was considered crucial for the final impact on the functional outcome and DCI risk19. Interestingly, especially early spontaneous increased blood pressure and persisting endogenous hypertension was associated with or showed a trend towards poor outcome and increased probability of DCI19. Furthermore, in case of an ischemic stroke, blood pressure will increase in the majority of patients and take up to 10 days to reach baseline levels22. The multiple progenitors leading to DCI associated infarction development after aSAH may cause a constant state of prolonged ischemia in different areas of the brain. Comparable to ischemic stroke, this seems to lead to a constant upregulation of the blood pressure. Moreover, aSAH leads to an impairment of cerebral autoregulation24. This might additionally contribute to poor outcome in patients that exceed the aimed MAP values.An autonomous increase of the blood pressure levels during acute cerebrovascular events, including aSAH, was described beforeThe differences in the vasopressors dosage between SMAP and IMAP patients observed in the NO VS group might be related to the need for optimization of the CPP in the individuals with IMAP, since there was no vasospasm-triggered autonomous upregulation of the blood pressure in these patients. In contrast, IMAP pattern was not driven by higher dosage of vasopressors in aSAH individuals with cerebral vasospasm, as MAP increase over the therapeutic aim might be, at least partially, related to autoregulatory mechanisms in this cohort group.The erring link between autonomous upregulation and IMAP in the NO VS group and the missing positive impact on DCI associated infarction and outcome, questions induced hypertension in the treatment of aSAH in patients without clinical vasospasm.16. As mentioned before, an increase in cardiac complications was the main reason for early termination of the HIMALAIA trial16. However, in our cohort no correlation between cardiac and nephrological complications and IMAP could be shown. It might be due to the fact that the MAP values aimed in our cohort were not driven as high as in other studies on intention and that both study cohorts might not be equal regarding preexisting kidney and heart function. In summary, our results are in line with the previous studies and show that a moderate MAP management without utilization of extreme hypertension is a useful measure against increased risk of systemic cardiac, pulmonary and kidney-related complications.A higher blood pressure has been connected with increased cardiac and kidney-related complications, especially if caused iatrogenic in the acute phase of aSAHThis is a retrospective study that includes the common drawbacks of this study design. Even if based on a prospectively enrolling electronic database, the completeness and accuracy of the available dataset was lower as compared to studies with a complete prospective design. For two variables (GFR and pulmonary congestion) a relevant portion of the data was missing. Furthermore, due to the retrospective setting of the study without a standardized study protocol, we were unable to analyze the possible association between the MAP values at the specific timepoints and the subsequent clinical events. Accordingly, we limited the analyses to the mean values over a predefined time interval of two weeks that might better reflect the autoregulatory reaction of patients during aSAH. Of note, the analyses based on the maximum, minimum and delta values of the blood pressure did not show any significant results. Finally, there is a risk of bias regarding the treatment intention and outcome confounding which was addressed by utilizing subgroup analyses with further correction for relevant confounders in the multivariable analysis.Sustained increase of MAP beyond the therapeutic targets in patients with aSAH and cerebral vasospasm independently correlates with the risk of DCI associated infarction and poor functional outcome. Therefore, the presence of MAP exceeding the aimed values without additional iatrogenic intervention on the blood pressure might reflect the autoregulatory mechanisms against the impending cerebral ischemia in patients with cerebral vasospasm. aSAH patients with such vasospasm-triggered blood pressure upregulation might profit from intense diagnostic and preventive therapeutic interventions against cerebral vasospasm.Supplementary Information.


