To examine the dose-response relation between reduction in dietary sodium and blood pressure change and to explore the impact of intervention duration.Systematic review and meta-analysis following PRISMA guidelines.Ovid MEDLINE(R), EMBASE, and Cochrane Central Register of Controlled Trials (Wiley) and reference lists of relevant articles up to 21 January 2019.Randomised trials comparing different levels of sodium intake undertaken among adult populations with estimates of intake made using 24 hour urinary sodium excretion.Two of three reviewers screened the records independently for eligibility. One reviewer extracted all data and the other two reviewed the data for accuracy. Reviewers performed random effects meta-analyses, subgroup analyses, and meta-regression.v usual sodium) of 24 hour urinary sodium, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were 130 mmol , 4.26 mm Hg , and 2.07 mm Hg , respectively. Each 50 mmol reduction in 24 hour sodium excretion was associated with a 1.10 mm Hg  reduction in SBP and a 0.33 mm Hg  reduction in DBP. Reductions in blood pressure were observed in diverse population subsets examined, including hypertensive and non-hypertensive individuals. For the same reduction in 24 hour urinary sodium there was greater SBP reduction in older people, non-white populations, and those with higher baseline SBP levels. In trials of less than 15 days’ duration, each 50 mmol reduction in 24 hour urinary sodium excretion was associated with a 1.05 mm Hg  SBP fall, less than half the effect observed in studies of longer duration . Otherwise, there was no association between trial duration and SBP reduction.133 studies with 12 197 participants were included. The mean reductions (reduced sodium The magnitude of blood pressure lowering achieved with sodium reduction showed a dose-response relation and was greater for older populations, non-white populations, and those with higher blood pressure. Short term studies underestimate the effect of sodium reduction on blood pressure.PROSPERO CRD42019140812. High blood pressure is a leading modifiable risk factor for cardiovascular disease, which caused at least 17.8 million deaths worldwide in 2017.The effect of sodium reduction on blood pressure and the risk of cardiovascular disease has been examined in numerous studies. Although there is a consensus among health and scientific organisations to reduce intake of dietary sodium in the general population,The nature of the association between change in sodium intake and blood pressure is key to understanding the potential for health interventions based on sodium reduction. Previous overviews of the data were limited because a definitive dose-response relation could not be determined, especially for participants with normal blood pressure.We carried out a search following a strategy developed for a previous meta-analysisTwo of three reviewers  independently assessed records identified from the search for eligibility. We resolved any discrepancies by consensus. We included only trials with random allocation of participants to reduced dietary sodium intake and usual/higher dietary sodium intake . Trials with concomitant interventions  were included only if the other interventions were applied equally to all randomised groups of interest. We included only studies with sodium intake estimated by 24 hour urine collection that also had data on systolic blood pressure or diastolic blood pressure measurements. Studies with only mean arterial blood pressure reported were not included unless we could retrieve relevant data from the authors. We excluded studies conducted in children (age <18 years), pregnant women, or individuals with confounding chronic conditions such as chronic kidney disease or heart failure.Study quality was assessed independently by two reviewers based on the five domains defined by the Cochrane Collaboration’s tool for assessing risk of bias version 5.0.1One author (LH) extracted all data and two authors (KT and SY) reviewed the data for accuracy. Data sought for extraction included: characteristics of the study; demographics of the participants ; study design ; risk of bias; duration of the intervention ; 24 hour urinary sodium and blood pressure at baseline; and intervention effect on 24 hour urinary sodium and blood pressure.For studies that only reported results as subgroups , we obtained overall estimates as weighted averages based on the size of the subgroups for the primary analysis, following the Cochrane Handbook for Systematic Reviews of Interventions.Where available, data were extracted for subgroups defined by age, sex, ethnic group, and presence or absence of hypertension. We used casual blood pressure measurements rather than 24 hour ambulatory blood pressure if both were reported, and supine blood pressure was used ahead of standing blood pressure. In crossover studies, the last measurement of 24 hour urinary sodium and blood pressure at the highest sodium intake period was taken as the baseline.The intervention effects on the 24 hour urinary sodium, systolic blood pressure, and diastolic blood pressure, were extracted directly from the studies, if reported. If not, we calculated them in crossover studies as the differences between the end of lowest sodium intake period (intervention) and the end of the highest sodium intake (control) period. For parallel studies, we calculated these as the differences between groups in the change from baseline to the last follow-up measurement.Corresponding standard errors of each outcome were either extracted directly or calculated from standard deviations, confidence intervals, or exact P values following the Cochrane Handbook for Systematic Reviews of Interventions.2 statistic to examine the heterogeneity between trial results and used funnel plots and Egger’s regression test to detect potential publication bias.We used random effects meta-analysis, based on the DerSimonian and Laird method, to generate pooled estimates of the intervention effect on 24 hour urinary sodium excretion, systolic blood pressure, and diastolic blood pressure. We used the ITo explore the dose-response relation, we summarised the changes in systolic and diastolic blood pressure by categorising trials into five equal groups (quintiles) based on the change in 24 hour urinary sodium excretion. We plotted studies with more than two levels of dietary sodium intake in a connected line graph.We assessed the effect of intervention duration on changes in blood pressure by grouping trials into five categories . In addition, for studies with multiple follow-up measurements, we plotted the effects on blood pressure standardised to 50 mmol reduction of 24 hour urinary sodium against the duration of intervention in a connected line graph.Age, sex, ethnic group, baseline sodium intake, and baseline blood pressure have been previously identified as potential modifiers of the effect of sodium reduction on blood pressure.To further explore the effects of these variables on blood pressure response to sodium reduction, we conducted unadjusted meta-regression for change in 24 hour urinary sodium, duration of intervention, mean age, percentage of white ethnicity, percentage of female sex, mean baseline blood pressure, and mean baseline 24 hour sodium excretion. We also did multivariable meta-regression including all covariates except for baseline 24 hour sodium excretion, which showed strong collinearity with the change in 24 hour urinary sodium (r=0.83). The following steps were undertaken to minimise missing data in the meta-regression analyses: if the studies did not report the participants’ race, this was imputed based on the study country (66 data points); if the percentage of female participants was not reported, the mean percentage of female of all other studies was used (8 data points); if only the age range was reported, the mean age was estimated as the mean of the minimum and maximum (12 data points); if neither mean age nor age range were reported, the mean age of all other studies was used (3 data points); and if baseline blood pressure was not reported, the mean value of all other studies was used (2 data points).Short term studies are usually done using very restricted diets and could result in sudden large reductions in sodium intake that do not reflect medium or long term effects of sodium reduction on blood pressure.Patients and the public were not involved in the design and conduct of this review.The search identified 17 477 records. After screening titles and abstracts, we selected 462 publications for full text review, of which 329 were excluded for the reasons summarised in 2=99.1%) in 24 hour urinary sodium, −4.26 mm Hg  in systolic blood pressure, and −2.07 mm Hg  in diastolic blood pressure . The univariable meta-regression analyses restricted to longer trials and trials with smaller reductions in sodium excretion showed inverse associations between intervention duration and magnitude of systolic blood pressure reduction but these were not apparent in the corresponding multivariable meta-regression analyses  or appropriate allocation concealment . Only 41.9% (57/136) studies were double blinded and 52.9% (72/136) had inadequate blinding . Most studies had low rates of loss to follow-up and there was low risk of selective outcome reporting . Although the information for assessing individual study quality was limited, the overall evidence should be considered of fairly high quality since only randomised trials were included and only a small proportion of studies had significant missing data.Egger’s regression test suggested asymmetry of funnel plots for both systolic blood pressure change (P<0.001) and diastolic blood pressure change (P=0.005) (supplementary figure 4). Use of the trim and fill method did not change the results and the contour enhanced funnel plots did not suggest under-reporting of studies with less statistical significance (supplementary figure 5). Asymmetry of the funnel plots is more likely to arise from other differences in study characteristics.This meta-analysis shows that sodium reduction leads to a significant reduction in systolic blood pressure in adults, both female and male, all ethnic groups, and in both hypertensive and normotensive populations. Diastolic blood pressure also decreased significantly in most participant subgroups. There was a dose-response relation with a greater reduction in sodium intake producing a greater fall in blood pressure. Populations with older age and higher baseline blood pressure achieved greater blood pressure lowering from the same amount of sodium reduction and so did non-white compared with white populations.Overall, the duration of the sodium reduction intervention was not associated with the size of the change in blood pressure, although short term studies of less than 15 days’ duration appear to underestimate the effect of sodium reduction on blood pressure. With few long term studies available, additional research is required to draw a definitive conclusion about whether prolonged sodium reduction influences the magnitude of the blood pressure lowering.2 statistics for heterogeneity reduced to 24.9%. The use of study level data rather than individual participant data greatly reduced the power of