The Methotrexate (MTX) Intolerance Severity Score (MISS) questionnaire has been developed to identify MTX adverse events in juvenile idiopathic arthritis (JIA). The objective of this study was to translate and validate MISS into Brazilian Portuguese for children and adolescents. The MISS was translated into Portuguese following the standardized guidelines. We analyzed the following psychometric properties: acceptability, internal consistency, test–retest reproducibility, relative–child reliability, and external criterion and discriminant validity. We included 138 JIA patients (age: 8–18 years) and 108 relatives who took less than 5 min to answer MISS. Reproducibility tested after 15 days was good, with a kappa > 0.76. We observed good internal consistency (Cronbach’s coefficient 0.75–0.87 (patients) and 0.75–0.79 (relatives)). Reliability between patients and relatives was good except for stomachache and restlessness. Cut-off points of 5 and 6 had good sensitivity  and specificity . Using a cut-off value of 6, we observed 86 (62.3%) MTX-intolerant patients. In conclusion, MISS is a viable and practical tool for routine clinical care to identify MTX intolerance in JIA. Parents do not easily identify stomachache and restlessness as adverse MTX events. For srovement . MTX canrovement . The MISrovement ,14,15,16rovement . Therefo2/week) for more than 3 months independent of the route of administration. It was a convenience sample, and we aimed to include a minimum of 10 JIA patients per MISS questionnaire item [We invited consecutive JIA patients and their relatives to participate in the study during their regular visit at the pediatric rheumatology unit at the Clinics Hospital of the University of Campinas between August 2017 and June 2019. To be included, children and adolescents had to be between 8 and 18 years of age, fulfill ILAR criteria for JIA [In total, 138 patients and 108 relatives filled out the questionnaires in different rooms, and we measured the time spent to answer it. A total of 36 patients repeated the questionnaire after 15 days to test the reproducibility.Demographic and disease-related characteristics were collected through medical chart review.The study was approved by the local IRB (CAAE = 69672717.5.0000.5404), and informed written consent was obtained from each subject and/or legal guardian.Prior authorization was obtained from the authors of the original questionnaire (NW) . The MIS® software, version 21, and R, version 4.0.3. The sample size was calculated on the basis of the formula by Kothari [Data analysis was performed on SPSS Kothari . Conside Kothari . The fol Kothari ,25,26. FWe plotted the ROC curve to evaluate the discriminant validity of the translated MISS questionnaire compared to the gold standard, which is based on clinical interviews and symptoms of adverse events of MTX recorded in medical charts. The cut-off score for intolerance was determined by analyzing sensitivity and specificity.Principal component analysis (PCA) was conducted to determine variability in the principal components.We included 138 subjects with JIA (101 (73.2%) women) with a median age of 12.3 years . Parenteral MTX was used by 111 individuals (80.4%). The median time of MTX use was 4.87 years We found minor difficulties in translate the MISS, especially in the description of “several hours to 1 day before taking MTX”. Minor transcultural adaptation was needed regarding the description of the frequency of intolerance symptoms in Portuguese (Brazil). The original version of “no, mild, moderate, and severe symptoms” was changed into “never, sometimes, often and always”. These words facilitated the understanding of the scores by our target population.Psychometric characteristics of the translated version are reported below:Acceptability: patients and relatives required less than 5 min to respond to the questionnaire.Internal consistency: The translated questionnaire had a Cronbach’s alpha of 0.88. Individual items had Cronbach’s alpha values ranging from 0.75 to 0.87 for JIA patient responses .Criterion validity: We plotted the ROC curve, and the area under the curve (AUC) was 0.90  for the total sample (children and relatives) (latives)  and 0.89latives)  for JIA Principal component analysis: the two first components were responsible for 54% of the observed variability, as shown in Variables associated with MTX intolerance: The median score for the MISS in our JIA cohort was 6.0 points (p = 0.03). No association with age (p = 0.35) and MTX treatment duration (p = 0.54) was observed.0 points . We obseThe MISS was used in both JIA and RA patients using MTX ,15,16,17A cut-off score of 5 or 6 for children and relatives yielded the best sensitivity and specificity to discriminate between MTX intolerance and tolerance, similar to the original version . The FreWe observed that approximately 62% of our JIA patients had a degree of MTX intolerance. In the literature, MTX intolerance in JIA varies in the range of 40–70% ,28. The This study has some limitations. It was a single-center, cross-sectional study, and a convenience sample was used for validation. This validation was performed during the same period of the validation of the adult RA MISS scores . The texLongitudinal studies are of interest to determine clinical and psychological factors associated with MTX intolerance, and strategies to reduce its occurrence and increase adherence.In conclusion, the MISS can be used in clinical practice to determine the prevalence of MTX intolerance in JIA patients and improve patient adherence.


Background: Thyroid hormones have substantial effects on blood pressure (BP) and renal function as they influence the glomerular filtration rate (GFR). Maintaining healthy BP and preventing premature development of nephropathy necessitates taking steps.Objectives: The aim of this study was to explore the association between BP, GFR, and thyroid-stimulating hormone (TSH) levels in hypothyroid patients at King Abdulaziz University Hospital, Jeddah, Saudi Arabia.Methods: A retrospective record review study of all hypothyroid patients from June 1, 2010 to June 6, 2020. The medical records of 1,181 adult patients were reviewed, and 157 met the criteria. All patients aged >18 years who were diagnosed with hypothyroidism and were on levothyroxine therapy, were included in this study.Results: More than half of the participants were female (83.4%). There was no significant correlation between TSH and systolic BP (P= 0.6), or TSH and diastolic BP (P=0.8), while there was a positive correlation between TSH and creatinine  and a negative correlation between TSH and GFR .Conclusions: We found no association between BP and TSH, while creatinine correlated directly and GFR inversely with TSH. Follow-up renal function should be a target for physicians in hypothyroid patients to prevent premature complications. Thyroid hormones have substantial effects on blood pressure (BP), lipids, and energy consumption . They afHypothyroidism is a common disease. Overt hypothyroidism is defined as serum thyroid-stimulating hormone (TSH) concentration above, and free thyroxine FT4) below, the normal reference range . A retro below, tNumerous studies have investigated the correlation between TSH levels and BP. In 2019, a study demonstrated that thyroid disorders, including hypothyroidism, can increase the risk of hypertension . AnotherAdditionally, long-standing hypothyroidism can cause significant changes in renal function, serum creatinine levels are elevated, and GFR values are reversibly reduced in overt hypothyroid patients compared to euthyroid subjects .Maintaining healthy BP is a major goal for all individuals, and preventing premature development of nephropathy necessitates intervention. The aim of this study was to explore the association between BP, GFR, and TSH in hypothyroid patients at King Abdulaziz University Hospital (KAUH), in Jeddah, Saudi Arabia.This was a retrospective record review study of all hypothyroid patients attending KAUH from June 1, 2010 to June 6, 2020. The medical records of 1,181 adult patients were reviewed, and 157 met the criteria. Ethical approval was obtained from the Unit of Biomedical Research Ethics Committee, KAUH, College of Medicine (303-20).Study population and data collection Patients aged >18 years who were diagnosed with hypothyroidism and were on levothyroxine therapy were included in this study. We excluded patients who were known to have secondary hypothyroidism or other endocrine disorders such as Cushing’s syndrome, and acromegaly; patients on treatment with drugs that are known to influence renal function, such as amiodarone, lithium, angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, diuretics, allopurinol, steroids, phenytoin, carbamazepine, salicylates, beta-blockers, rifampicin, or cytotoxic drugs; patients with renal disorders, liver disorders, diabetes, hypertension, other autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, or malignancy; and pregnant women.Data were extracted during July 2020 from the electronic medical record system by using a data collection sheet that collected demographic data including age, sex, nationality, height, weight, BP, TSH, FT4, free triiodothyronine (FT3), and creatinine laboratory test results. A BP < 120/80 mmHg was considered to be normal [2). BMI was categorized based on the World Health Organization’s cut-offs: underweight (<18.5 kg/m2), healthy weight (18.5-24.99 kg/m2), overweight (25.0-29.99 kg/m2) or