the analyses, although this limitation was somewhat offset by the large number of studies available. Different studies defined hypertension using different criteria and there was limited capacity to quantify the effects of pharmaceutical treatments on baseline diagnoses of hypertension or baseline blood pressure measurements. Finally, our use of random effects meta-analysis has resulted in wider confidence intervals but might better reflect uncertainty about the true constancy of effects across included trials.This meta-analysis represents a substantial update and enhancement compared with previous overviews. The selective inclusion of studies that used 24 hour urine collections to estimate intervention effect on sodium intake reduced the risk of bias, while the inclusion of studies with concomitant interventions applied in the same way to both intervention and control arms maximised the available data and the statistical power of the analyses. Likewise, the inclusion of studies regardless of the length of the duration of intervention enabled a robust and powerful examination of the effects of intervention duration on outcomes. The extensive sensitivity analyses provided for a full understanding of the strengths and weakness of the findings. There were, however, limitations in regard to our capacity to assess the quality of the studies. There was substantial heterogeneity across the included studies, but this was largely explained by some the of study characteristics in the meta-regression analyses. When the analyses were limited to studies with duration of more than 14 days and a sodium reduction of up to 100 mmol, the IThe overall effect of sodium reduction on blood pressure has been observed in several previous meta-analyses, despite different trial selection criteria.Previous overviews have generated uncertainty regarding the effects of sodium reduction among individuals with different levels of starting blood pressure. Some reports have suggested much larger effects in hypertensive individuals compared with non-hypertensive individuals,Analyses that simply separate studies based upon those that included hypertensive, non-hypertensive, or mixed populations are weak because the definition of hypertension is arbitrary and there is a rationale for expecting a graded interaction between sodium reduction, blood pressure reduction, and starting blood pressure. In the present analysis, meta-regressions based on mean starting blood pressure levels of participants in each study provided for a much more nuanced evaluation of the effects of starting blood pressure on the size of the blood pressure fall achieved with sodium reduction. These analyses showed that sodium reduction produced a progressively greater reduction in blood pressure among those with higher starting blood pressure levels, but also that sodium reduction substantially lowered blood pressure, even among those with starting systolic blood pressure levels as low as 120 mm Hg. These findings indicate potentially important health benefits from sodium reduction among normotensive as well as hypertensive individuals. More importantly, sodium reduction among normotensive individuals could potentially avert or delay the development of hypertension with ageing as the association between sodium intake and blood pressure is greater at older age.The differential blood pressure lowering effect of sodium reduction across different ethnic groups has been observed in various studies and meta-analysesThe findings from this overview of randomised trials conflict directly with findings from the Prospective Urban Rural Epidemiology (PURE)The optimal method to assess the impact of duration of sodium reduction on the magnitude of blood pressure reduction would be to collate data from studies that measure change of sodium and change of blood pressure at multiple time points. In practice, however, most studies made measurements only once at completion of follow-up. Neither the univariable nor the multivariable meta-regressions identified an effect of intervention duration on the size of the blood pressure fall achieved with sodium reduction. The power of the analyses was strengthened by the wide range of intervention durations recorded (from three days to five years) but limited by the highly skewed distribution of the studies, with most being of short or very short duration. Among the 133 studies included, 77 were shorter than 15 days’ duration and only five extended beyond six months. The DASH sodium trial that assessed effects at four time points over one month showed with some rigour that blood pressure effects from sodium reduction were clearly greater at week 4 compared with earlier weeks.Sodium reduction resulted in lower blood pressure among a very broad group of populations with a strong dose-response relation between the magnitude of the sodium reduction achieved and the magnitude of the fall in blood pressure. The effects of sodium reduction were more evident at higher starting blood pressure levels, older ages, and among non-white populations, but almost every population group examined achieved a reduction in blood pressure. In trials of more than two weeks’ duration, the dose-response relation between sodium reduction and blood pressure fall was greater than that in trials of shorter duration, but there was limited evidence that interventions of longer duration further increased the effects of sodium reduction on blood pressure. Longer term trials that achieve sustained sodium reduction and make multiple assessments of blood pressure are required to properly assess this issue.An extensive body of evidence has shown that a higher level of dietary sodium intake is associated with a higher blood pressure There are clear effects of sodium reduction on blood pressure in those with hypertension, but uncertainty persists about the comparability of effects in different population subsets. In addition, the impact of intervention duration is not fully understoodEvidence shows that sodium reduction lowers blood pressure in both hypertensive and non-hypertensive individuals, with greater effects in high risk subsetsThe magnitude of blood pressure lowering achieved with sodium reduction showed a dose-response relation Very short term trials could substantially underestimate the effect of sodium reduction on blood pressure


This study aims to evaluate the risk factors and the association of acute kidney injury with treatments, complications, outcomes, and resource utilization in patients hospitalized for heat stroke in the United States. Hospitalized patients from years 2003 to 2014 with a primary diagnosis of heat stroke were identified in the National Inpatient Sample dataset. End stage kidney disease patients were excluded. The occurrence of acute kidney injury during hospitalization was identified using the hospital diagnosis code. The associations between acute kidney injury and clinical characteristics, in-hospital treatments, outcomes, and resource utilization were assessed using multivariable analyses. A total of 3346 hospital admissions were included in the analysis. Acute kidney injury occurred in 1206 (36%) admissions, of which 49 (1.5%) required dialysis. The risk factors for acute kidney injury included age 20–39 years, African American race, obesity, chronic kidney disease, congestive heart failure, and rhabdomyolysis, whereas age <20 or ≥60 years were associated with lower risk of acute kidney injury. The need for mechanical ventilation and blood transfusion was higher when acute kidney injury occurred. Acute kidney injury was associated with electrolyte and acid-base derangements, sepsis, acute myocardial infarction, ventricular arrhythmia or cardiac arrest, respiratory, circulatory, liver, neurological, hematological failure, and in-hospital mortality. Length of hospital stay and hospitalization cost were higher in acute kidney injury patients. Approximately one third of heat stroke patients developed acute kidney injury during hospitalization. Acute kidney injury was associated with several complications, and higher mortality and resource utilization. The impact of heat stress has become an important issue as the average global temperature has increased. Heat stroke is the most severe form of heat-related illness. It is characterized by a core body temperature of more than 40 °C, combined with hot, dry skin and alteration of the central nervous system . Heat stRapid and effective cooling, along with supportive measures, are the standard treatment of heat stroke. The targets of cooling are to reduce the body core temperature to 39 °C within 10–40 min and to 38.5 °C or below within 2 h [Acute kidney injury is one of the most common complications of heat stroke; previous studies demonstrated that it is more common in exertional heat stroke ,7. The pAlthough acute kidney injury has been known as a common complication of heat stroke for several years, knowledge of its impact on patient outcome is limited due to a lack of large cohort studies. We conducted this study to assess the risk factors and association of acute kidney injury with treatments, complications, outcomes, and resource utilization in patients hospitalized for heat stroke in the United States.This cohort study was conducted using the National Inpatient Sample (NIS) database. The NIS is the largest all-payer inpatient database in the United States. Discharge datasets from a 20% stratified sample of hospitals in United States with patient encounter-level information are recorded in the NIS. The dataset includes primary and secondary diagnosis codes as well as procedure codes in the form of the International Classification of Diseases, Ninth Revision (ICD-9). Sample weight is used to generate national estimates for hospitalization nationwide. Approval from the institutional review board was exempted, as the information was obtained from a deidentified public database.All hospitalized patients with the primary ICD-9 diagnosis code for heat stroke (992.0) were included. Patients with ICD-9 diagnosis codes for chronic kidney disease stage 5 (585.5) and/or end stage renal disease (585.6) were excluded. In addition, patients with the ICD-9 procedure code for dialysis (39.95) but without that for acute kidney injury (584.