obese (≥30.0 kg/m2) [Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared  Study Equation :GFR = 175 × (sCr − 1.154) × (age − 0.203) × where sCr is serum creatinine in mg/dL. Power calculation was based on estimations of differences in GFR in an adult population with and without hypothyroidism . With alStatistical analysisMicrosoft Excel 2019 was used for data entry, and statistical analysis was performed using IBMSPSS Statistics version 21 . Continuous variables were reported using means and standard deviations, while categorical variables were reported using frequencies and percentages. Differences between TSH, F4 and F3 levels between anthropometric and demographic groups were analyzed using students' t-test or one-way ANOVA. Correlations between continuous data were analyzed using Pearson’s correlation. P-values < 0.05 were considered statistically significant.2  were female, and 110 (70.1%) were Saudi nationals. Their mean age was 45 ± 15 years, and their mean BMI was 32.1 ± 19.5 kg/mThe data in Table 2). This was significantly lower than patients with TSH 2.01-4.20 µIU/L who had GFR 123.9 ± 49.8 24 (mL/min/1.73 m2) (p = 0.019).Table In this retrospective record review, 131 83.4%) of the participants were women. Similar results were reported by Aldossari et al. (84.5%) and Aljabri et al. (77.2%) % of the . This coOur study did not show an association between BP and TSH, which is similar to studies done by Walsh et al. and De Pergola et al. ,14. ThisAs in previous studies, TSH was directly correlated with creatinine and inversely correlated with GFR ,26. MoreThis study had some limitations. First, we took one reading for each SBP and DBP because of a lack of documentation. Second, the generalizability may be limited because the study was conducted in only one healthcare center. One of the strengths of this study is that we excluded patients who were taking medications that might affect the thyroid hormones.We found no association between BP and TSH, while TSH was correlated directly with creatinine and inversely with GFR. Physicians should monitor renal function in hypothyroid patients to prevent premature complications. Prospective population-based studies should be conducted to assess whether these associations are sufficiently strong to influence the future risk of nephropathy and to provide an accurate estimate of the association between BP and TSH. A multi-center study with large sample size is warranted.


Long noncoding RNA ANRIL has been found to be involved in the pathogenesis of diabetic kidney disease (DKD) and is expected to be a new target for prevention of DKD. However, the circulating expression and clinical significance of ANRIL in DKD patients is uncertain. This study aims to explore this issue.The study consisted of 20 healthy controls, 22 T2DM patients  and 66 DKD patients . The expressions of ANRIL in peripheral whole blood of all participants were measured by RT-qPCR.The expression of ANRIL was significantly up-regulated in DKD patients  than that in healthy control group. ANRIL was also over-expressed in macroalbuminuria and renal dysfunction groups in comparison with normalbuminuria group. ANRIL expression was positively correlated with Scr, BUN, CysC, urine β2-MG and urine α1-MG; while negatively correlated with eGFR in DKD patients. In addition, ANRIL was the risk factor for DKD with OR value of 1.681. The AUC of ANRIL in identifying DKD was 0.922, and the sensitivity and specificity of DKD diagnosis were 83.3% and 90.5%, respectively.Our results indicated that highly expressed ANRIL in peripheral blood is associated with progression of DKD. Circulating ANRIL is an independent risk factor of DKD and has a highly predictive value in identifying DKD. Diabetic kidney disease (DKD) is a serious microvascular complication of diabetes mellitus (DM) and a primary etiology for end-stage renal disease (ESRD) worldwide –3. The pCurrently decreased GFR and