×) were excluded, because these patients were assumed to have received renal replacement therapy due to end stage renal disease.Acute kidney injury was identified by the presence of ICD-9 diagnosis codes 584.5 (acute kidney failure with lesion of tubular necrosis), 584.6 , 584.7 , 584.8 , or 584.9  in any of the listed diagnoses. The identification of acute kidney injury using the ICD-9 diagnosis codes has a specificity of 98% but a sensitivity of 17%, and is likely to capture a more severe spectrum of acute kidney injury, compared with KDIGO serum creatinine-based criteria ,11. DialPatient characteristics, treatments, complications, and outcomes during hospitalization were similarly identified using ICD-9 codes . Patientp-value was <0.05. SPSS statistical software  was used for all analyses.The total number of heatstroke patients was estimated using discharge-level weights. The clinical characteristics associated with acute kidney injury were identified using multivariable logistic regression with forward stepwise selection. The association of acute kidney injury with treatments, complications, and outcomes was evaluated using logistic regression analysis, and with length of hospital stay and hospitalization cost using linear regression analysis, with prespecified adjustment for the clinical characteristics reported in There were 3372 admissions with a primary diagnosis of heat stroke during the study period. After 26 end-stage renal disease patients were excluded, a total of 3346 admissions were included in the study. Of these, 1206 (36%) had acute kidney injury, including 49 (1.5%) and 1157 (34.6%) who did and did not require dialysis in hospital, respectively. Patients with acute kidney injury had greater need for invasive mechanical ventilation and blood component transfusion than patients without acute kidney injury. Acute kidney injury was significantly associated several electrolyte and acid-base derangements, sepsis, acute myocardial infarction, ventricular arrhythmia or cardiac arrest, respiratory failure, circulatory failure, liver failure, neurological failure, hematological failure, and in-hospital mortality .Among 49 patients with dialysis-requiring acute kidney injury, the mean age was 40 ± 16 years, and 90% were male; 45% were Caucasian, 33% were African American, and 12% were Hispanic. The in-hospital mortality was 2.7% in patients without acute kidney injury, 7.9% in patients with nondialysis-requiring acute kidney injury, and 38.8% in patients with dialysis-requiring acute kidney injury. Nondialysis-requiring and dialysis-requiring acute kidney injury was significantly associated with increased risk of in-hospital mortality, with odds ratios of 3.50 (95% CI 2.45–5.01) and 19.77 (95% CI 9.94–39.33), respectively.The mean length of stay in acute kidney injury patients was two days longer than for nonacute kidney injury patients. The mean hospitalization cost for patients with acute kidney injury was significantly higher than that of patients without acute kidney injury, with an additional adjusted mean of $26,974 .Our large cohort of heat stroke patients demonstrated an incidence of acute kidney injury of 36%, and acute kidney injury requiring dialysis of 1.5%. Chronic kidney disease, rhabdomyolysis, obesity, male gender, congestive heart failure, young age, and African American race were factors associated with a higher risk of developing acute kidney injury. The impact of acute kidney injury on heat stroke was significant. The mortality rate in acute kidney injury patients was higher, particularly in patients requiring dialysis. Furthermore, acute kidney injury was associated with several complications, including metabolic derangements, sepsis, ventricular arrhythmia, cardiac arrest, acute myocardial infarction, and end-organ failures. Finally, acute kidney injury led to longer hospitalization and higher hospitalization costs.Acute kidney injury is a common complication in heat stroke patients. Previous studies reported a high incidence of acute kidney injury in heat stroke, ranging from 35 to 60% ,13,14,15The impact of acute kidney injury on outcomes in heat stroke patients is considerable. It increases the mortality rate of heat stroke patients, particularly for those requiring dialysis. However, the mortality rate in renal failure patients is comparable between heat stroke and general hospitalized patients . Acute kFurthermore, we found that acute kidney injury was also associated with liver failure. Kidney–liver crosstalk has been proposed as an explanation for this association. Several complications of renal dysfunction, including metabolic acidosis, azotemia, hyperphosphatemia, and inflammatory cascades, might interfere with the function of the liver and result in liver dysfunction. In addition, acute kidney injury also had crosstalk with other remote organs associated with multiorgan failures . The indIt is widely known that baseline renal function is an independent risk factor for acute kidney injury. A lower baseline eGFR was associated with a higher risk of acute kidney injury . The assSome limitations of our study need to be considered. First, NIS is an inpatient-only database. Therefore, we could not evaluate long-term outcomes, such as the recovery from acute kidney injury. Second, we could not conclude whether acute kidney injury was the cause or consequence of other complications in heat stroke patients. Third, the diagnosis of acute kidney injury in this study was only based on ICD codes, and not on kidney function parameters  . The diaIn conclusion, acute kidney injury is one of the most common complications of heat stroke, resulting in worse patient outcomes. The impacts of acute kidney injury include mortality and other complications. Baseline kidney dysfunction, obesity, male gender, congestive heart failure, young age, and African American race are factors associated with a higher risk of developing acute kidney injury.


It is a multiform event resulting from the hydrodynamic action of a complex fluid that exerts force on a compliant vascular wall, with elastic properties governed by multiple forces arising from vascular structures . This vector wave of blood follows the laws of sympathetic and parasympathetic interaction, varying more or less depending on the conditions of each patient. Therefore, the simple measurement of blood pressure in the office does not reflect the reality of the patient's true daily life.2The several measurements obtained during the 24-hour period have a better meaning in the global context evaluation and can discriminate the blood pressure behavior in the different phases of the day and, especially, during sleep. Ambulatory blood pressure monitoring (ABPM) has a better correlation with all cardiovascular events compared to simple office measurements. The pressure varies widely during the 24-hour period, and during sleep, in individuals considered normal, there is a reduction between 10 and 20% of daytime values. In some studies, the absence of this physiological dipping during sleep, which we call “non-dipper”, shows a positive association with an increase in target-organ damage and mortality.5 In clinical practice, hypertensive patients with the non-dipper pattern usually have some other comorbidity, leading to the suspicion of secondary causes for blood pressure elevation and/or obstructive sleep apnea.This concept that hypertensive individuals (as well as normotensive ones) who do not have a physiological pressure dipping during sleep (non-dipper), are subject to a worse prognosis has been a consensus for some time, well evidenced in several studies; however, the intrinsic mechanisms that lead to a higher risk are not yet fully known.6The usefulness of ABPM is quite broad. The European guideline for hypertension very objectively suggests performing out-of-office blood pressure measurements, supplemented with in-office measurements for diagnosing hypertension.The phenomenon of white coat hypertension and the white coat effect are common and may lead to case wrong conduct and management. In the first, there is an erroneous diagnosis of hypertension and in the presence of the white coat effect, one can start the medication incorrectly, with harm to the patient. These are situations where ABPM is critical for the diagnosis.7Another important and not infrequent aspect is masked hypertension, where the patient has normal office pressure values and levels outside the office are elevated. This situation can be correctly assessed only by out-of-office measurements and represents a huge danger, as target-organ damage is more intense and occurs earlier.8The assessment of out-of-office BP can also be attained by the much more inexpensive and affordable home blood pressure measurement (HBPM), which shows a good correlation with ABPM measurements. However, it does not evaluate BP during sleep, the fundamental period of the biological cycle. That is why ABPM remains the gold standard for out-of-office measurements.9The assessment of sleep, of the phenomena that occur before waking up and after awakening are also very important for a more precise stratification of the risk of cardiovascular events. Kazuomi Kario was one of the pioneers in assessing the effect of arousal on cardiovascular risk, finding greater target-organ damage in those with more intense and sustained blood pressure response.10 found that patients who experienced BP increases during sleep rather than the physiological reduction were more subject to cardiovascular events. Studying a population of 828 patients through brain imaging tests, such as MRI, they found that elevated systolic pressure during sleep was associated with subclinical brain disease.10Nakanishi et al.11 they found that patients who had aortic arch calcification on routine chest X-ray showed an association with the non-dipper pattern in ABPM.11 The authors started by detecting a target-organ lesion, aortic atherosclerosis, in a routine exam to look for changes in blood pressure physiology and eventually diagnose or correct other problems.In the study by Adar A. et al.,This association is important, because we can verify the entire aortic arch calcification in the search for non-dippers and, thus, better typify the behavior of arterial hypertension.Chest X-ray, used in many clinical situations, is an inexpensive, practical exam and, when performed for some other reason, it can also be used to screen for non-dippers, as demonstrated in this study. This may be beneficial to the patient, since secondary causes of hypertension, including obstructive sleep apnea, are frequent.12 Moreover, it also participates in the autonomic regulation with greater accentuation of the parasympathetic system with direct vasodilating effects, thereby reducing blood pressure.13The pathophysiological justification of this association, between the aortic artery calcification and the non-dipping of pressure during sleep, remains unknown. The authors mention the association of this calcification with pineal gland calcification and melatonin reduction, which plays an important role in sleep regulation.The finding of calcification in the aortic arch allowing the diagnosis of the presence of atherosclerosis immediately changes the cardiovascular risk, wherein clinical care should be more intense and thus improve our clinical practice.15Although there are not robust clinical trials evaluating chromotherapy and the influence of antihypertensive drugs on the non-dipper pattern yet, it is sensible and intuitive that this should be done, when there is no objective cause for no reduction of blood pressure during sleep. In hypertensive patients, the change in the time of medication may change the pattern of nocturnal dipping and possibly benefit the patient.An adequate BP control in the early hours of the morning means the use of drugs with adequate 24-hour coverage; however, some medications do not provide this effect. Changing the time when the patient takes the drug, particularly to the afternoon, may result in a pressure behavior closer to the normal circadian rhythm.We still have a long road ahead to assess blood pressure behavior, but some light is already emerging to unravel the mechanisms implicated in this complex web of hypertensive disease etiology.