albuminuria have long been considered significant manifestations of DKD , 9. HoweLong-chain non-coding RNAs (lncRNAs), a novel class of nonprotein-coding functional RNA molecules with a transcript length greater than 200 nucleotides, are recognized as gene expression regulators and involved in a variety of physiological and pathological processes , 11. IncAntisense RNA to INK4 locus (ANRIL), also known as CDKN2B-AS1, is transcribed from the short arm of human chromosome 9 on P21, which is involved in cell proliferation, migration, apoptosis, inflammation, immune responses and DNA damage . ANRIL hPatients diagnosed with type 2 diabetes mellitus (T2DM) based on the criteria of the American Diabetes Association (ADA) were collected from Shaanxi Provincial People’s Hospital  between September 2020 and December 2021. Patients with type 1 diabetes, secondary diabetes, urinary tract infection, urolithiasis, pregnancy, superimposed systemic diseases and other glomerular diseases were excluded. 88 diabetic patients were enrolled in this study in which 22 were T2DM patients  < 30 mg/g) and 66 were DKD patients. DKD patients were divided into three groups according to UACR and serum creatinine: (1) Patients with microalbuminuria (UACR 30–300 mg/g), n = 23; (2) Patients with macroalbuminuria (UACR > 300 mg/g), n = 22; and (3) Patients with increased serum creatinine  (serum creatinine > 120µmol/L), n = 21. Meanwhile, 20 non-diabetic healthy volunteers were enrolled as control group. The present study was approved by the ethical committee for human investigation of Shaanxi Provincial People’s Hospital and was conducted according to the Declaration of Helsinki. Informed consent was obtained from all participants.2 (kg/m2). The following laboratory parameters were obtained from each patient: glycated hemoglobin A1c (HbA1c), fasting serum glucose (FSG), UACR, urine β2-microglobulin (β2-MG), urine α1-microglobulin (α1-MG), serum creatinine (Scr), blood urea nitrogen (BUN), Cystatin C (CysC), neutrophil gelatinase-associated lipocalin(NGAL), uric acid (UA), white blood cell (WBC), hemoglobin (HGB), albumin (ALB), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL) at the time of enrollment. UACR was calculated using the formula: UACR = urine albumin/creatinine. The eGFR was calculated according to the modified MDRD formula: eGFR = 186 × Scr− 1.154 × Age− 0.203 × gender .General data were collected as follow: gender, age, body height, body weight, systolic blood pressure (SBP), diastolic blood pressure (DBP) and duration of diabetes on admission. Body mass index (BMI) was calculated using the formula: BMI = body weight/body heightPeripheral whole blood was collected by venipuncture with an ethylenediaminetetraacetic acid (EDTA) anticoagulant vacutainer from all patients and stored at − 80 °C until analysis. Total RNA was extracted as soon as possible.−ΔΔCt method. ANRIL primer sequences were shown as follows: upstream: 5’-AGGGTTCAAGCATCACTGTTAGG-3’; downstream: 5’-GAAACCCCGTCTCTACTGTTACCT-3’.1mL peripheral whole blood was centrifuged at 3000rpm for 5min and the supernatants were removed. Then 3mL red cell lysis buffer added and mixed before being centrifuged at 3000rpm for 5min. The supernatant was then discarded and the extracted leukocytes were collected. The total RNA of leukocytes was extracted using Trizol reagent , and then dissolved in RNase-free water. The concentration of RNA was determined using NanoDrop 2000 . Extracted RNA was reversibly transcribed into complementary DNA (cDNA) using Servicebio®RT First Strand cDNA Synthesis Kit . Quantitative real-time polymerase chain reaction was performed using SYBR Green qPCR Master Mix  on a CFX RT-PCR system (Bio-Rad). PCR reaction system was as follows: pre-degenerated at 95°C for 10min, followed by 40 cycles of 95°C for 15s and 60°C for 30s. The expression level of lncRNA ANRIL was normalized to the expression level of GAPDH as a housekeeping