The most accurate prognostic approach for follicular lymphoma (FL), progression of disease at 24 months (POD24), requires two years’ observation after initiating first-line therapy (L1) to predict outcomes. We applied machine learning to structured electronic health record (EHR) data to predict individual survival at L1 initiation. We grouped 523 observations and 1933 variables from a nationwide cohort of FL patients diagnosed 2006–2014 in the Veterans Health Administration into traditionally used prognostic variables (“curated”), commonly measured labs (“labs”), and International Classification of Diseases diagnostic codes (“ICD”) sets. We compared performance of random survival forests (RSF) vs. traditional Cox model using four datasets: curated, curated + labs, curated + ICD, and curated + ICD + labs, also using Cox on curated + POD24. We evaluated variable importance and partial dependence plots with area under the receiver operating characteristic curve (AUC). RSF with curated + labs performed best, with mean AUC 0.73 (95% CI: 0.71–0.75). It approximated, but did not surpass, Cox with POD24 (mean AUC 0.74 [95% CI: 0.71–0.77]). RSF using EHR data achieved better performance than traditional prognostic variables, setting the foundation for the incorporation of our algorithm into the EHR. It also provides for possible future scenarios in which clinicians could be provided an EHR-based tool which approximates the predictive ability of the most accurate known indicator, using information available 24 months earlier. Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma , accountSpecific patient-, disease-, and environment-related variables that can serve as prognostic factors in oncology play a pivotal role in understanding disease trajectory, designing clinical trials, making treatment decisions, and providing individual patients with accurate information about their health risks . The mosIn recent years, disease progression within two years following initiation of first-line (L1) treatment with chemoimmunotherapy (aka progression of disease within 24 months [POD24]) ,15 has rEHR systems, now widespread, could theoretically automatically extract and calculate prognostic indices and present the resulting risk prediction to practicing physicians. Absent the need to provide for easy recollection and calculation by clinicians, EHR-based indices could instead be optimized for predictive performance and ease of extraction. Such an optimization would favor the use of structured or semi-structured information over information presented in unstructured narratives.For survival analysis in oncology, the traditional Cox proportional hazard model investigates the relationship between survival time and one or more variables. Although well established and easily interpretable, the Cox model carries key limitations. It must satisfy linearity and proportional hazards assumptions and can be applied only to a restricted number of predictors, thus limiting its ability to account for interactions between variables. Compared with the Cox model, machine learning can better manage large numbers of predictors—even handling cases with more predictors than observations—and can account for both non-linearity and variable interactions . The ranMachine learning techniques have been widely used in cancer research ,24,25. HIn this study, we applied RSF to predict FL patients’ survival and risks using EHR data from a cohort of patients treated within the Veterans Health Administration (VHA). Our findings supported our hypothesis that this method would outperform traditional approaches and achieve predictive performance similar to that of POD24. Our results provide clinicians with insights into the relative importance of traditional and new variables in predicting FL patients’ risk.This research was approved by the University of Utah Institutional Review Board (#00083982) and the VA Salt Lake City Human Research Protection Program.Using data from the Veterans Affairs (VA) Cancer Registry System and pharmacy dispensation records from the VA Corporate Data Warehouse, we identified a nationwide cohort of patients with grade 1–3a, stage II–IV FL diagnosed from 1 January 2006 to 31 December 2014 in the VHA, who received any of three widely used L1 therapies—rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone; rituximab combined with cyclophosphamide, vincristine, and prednisone; or bendamustine combined with rituximab . Table 1n = 28) or died (n = 61) from causes unrelated to their disease relapse, to the best of our knowledge. The dataset included 523 patients, with 1933 variables. Median follow-up time was 5.03 years, and median survival time 9.32 years. At five years, 150 (28.68%) patients had died, 111 (21.22%) patients had been administratively censored at the end of the study observation period, and 262 (50.10%) patients were alive. Within two years, 99 patients (22.81%) experienced progression of disease (POD24). As in Casulo et al. ). Given the hazard function, survival function in terms of time, the probability that an observation survives beyond a specific time, can be obtained. The model parameters are estimated through optimization of the cox partial likelihood.The Cox model estimates the hazard function t given covariates The hazard at time F is the set of indices of failures, R is the set of indices of individuals at risk at time t, and t.From (1), we can also obtaini, with covariate vector For individual risk prediction in Cox model, the estimated hazard for individual i at time t is then is calculated with the following formula:t )  and withIn this study, we sought to compare the predictive performance of an RSF model versus a Cox model on datasets including traditional curated variables  along with higher-dimensional data  from the EHRs of a population of patients with FL. We also compared performance of the RSF model against the most accurate known indicator of FL risk, POD24. Note that this comparison is inherently biased towards POD24 since our model considered information available at the time of L1 treatment initiation, while POD24, based on progression-free survival in the 24 months following L1 treatment initiation, incorporates information about the disease course a full two years after our model’s prediction is made.We demonstrate that a limited set of EHR-extractable variables can provide similar performance to those generated from clinical trials . While tOur findings show that a broader set of data extracted from the EHR can improve prediction performance. However, not all data achieved such an effect. ICD codes added relatively little to prediction performance. On the other hand, lab values, available in EHRs but not other claims-based healthcare databases, substantially improved prediction. These results suggest that studies of the utility of EHR data in predicting clinical endpoints should emphasize EHR information that reflects the biology of patient and disease and deemphasize administrative data.In addition, while our model included disease-specific variables commonly considered a reflection of disease risk , the variable importance analysis identified none of these variables as being among the most useful variables for prediction. Instead, the top variables reflected age, along with kidney, liver, and marrow function. This finding suggests that change in these variables may serve as the best indicator of disease risk, host fitness, and the host’s ability to tolerate the stresses introduced by the disease and its associated treatments. The correlation between age and survival probability in our model corroborates findings that a model including age in three categories (≤60 years old/61–70 years old/>70 years old) improves the predictive performance of FLIPI .The best-performing combination of model and data we found, RSF applied to the curated + labs dataset, could not outperform POD24, although we found no significant difference between the AUCs of the two models. We report a relative risk for the predicted high- vs. low-risk groups using our best model of 4.39, lower than the previously reported risk ratio of POD24 vs. reference group of 6.44 . HoweverThere are several limitations in this study. First, the majority of VHA patients are male, limiting generalizability of our results to a subset of the population of patients with FL. However, we implemented our methods in a nationwide cohort using structured EHR data commonly available in any EHR system, making it feasible to widely adopt our methods to automatically predict individual patient risk at the beginning of L1. Secondly, we limited our analysis to variables available in structured format; we did not include unstructured data in radiology reports, which might improve prognostic ability. With advances in precision medicine, genetic profiling and deep sequencing of tumors could likely become a widely adopted standard of care. Such information would probably improve prognosis as well. Lastly, we did not include deep learning approaches in our preliminary comparison of machine learning methods due to their computational cost and the need for specialized hardware.RSF using EHR data achieved better performance than traditional prognostic variables. Our method sets the foundation for the incorporation of our algorithm into the EHR. It also allows for possible future scenarios to provide clinicians with an EHR-based tool which approximates the predictive ability of the most accurate known indicator, using information available 24 months earlier in the disease trajectory.


Excess aldosterone has been shown to be associated with obesity; however, there is currently a lack of data regarding the relationship between percentage of body fat and primary aldosteronism (PA), particularly pertaining to Asian populations. Furthermore, essential hypertension may mimic the condition of PA and there needs to be differentiation between the two. This study aimed to assess the association between percentage of body fat and PA.A cross-sectional study was conducted in the outpatient department of the Endocrine and Metabolism Unit of the tertiary care medical center in Thailand. Data was obtained from 79 patients who had been screened for PA due to hypertension in young-onset, hypokalemia, adrenal incidentaloma or resistance hypertension. Essential hypertension was defined as patients who had high blood pressure and were negative for PA screening. Body fat percentage was assessed by bioelectrical impedance analysis. The relationship between percentage of body fat and a diagnosis of PA was assessed using logistic regression analysis, including adjustment for confounding factors.n = 41) and an essential hypertension group (n = 38). After controlling for confounding variables , the odds ratio of having PA in males with a percentage of body fat > 25% and females with percentage > 30% was 1.82 .The participants were divided into a PA group (A higher percentage of body fat is associated with an increased risk of PA. Further studies need to be conducted to confirm the relationship between body fat percentage and PA. Overweight and obesity have been reported to be strongly associated with hypertension . A stronThe most common form of secondary hypertension is primary aldosteronism (PA) which has a reported prevalence in the general population of > 10% . The preThe established criteria for detection of possible PA is ratio between plasma aldosterone concentration (PAC) and plasma renin activity (PRA) known as the ARR. An ARR of more than 20 to 30 ng/dl per ng/(ml·h) is an indication of possible PA . AldosteThe objective of this study was to assess the association between percentage of body fat and primary aldosteronism in both men and women.2), Cushing’s syndrome, metastatic cancer, heart failure, or patients who used glucocorticoids.This cross-sectional study was conducted at the outpatient Endocrine and Metabolism Unit of a tertiary medical center in the northern part of Thailand from September 2018–August 2019. The project protocol was approved by the Faculty of Medicine, Chiang Mai University Ethical Board (No.337/2018). We adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement. Seventy-nine patients aged over 18 and suspected of having PA were included in this study. The indications for PA screening were based on the Endocrine Society Clinical Practice Guidelines which included hypertension in the young, hypokalemia, adrenal incidentaloma or resistance hypertension [Screening for PA was performed according to the Endocrine Society 2016 standard guidelines . In addi2). Waist circumference was measured midway between the lowest rib and the iliac crest in the horizontal plane with the patient at the end of a normal breath expiration.Weight was measured using a standardized digital scale with patients dressed in light clothing without shoes. A wall stadiometer was used to measure standing height of the patients without shoes. Weight and height were recorded to one decimal place. BMI was calculated as weight (kg)/height  was used to assess bioelectrical impedance. Measuring current was standardized at 50 Hz; the age, sex and height of the patients were input manually. The patients were instructed to wear light clothing, stand barefoot and remain still while being measured. The percentage body fat was computed using the software installed in the machine. The contact electrode system was validated against dual-energy X-ray absorptiometry (DEXA) measurements with good correlation for fat mass .PAC was measured using direct ELISA assays  with a reference range in the upright position of 3–40 ng/dL and in the supine position of 2–18 ng/dL. The intra-assay variability was < 9.7% and the inter-assay variability was < 11%. PRA was determined using direct ELISA assay  with a reference range of 0.06–4.69 ng/mL/hr. For assessment of insulin, electrochemiluminescence immunoassay  was employed with reference values of 2.6–24.9 μU/mL and with intra- and inter-assay coefficients of variation for serum insulin of < 10%.A high percentage body fat was defined as > 25% in males and > 30% in females . A diagnp < 0.05.Data were analyzed using STATA . Categorical variables are presented as numbers and percentages, continuous variables as mean values and SDs. Comparison between categorical variables and PA was done using the Fisher’s exact test; in the case of continuous variables, the Student t-test was used for normally distributed variables and the Mann-Whitney U test for non-normally distributed variables. Multivariable logistic regression analysis was used to assess the association between percentage body fat and PA. The data are presented as crude and adjusted odds ratios (OR). The model was adjusted for age, BMI, serum cholesterol and HOMA-IR level and clustered by sex. Statistical significance value was set at . Data are categorized by results of PA screening which were PA and essential hypertension (negative result of PA screening). The mean age of the study subjects was 38.4 ± 14.5 years; 51% were male. Those with PA were significantly older than those without (p = 0.04). The majority of the males were in the group with PA. Average body fat percentage was higher in males than in females, but there was no significant difference in body fat percentage between the PA and essential hypertension groups . The most common indication for PA screening was hypertension diagnosed at age <40; the majority of screened patients were negative for PA. With the exception of serum triglycerides, there were no significant differences between groups in the case of any of the demographic, biochemical or anthropometric characteristics measured.Demographic, anthropometric and biochemical characteristics of the 79 patients are shown in Table 1p < 0.001. After adjusting for confounding variables  in the multivariable analysis, the OR of a PA diagnosis was 1.82, 95%CI (1.79–1.86), p < 0.001 , This study highlights that a higher percentage body fat in hypertensive patients is significantly associated with an increase in risk of PA by a factor of 1.82. This finding suggests that physicians should consider screening for PA in hypertensives with a high percentage of body fat.The study also identified some indirect evidence pertaining to the relationship between body fat percentage and the renin-angiotensin-aldosterone system. A prior study reported a correlation between PAC and obesity status in non-PA patients. In that study, PAC was found to be significantly higher in obese hypertensive than in non-obese normotensive patients . Rossi eOne study in PA patients reported a U-shaped correlation between PAC and BMI . However2+ levels and can lead to CYP11B2 expression which helps regulate adrenal aldosterone production [The results of the present study suggest a possible interaction between adipose tissue and the production and/or secretion of aldosterone. A study in an animal model proposed that adipose tisuues contain ARFs which can stimulate aldosterone synthesis and secretion in adrenocortical cells . On the oduction .In accordance with the Endocrine Society Clinical Practice Guidelines, obesity was not included as one of the indications for PA screening . HoweverStrengths of this study include that, unlike prior studies , this stThis study does have some limitations. First, bioelectrical impedance was used to calculate body fat percentage. This method of measurement has not generally been considered the gold standard for measuring this value, so the results may not be as accurate in comparison with some other methods, e.g., dual-energy X-ray absorptiometry (DEXA). Additional validation is needed. Another limitation is that only members of the Thai population were included, so the results may not be representative of other Asian ethnic groups. Finally, the subtypes of PA  in this cohort were not determined by adrenal venous sampling due to resource limitation and some patients have chosen medical management over surgical management.There is a signficant relationship between higher body fat percentage  and PA. Physicians should be very aware of the potential for PA in those patients. Early screening tests, including for levels of plasma aldosterone and renin, should be performed, as delay in diagnosis can lead to negative outcomes which include congestive heart failure, myocardial infarction and chronic kidney disease.