gene. The relative quantitative value was expressed by the 2SPSS software, version 18.0  was used for statistical analysis. Normally distributed data were presented as mean ± S.D. and non-normally distributed data were expressed as median range. Differences between two groups for quantitative data and qualitative data were compared using a t test and chi-square test, respectively. Comparisons of normally distributed data in 3 or more groups were analyzed using one-way ANOVA, while non-normally distributed data were analyzed using nonparametric counterpart Kruskal-Wallis test. Correlations were examined using Pearson’s correlation analysis. Binary regression analysis was used to determine the influence factors of the presence of DKD. The diagnostic value of ANRIL was evaluated by ROC curve analysis. Area under the ROC curve (AUC) was calculated. When AUC = 0.5, diagnostic value was denied. The cut-off value and corresponding sensitivity and specificity were determined according to ROC curve analysis. P < 0.05 was considered to represent significant differences between groups.The basic characteristics of individuals involved in this study and results of biochemical analyses within the study groups were displayed in Table The differential expression of ANRIL in normalbuminuria, microalbuminuria, macroalbuminuria, renal dysfunction groups and healthy control group are shown in Fig. The correlations between ANRIL expression and clinical parameters in DKD patients were analyzed using Pearson’s linear correlation. Fig. Binary regression analysis showed that ANRIL, SBP, α1-MG and disease duration were the risk factors of DKD, with OR value of 1.681, 1.248, 1.142 and 1.599 (P < 0.05); while, HGB was found to be the protective factor of DKD, with OR value of 0.838 (P < 0.05) (Table To confirm the predictive value of circulating ANRIL as the biomarker for the early diagnosis of DKD, the diagnostic value of ANRIL was evaluated by ROC curve analysis. As depicted in Fig. DKD is a common DM complication characterized by a progressive damage of kidney structure and deterioration of renal function, which has become the primary cause of ESRD worldwide –3, 8. ReANRIL in peripheral whole blood, mainly expression in leukocytes, is relatively stable. This present study examines the expression of ANRIL in peripheral whole blood and shows that the expression of ANRIL in peripheral whole blood was significantly upregulated in DKD patients than those in healthy controls and T2DM patients. ANRIL expression showed a positive correlation with Scr, BUN, CysC, urine β2-MG and urine α1-MG, while negatively correlated with eGFR in DKD patients. These above clinical parameters were used to evaluate kidney function status of DKD. Binary regression analysis showed that ANRIL was the risk factor of DKD. The results indicated that ANRIL might be involved in the kidney impairment of DKD; might be play a key role in the pathogenesis of DKD and might be an efficient target for DKD prevention and treatment.In the early stage, DKD begin from glomerular hyperfiltration, without any clinical symptoms, followed by the development of microalbuminuria. Along with gradual progression, DKD manifests as a clinical syndrome including persistent albuminuria, increased blood pressure, sustained reduction in GFR and increased cardiovascular events , 9, 34. This is the first study that revealed an independent relationship between ANRIL expression in peripheral whole blood and DKD patients. While, there is still a limitation in this study. Sample size in this study was small, which might result in bias of the results evaluating the association of ANRIL expression and DKD.In conclusion, our findings provided new evidence that the presence and progression of DKD is associated with an over-expressed ANRIL in peripheral whole blood. Circulating ANRIL is an independent risk factor of DKD and has a highly predictive value in identifying DKD.