Patients with chronic kidney disease (CKD) are at a high risk for cardiovascular disease (CVD), and approximately half of all deaths among patients with CKD are a direct result of CVD. The premature cardiovascular disease extends from mild to moderate CKD stages, and the severity of CVD and the risk of death increase with a decline in kidney function. Successful kidney transplantation significantly decreases the risk of death relative to long-term dialysis treatment; nevertheless, the prevalence of CVD remains high and is responsible for approximately 20-35% of mortality in renal transplant recipients. The prevalence of traditional and nontraditional risk factors for CVD is higher in patients with CKD and transplant recipients compared with the general population; however, it can only partly explain the highly increased cardiovascular burden in CKD patients. Nontraditional risk factors, unique to CKD patients, include proteinuria, disturbed calcium, and phosphate metabolism, anemia, fluid overload, and accumulation of uremic toxins. This accumulation of uremic toxins is associated with systemic alterations including inflammation and oxidative stress which are considered crucial in CKD progression and CKD-related CVD. Kidney transplantation can mitigate the impact of some of these nontraditional factors, but they typically persist to some degree following transplantation. Taking into consideration the scarcity of data on uremic waste products, oxidative stress, and their relation to atherosclerosis in renal transplantation, in the review, we discussed the impact of uremic toxins on vascular dysfunction in CKD patients and kidney transplant recipients. Special attention was paid to the role of native and transplanted kidney function. Patients with chronic kidney disease (CKD) are at a high risk for cardiovascular disease (CVD), and approximately half of all deaths among patients with CKD are a direct result of CVD. Premature cardiovascular disease extends from mild to moderate stages of CKD, and the severity of CVD and the risk of death increase with a decline in kidney function –3.Moreover, the nature and spectrum of cardiovascular disease in CKD are recognized to be different from that in people without kidney disease including atherosclerosis, arteriosclerosis, calcific arterial and valve disease, left ventricular remodeling and dysfunction, arrhythmia, and sudden cardiac death.Successful kidney transplantation significantly decreases the risk of death relative to long-term dialysis treatment . NeverthThe prevalence of traditional and nontraditional riskfactors for CVD is higher in patients with CKD compared with the general population; however, it can only partly explain such sorely increased cardiovascular burden in CKD patients , 6. Nont2 increase in eGFR is associated with a 15% reduction in cardiovascular disease and mortality [Kidney transplantation can mitigate the impact of some of these nontraditional risk factors, but they typically persist to some degree following transplantation. The restoration of renal function favorably modifies cardiovascular risk in transplant recipients, and each 5 ml/min/1.73 mSmall, water-soluble molecules with a maximum molecular weight (MW) of 500 Da which can be easily removed by dialysis; molecules in this group include, i.a., guanidines (asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA)), oxalate, methylamines (trimethylamine-N-oxide (TMAO)), polyamines, urea, carbamylated compounds, and purinesβ2-microglobulin, complement factors D and Ba, cytokines , endothelin, fibroblast growth factor-23 (FGF-23), modified lipids and lipoproteins, pentraxin-3, VEGF, and parathyroid hormoneMiddle molecules—small proteins or peptides with MW ≥ 500 Da, although most of them have MW > 10000 Da. They are often expressed in response to other toxins , and their concentration depends both from retention and on endocrine and paracrine mechanisms. Dialytic removal of middle molecules is possible with membranes with a large enough pore size used in either diffusive or convective mode. Compounds in this group include angiogenin, atrial natriuretic peptide (ANP), Protein-bound molecules—the heterogeneous group of generally low MW solutes, which due to their protein binding are difficult to remove by dialysis; many of these molecules are generated by the intestine microbiota; the main compounds in this group are advanced glycation end products (AGEs), cresols , hippurates, homocysteine, indoles , kynurenines, and phenols (phenylacetic acid) The progressive loss of kidney function is accompanied by the retention of plenty of metabolites, due to a decrease in their renal clearance and/or a rise in production. Many of these solutes have been shown to exert biological activity, thereby affecting the functioning of cells and affecting metabolic processes, resulting in the uremic syndrome. Generally, they may originate from endogenous metabolism, be produced by microbial metabolism, or be ingested from an endogenous source. According to the European Uremic Toxin Work Group (EUtox) organic uremic toxins are classified according to their physicochemical properties and possibilities of removal by dialysis :Small, Accumulating data suggest that uremic toxins contribute substantially to the development and severity of cardiovascular disease in CKD patients. Accumulating data suggest that atherosclerosis starts from early stages of CKD and remaining high as CKD progresses . CKD-relThe vascular toxicity of uremic toxins has been demonstrated in clinical studies among chronic kidney disease, dialysis, and kidney transplant patients. Decreased kidney function impacts the levels of these solutes and may be a relevant confounder when the association between uremic toxins and hard cardiovascular outcomes is studied. The factors potentially contributing to atherosclerosis in CKD patients are presented in Protein-bound uremic toxins (pbUTs)—p-cresyl sulfate (p-CS), p-cresyl glucuronide (p-CG), indoxyl sulfate (IxS), and indole-3-acetic acid (IAA)—originate from the metabolism of the intestinal microbiota of aromatic amino acids  –44. In tThe serum levels of pbUTs are inversely related to renal function, and the serum concentrations increase progressively with the progression of CKD in adults and pediatric CKD patients , 46–51. Few studies evaluated the levels of pbUTs in transplanted patients , 54–56. Serum levels of ADMA and SDMA are elevated in patients with CKD , 59. ForThe data on levels of ADMA and SDMA in renal transplant patients are scarce and somewhat inconsistent. Most often, plasma ADMA levels demonstrated a biphasic course after successful kidney transplantation with a transient rise in the immediate postoperative period followed by a subtle decline in the weeks; however, the change did not correlate with improvement of graft function. ADMA levels remained elevated compared with CKD patients, matched for age and comorbidities –64. A poAdvanced glycation end products (AGEs) are a heterogeneous group of compounds derived from the nonenzymatic glycation of proteins, lipids, and nuclear acids through a complex sequence of reactions referred to as the Maillard reaction . N-CarboAccumulation of AGEs in CKD patients is a result of oxidative stress and inflammation and comes from external sources such as diet and cigarette smoke , 73. AGEKidney transplantation is the most effective therapy to reduce elevated levels of AGEs. Nevertheless, in renal transplant recipients, AGEs remain higher than in normal subjects and disproportionally higher than the GFR alone would imply , 81. It α-Klotho, FGF23, 1,25-dihydroxyvitamin D, diet, and medications, interacting with each other in complicated ways.Abnormalities of mineral metabolism are universal complications of CKD associated with accelerated atherosclerosis and vascular calcification and correlated with increased mortality across all stages of CKD, independent of traditional risk factors –86. The α-Klotho not only functions as one of the regulators of mineral homeostasis but also exerts pleiotropic biological effects including antioxidative stress, antiapoptosis, and antiaging [α-Klotho is expressed in multiple tissues; however, the strongest expression is in the kidney [α-Klotho production. It has been shown that serum α-Klotho starts to decline in stage 2 CKD, and urinary α-Klotho even earlier, in stage 1 CKD [2 eGFR decrease, an adjusted mean decrease of 3.2 pg/ml of serum α-Klotho was revealed [α-Klotho expression [α-Klotho is positively associated with eGFR [ntiaging , 88. α-Ke kidney . Kidney ge 1 CKD , and forrevealed . Furtherpression . Clinicaith eGFR , 93, 94.α-Klotho [Fibroblast growth factor 23 (FGF23) is a bone-derived phosphatonin, which acts in the kidney to induce urinary phosphate excretion and suppress 1,25-dihydroxyvitamin D synthesis, in the presence of FGF receptor 1 (FGFR1) and its coreceptor α-Klotho , 96. It α-Klotho –99.α-Klotho to maintain mineral homeostasis, so in early stages of CKD, serum phosphates are not elevated. In turn, increased levels of FGF23 decrease α-Klotho expression, and finally, dietary phosphorus overload cannot be compensated and contributes to overt hyperphosphatemia in advanced stages of CKD [The increase in FGF23 is a compensatory reaction in response to decreased expression of transmembrane s of CKD . FGF23 ls of CKD . In Isaks of CKD  study, 3s of CKD . Moreoves of CKD , 101.Close to 90% of patients with 3-4 CKD stage have normal phosphate levels, and with the progressive loss of functional nephrons, the compensatory mechanism is overwhelmed, and most patients with ESRD have overt hyperphosphatemia. Hyperphosphatemia is considered to be a risk factor for cardiovascular and all-cause mortality, and for each 1 mg/dl increase in serum phosphate, the risk of death is increased by 18-20% , 103.α-Klotho and FGF23 in transplant recipients are scarce, and sometimes inconsistent. During the first week after kidney transplantation, the decrease in serum levels of α-Klotho was noted [α-Klotho was observed with the highest levels exhibited at 52 weeks posttransplantation and compared with pretransplant levels [α-Klotho levels and kidney function has not been demonstrated in Tan et al.'s study, as well as in three other cross-sectional studies [The data on levels of as noted , 105. Thas noted , 106. Int levels . However studies –109.FGF23 levels decline in the postrenal transplantation period; however, they remain higher than in CKD patients matched for eGFR , 110–113In up to 90% of transplant recipients, mild to moderate hypophosphatemia is present. Phosphate levels remain low for longer than in patients with CKD matched for the eGFR . Kidney κB, AP1, and SP1) executed via RAGE, and activation of NADPH oxidases (NOXs) which directly generate free radicals [Accumulation of AGEs activating transcription factors  is defined as a state of imbalance between excessive prooxidant activities relative to antioxidant defense mechanisms. Oxidative stress leads to metabolic dysregulation and oxidation of lipids, proteins, and nucleic acids and oxidative damage in cells, tissues, and organs caused by ROS and reactive nitrogen species (RNS) , 117. OSThe imbalance in oxidant-antioxidant status begins early in the course of CKD. It was shown that increased levels of NADPH-generated ROS and lower levels of the antioxidant enzymes can be revealed in patients with 1 and 2 CKD stage , 131–133Successful kidney transplantation leads to a reduction in metabolic abnormalities and significant improvement in OS-related markers. Normalization of graft function seems to be a key factor in the restoration to near-normal levels of OS biomarkers. Despite the fact that surgical procedure of kidney transplantation and ischemic injury during the procurement and organ transfer cause an oxidative burst, the improvement of OS can start immediately after transplantation . Sudden CKD-associated OS in pretransplant phase, reperfusion injury, and increased immunosuppression are considered the key factors of continual OS during the early phase of transplantation –153. OveMost studies have shown that reestablishment of kidney function improves the OS over few weeks after transplantation , 154–162In CKD, endothelial dysfunction and atherosclerosis are almost universal, as well as cardiovascular complications as first reported by Lindner et al. , who dreInflammation is considered one of the main mechanisms of atherosclerosis, and CKD is a state of systemic inflammation , 168. ItUremic toxins promote the production of ROS and decrease antioxidant defenses, resulting in oxidative stress , 127. ROUremic toxins contribute to structural damage of ECs resulting in increased endothelial permeability. In vitro studies demonstrated that uremic toxins (pbUTs and phosphate) induce cytoskeletal remodeling, resulting in the changes in EC morphology, and lead to the rupture of cell-cell junctions damaging endothelial barrier and contributing to increased permeability –175. EndUremic toxins also cause a reduction in the number and function of endothelial progenitor cells. Protein-bound UTs and AGEs suppress the expression of transcription factors, SIRT1 and KLF2, responsible for the maintenance of endothelial homeostasis, inhibiting oxidative stress and cell senescence , 185.Uremic toxins contribute to the prothrombotic state of endothelium leading to an increased risk of thrombotic events, such as thromboembolism and ischemia. Furthermore, in CKD, the processes of coagulation and fibrinolysis are impaired with increased levels of tissue factor (TF), von Willebrand factor (vWF), thrombomodulin, factor VIII, and D-dimer . In vitrEndothelial cell integrity and function are critical to the prevention of atherosclerosis; therefore, dysfunction of endothelium is critical in the development of vascular dysfunction and progression of CVD. Nevertheless, uremic toxins participate in atherosclerosis development in many steps. They influence proliferation, migration, calcification, and senescence of VSMC , 34, 35.It would be worth to mention that AKI contributes to the initiation and progression of CKD, and vice versa CKD predisposes to AKI –192. AKIIn CKD, the retention of a variety of metabolites, due to a decrease in their renal clearance and/or a rise in their synthesis, is found. These compounds could be small and water soluble, lipophilic and/or protein bound, or larger and in the middle-molecule range. Several solutes have been shown to exert biological activity, on cells and metabolic processes, leading to uremic syndrome. Moreover, dietary protein breakdown, alternative sources such as environmental contact, food additives, natural stimulants (coffee and tea), herbal medicines, or addiction to psychedelic drugs, may also play a role in uremic toxicity. Slowing of the progression of CKD thereby preservation of kidney function is crucial in the removal of uremic toxins. Successful kidney transplantation with good graft function offers the best possibility to lower the levels of uremic toxins. In addition, uptake of uremic toxins in the intestine could be decreased by influencing dietary uptake, oral administration of sorbents, or administration of prebiotics or probiotics influencing intestinal flora. Moreover, changing the source of protein intake from animal-based to plant-based diet may also reduce intestinal production of uremic toxins. Other therapeutic intervention includes administration of drugs countering the biological impact of uremic solutes such as angiotensin-converting enzyme inhibitors (ACEi) which neutralize Ca influx due to SDMA . Moreoveα-Klotho and lower phosphate, FGF23, and other uremic toxins is of great importance as they may contribute to the decline in cardiovascular morbidity and mortality in CKD and after kidney transplantation.In addition, the development of therapeutic strategies to raise


The objective of the study was to evaluate the relation between occlusal loading and peri-implant crevicular fluid cytokine expression in patients with implant-supported complete fixed prostheses in both arches.A prospective longitudinal clinical study was performed at a university clinic. Fifteen patients were selected and 11 were included. All patients had bimaxillary implant-supported complete fixed ceramo-metallic prostheses loaded at least 12 months before the beginning of the study. Allocation was established for each patient using a computerized occlusal analysis system. The test implant was the maxillary implant closest to the point of highest occlusal loading. The maxillary implant with least loading was the control implant. Occlusal adjustment was performed using a round diamond burr. This occlusal distribution was verified with the occlusal analysis system. Expression of cytokines from peri-implant crevicular fluid  were recorded and analyzed in both test and control implants before (baseline: T0) and 2 (T1) and 12 months (T2) after occlusal adjustment. The Brunner-Langer non-parametric test was performed.p = 0.018). Between T0 and T1, the expression of all the cytokines decreased in the implants of both groups with statistically significant differences, except for TNF (p = 0.271). When comparing both groups at T1, there was no statistically significant difference in any of the analyzed cytokines. At T2, TNF-α suffered when compared with baseline, a statistical decrease in both study and control implants . At T2, there were no statistically significant differences between groups in any of the cytokines analyzed.At T0, the expression of IL-10 was significantly higher in the test group implants (Implants with higher occlusal load presented higher expression of IL-10 in peri-implant crevicular fluid. Occlusal adjustment produced a decrease in the expression of all the analyzed cytokines, both in test and control implants. Occlusal forces affect the peri-implant bone. Mechanical stress can have both positive and negative consequences for bone tissue  and consWith animal experimental studies showing conflicting results, it is unclear whether occlusal overload might cause peri-implant marginal bone loss or total loss of osseointegration . While sIn a previous human study , our groSo, the aim of this prospective longitudinal study was to evaluate the relation between occlusal loading and peri-implant crevicular fluid cytokine expression in patients with implant-supported complete fixed prostheses in both arches.The study fulfilled Declaration of Helsinki principles for medical research involving humans. All patients gave their informed consent to take part, and the study was approved by the University of Valencia Ethics Committee (ref no. H1335344280712).This article is the second part of a prospective clinical study that took place at the Oral Surgery Unit at Valencia University  with a 1Patients had to fulfill the following inclusion criteria: adult patients (> 18 years) rehabilitated with fixed full-arch prosthesis loaded at least 12 months before the study and signature of informed consent document. The exclusion criteria were as