Therefore, we aimed to evaluate the trends of hemodynamics and biomarkers in a porcine bacteremia model treated with insusceptible antibiotics compared to those in susceptible models. Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli  was intravenously administered to 11 male pigs. One hour after bacterial infusion, pigs were assigned to two groups of antibiotics, ceftriaxone (n = 6) or ertapenem (n = 5). Pigs were monitored up to 7 h after bacterial injection with fluid and vasopressor support to maintain the mean arterial blood pressure over 65 mmHg. Blood sampling for blood culture and plasma acquisition was performed before and every predefined hour after E. coli injection. Cytokine  levels in plasma were analyzed using enzyme-linked immunosorbent assays. Bacteremia developed after intravenous injection of E. coli, and negative conversion was confirmed only in the ertapenem group. While trends of other biomarkers failed to show differences, the trend of sTREM-1 was significantly different between the two groups . Among hemodynamics and biomarkers, the sTREM-1 level at post 2 h after antibiotics administration represented a significant difference depending on susceptibility, which can be suggested as a biomarker candidate of in vivo antibiotics susceptibility. Further clinical studies are warranted for validation.Proper selection of susceptible antibiotics in drug-resistant bacteria is critical to treat bloodstream infection. Although biomarkers that guide antibiotic therapy have been extensively evaluated, little is known about host biomarkers targeting IMPORTANCE Early and appropriate antibiotic treatment is a keystone in treating patients with sepsis. Despite its importance, blood culture which requires a few days remains as a pillar of diagnostic method for microorganisms and their antibiotic susceptibility. Whether changes in biomarkers and hemodynamics indicate treatment response of susceptible antibiotic compared to resistant one is not well understood to date. In this study using extended-spectrum β-lactamase -producing E. coli bacteremia porcine model, we have demonstrated the comprehensive cardiovascular hemodynamics and trends of plasma biomarkers in sepsis and compared them between two groups with susceptible and resistant antibiotics. While other hemodynamics and biomarkers have failed to differ, we have identified that levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) significantly differed between the two groups over time. Based on the data in this study, trends of sTREM-1 obtained before the antibiotics and 2~4 h after the antibiotics could be a novel host biomarker that triggers the step-up choice of antibiotics. Early administration of adequate antimicrobials, ideally within 1 h of sepsis recognition, is one of the most efficient interventions to reduce mortality in patients with sepsis 13. In the4–Whether changes in hemodynamics and biomarkers could be indicative of antibiotic susceptibility and further reduction of bacterial load in sepsis is unknown . Direct in vivo antibiotic susceptibility. Thus, to understand the changes in hemodynamics and biomarkers after antibiotic treatment that can be further developed as an optimization of antibiotic selection in patients with sepsis, the differentiation of hemodynamic characteristics and biomarker dynamics according to susceptible antibiotics must be revealed  to fraction of inspired oxygen (FiO2) (P/F) ratio .To assess organ failure in this model, relevant values were measured . As the F) ratio , and albF) ratio  decreaseP < 0.0001 between times, P = 0.4674 between groups, P = 0.7086 between times × groups).Based on the hemodynamics and laboratory data, the sequential organ failure assessment score (SOFA) score without the central nervous system (CNS) was evaluated to reflect the objective response  . The totE. coli injection (Pre), no bacteria were identified in the blood culture test . E. coli were identified in all specimens at the time of completion of E. coli administration (0 h) and up to 1 h after administration. In the ertapenem group, all of the blood culture tests after 2 h of E. coli administration, which corresponds to 1 h after antibiotic treatment, presented negative results while the ceftriaxone group presented positive results.Before the E. coli injection ; a significant difference was observed 3 h after bacterial administration (2 h after antibiotics) .Biomarkers were measured 1, 3, 5, and 7 h before the completion of njection . Tumor nnjection  peaked anjection , IL-6  is expressed on neutrophils, mature monocytes and macrophages , 30. Lipin vivo antibiotic susceptibility in sepsis has not been previously evaluated, highlighting its novel role in this study. Further clinical studies addressing the association of trends of sTREM-1 levels and antibiotic susceptibility are needed.In previous studies, the differential value of sTREM-1 was evaluated in the diagnosis of sepsis in populations of systemic inflammatory response syndrome and outperformed CRP and procalcitonin , 34. Morn = 10) with ESBL-producing E. coli (5.0 * 10^9 CFU) administration was 10 h, complicating the acquisition of biomarkers over long periods of time.Long-term observation, including the prognosis of experimental subjects, was unfeasible due to the limitation of the study design. Instead, our study demonstrated negative conversion of blood cultures, which could be interpreted as a decrease in pathogen load to clinically undetectable levels as a consequence of effective antibiotics. In our previous pilot experiments, the median survival of pigs (E. coli) was utilized in this study, it cannot be extrapolated to other organisms, particularly Gram-positive bacteria (methicillin-resistant Staphylococcus aureus), which do not contain lipopolysaccharides. Although larger sample sizes and the use of multiple antibiotics against multiple bacteria species could broaden the scope of this study, practical limitations with the porcine model exist. However, although it may vary from region to region, we have selected the most common pathogens with significant issues with resistance to prescribed antibiotics, which are of widespread interest  may be limited. In fact, the level of sTREM-1 after the bacterial injection in this study was higher than the usual level in patients with sepsis , 34. Thiinterest , 37. FurE. coli-induced porcine sepsis model and identified the trends of hemodynamics and biomarkers with susceptible antibiotics compared to those of the insusceptible group. Among hemodynamic variables and biomarkers, the trend of sTREM-1 showed a significant difference with the use of susceptible antibiotics, which suggests sTREM-1 as a biomarker candidate of in vivo antibiotic susceptibility in Gram-negative sepsis. Further clinical study is warranted for the validation of sTREM-1 as a biomarker of in vivo antibiotic susceptibility in human bacteremia.We implemented the The experiment was approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (protocol No. BA-2104-317-029-03), and all animals received care according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.2O, and respiratory rate of 12 − 15/min. Further mechanical ventilation was adjusted to maintain arterial PaO2 above 65 mmHg and arterial PaCO2 between 40 and 45 mmHg. Meticulous preoperative povidone-iodine dressing on the region of intervention was conducted, followed by the application of a sterile surgical drape on the abdomen. Using ultrasonography, a 6-Fr arterial catheter  was inserted into both femoral arteries for invasive blood pressure monitoring and repetitive blood sampling. A Swan-Ganz catheter  was introduced into the right jugular vein to monitor cardiac output and PCWP. An additional central venous catheter  was applied to the left jugular vein for intravenous administration of fluid and vasopressors. Suprapubic cystostomy was conducted, and a Foley catheter was placed to monitor the urine output. After the above aseptic procedure, blood samples, including cultures, were obtained from the arterial catheter  domestic pigs (44 ± 3.5 kg) were utilized in this study. Before the experiment, subjects were accommodated in an animal facility with standardized access to food and water. Anesthesia was induced via an intramuscular administration of a mixture of zolazepam/tiletamine  and xylazine . After the pigs were scrubbed with povidone-iodine soap, the bilateral neck and inguinal areas were shaved. The pigs were monitored with a 3-lead electrocardiography monitor, a pulse oximeter, and a rectal temperature probe . Intubation was performed with a 7-Fr endotracheal tube that was applied to a mechanical ventilator  with an adequate concentration (2~3%) of inhalation anesthesia . Ventilation was set in the volume-controlled mode with a tidal volume of 6 mL/kg, fraction of inspired oxygen (0.21), positive end-expiratory pressure of 5 cmHcatheter .Escherichia coli  was diluted in 1000 mL of normal saline and intravenously administered over the course of an hour to subjects -producing subjects . After cThe animals were monitored up to 7 h after completion of bacterial infusion, and the primary goal was to maintain over 65 mmHg of MAP. Balanced crystalloid solution  was challenged when MAP was less than 65 mmHg, and vasopressors consisting of norepinephrine, vasopressin, and epinephrine were sequentially initiated when the cumulative fluid exceeded 30 mL/kg or target MAP was not achieved with bolus fluid.The following hemodynamic variables were acquired continuously and recorded every hour: arterial blood pressure, HR, lead II electrocardiographic variables, pulse oximetry variables, pulmonary arterial pressure, and central venous pressure. Measurement of cardiac output using the thermodilution technique was conducted every hour, and the median value is presented. Arterial blood gas analysis, including the P/F ratio , electrolyte and lactate variables , complete blood cell count , and chemistry panel test , were performed using blood samples obtained through the femoral arterial catheter at baseline and 1, 3, 5, and 7 h. The SOFA was calculated based on hemodynamic and laboratory variables . Blood sP values less than 0.05 were considered significant. Statistical analyses and graph generation were performed using Prism 9.2 .Variables are described as medians and interquartile ranges. Two-way analysis of variance for repeated measures with Sidak’s multiple-comparison test (two-way RM ANOVA) was performed to compare trends of variables between the two groups.