follows: use of local or system antimicrobials 3 months prior to the study, smokers, pregnant or lactating women, patients with a history of bisphosphonate therapy, patients who had malignant diseases or other diseases treated with chemotherapeutic agents (“chemotherapy”) or head and neck radiotherapy during the past 5 years, severe bruxism, poor oral hygiene, incomplete data gathering, or failure to attend scheduled control visits.Test implant—maxillary implant closest to the point of highest occlusal loading.Control implant—maxillary implant closest to the point of least loading. In the case of finding 2 implants with least loading, the furthest from the study implant was selected as the control implant Fig. a.After implant loading, the occlusal adjustment was checked with 12 μm articulating paper; then, all the patients had a follow-up at 1 week; 1, 3, and 12 months; and once per year in order to control the occlusal distribution. Patients received rigorous professional prophylaxis with Teflon curettes and rotary instrument brushing, and instructions were given for improving and maintaining oral hygiene at home. Patients then underwent an occlusal analysis with a computerized system , where the patient makes mastication movements that activate a sensor placed between the dental arches, while the computer registers and processes the data. Occlusal contacts are represented on screen by topographic images that describe the shape of the contact areas, the relative force, the surface area, and the time sequence of occlusal contacts. Differences in occlusal loading are shown as color changes, ranging from red (high loads), graduating through the colors of the spectrum, to blue (low loads). It is important to underline that before the start of the study, patients’ comfort and chewing were correct. Occlusal adjustment did not produce changes in the chewing perception of the patient. Implants presented healthy peri-implant conditions as an inclusion criterion, so changes in cytokine expression would be due to the effect of occlusal loading. Through the occlusal analysis system, two implants per patient were established:One month after oral hygiene, at baseline, PICF from test and control implants was collected (T0), and occlusal adjustment was performed to distribute occlusal loading evenly over the whole arch. Occlusal distribution was verified with the T-Scan®III Fig. b, followThe following cytokine expression  was measured from the PICF. Cytokine expression between test and control implants was compared in each timepoint and between timepoints. Rigorous professional prophylaxis, as described above, was performed 4 weeks before every PICF collection.PICF was collected from the implants selected for study by inserting sterile paper strips . The technique consisted of air-drying the mouth, isolating the area with cotton wool rolls, and gentle drying of the implant area where the paper strip was to be placed. PICF sample collection was performed by inserting the Periopaper Strip® in the peri-implant sulcus for 30 s.g for 5 min, and the supernatant was stored at − 80 °C until use.Each sample was diluted in an Eppendorf tube , with 200 mL of 50 mM phosphate buffer, pH 7.2, together with a pool of protease inhibitors  and 0.1 mM phenyl sulfony l fluorate, and incubated for 2 h. The samples were centrifuged at 1000TNF-α, IL-10, IL-6, IL-1β, and IL-8 were evaluated in the supernatants stored at − 80 °C. The evaluation was performed using the Human Inflammation Cytometric Bead Array system  and cytofluorometry analysis . The samples and positive controls (standard curve) were processed according to the instructions of the manufacturer, and the values for TNF-α, IL-10, IL-6, IL-1β, and IL-8 were calculated and reported as picograms per milliliter. Data were acquired with a fluorescence-activated cell sorter Microbiology Calibur flowcytometer .p < 0.05.The statistical analysis used SPSS statistical software for Windows . The Brunner-Langer non-parametric test was performed. Statistical significance was established as Fifteen patients were preliminarily enrolled in the study. Four of them did not fulfill the inclusion criteria: one for failing to attend scheduled appointment, 2 for plaque scores > 25%, and one for using mouthwashes. This left 11 included patients, 4 women and 7 men, with a mean age of 58.4 years. Figure p = 0.018).Before occlusal adjustment , test group implants expressed higher concentration of IL-6, TNF-α, and IL-10, but significant differences were only found in IL-10 (p = 0.271). At this timepoint (T1), IL-10, IL-6, and IL-1β were highly expressed in study implants but there were no statistically significant differences in none of the cytokines between test and control implants.Two months after occlusal adjustment (T1), all the cytokines analyzed decreased when compared with baseline , in both test and control implants except for TNF-α that decreased without significant differences . At this timepoint (T2), IL-6, IL-1β, and IL-8 showed higher values in study implants, but there were no significant differences between groups in all the cytokines analyzed. TNF-α showed a tendency to significance with higher values in the test group (p = 0.085). Figure Twelve months after adjustment (T2), all the cytokines including TNF-α significantly decreased when compared with baseline, in both test and control implants  in patients with a peri-implant disease established. These would help to understand, together with clinical and radiological parameters, the influence of loading in the peri-implant tissues.The present study concludes that implants with higher occlusal load presented higher levels of IL-10 in peri-implant crevicular fluid. Occlusal adjustment produced a decrease in the expression of all the cytokines analyzed, both in test and control implants.


Supplemental Digital Content is available in the text. The study compares urine recirculation (URC) to urine replacement (UR) with Ringer’s lactate in a porcine normothermic kidney machine perfusion (NMP) model using a preclinical prototype device.Kidney pairs were recovered uninjured (as live-donor nephrectomy) and perfused consecutively. Pig kidneys (n = 10) were allocated to either NMP with URC (n = 5) or NMP with volume replacement (n = 5). Cold ischemia time was either 2 or 27 hours for the first or second perfusion (URC or UR) of a kidney pair. An autologous blood-based perfusate, leukocyte-filtered, was used and NMP performed up to 24 hours. Perfusion parameters, biochemistry/metabolic parameters were monitored and samples collected.P = 0.001). Perfusate sodium levels were lower with URC, 129.6 ± 0.7 versus 170.3±2.7 mmol/L, P < 0.001). Stable physiological pH levels were only observed with URC. Perfusate lactate levels were lower with URC . Furthermore, the hourly rate of urine output was lower with URC and closer to physiological levels . Normothermic kidney perfusion with URC was associated with longer achievable durations of perfusion: the objective in all experiments was a 24-hour perfusion, but this was not achieved in every case. The mean perfusions were 17.3 ± 9.2 hours with URC versus 5.3 ± 1.3 hours NMP with UR; P = 0.02. There appeared to be no differences in baseline tubular condition with and without URC.Physiological mean arterial pressures and flows were achieved in both groups but were sustainable only with URC. Significantly higher arterial flow was observed with URC (326.7 ± 1.8 versus 242.5 ± 14.3 mL/min, URC facilitates long-term kidney NMP in a porcine model. Perfusate homeostasis and stability of renal arterial flow throughout the perfusion period was only achievable with URC, independent of cold ischemia time duration. For the first time, urine recirculation (URC) was used to control perfusate volume and electrolyte homeostasis: this has the added advantage of facilitating volume replacement in what, ideally, should be developed as a stand-alone transportable perfusion device.13Normothermic kidney machine perfusion (NMP) provides possibly several potential advantages over both static cold storage and hypothermic machine perfusion. There is evidence from large animal studies that NMP may be valuable in the assessment and preservation/reconditioning of damaged kidneys before transplantation. Although there appears to be significant benefit in the short periods of NMP studied by the Nicholson/Hosgood group,The aim of the currently described study was to investigate NMP on uninjured porcine kidneys, with the added advantage of comparing URC and urine replacement (UR) in pairs of kidneys from the same donors. This would enable a direct comparison between URC and Ringer’s lactate replacement.13This study was performed at the Oxford University Biomedical Services, University of Oxford in accordance with the United Kingdom Animal Protection Act 1986; home office project license number is PPL30/3303. Kidney procurement was performed on 5 white female landrace pigs with a weight of 40–50 kg. The 10 kidney grafts recovered (open live-donor nephrectomy procedure) underwent normothermic perfusion at the Institute of Biomedical Engineering, University of Oxford, using the preclinical prototype device described previously.Both kidneys from each donor animal were perfused normothermically, with 1 kidney being perfused without URC (UR group) and 1 with URC (URC group). The 2 kidneys from each donor animal were perfused consecutively, with the second kidney being stored on ice until completion of the first perfusion, resulting in an NMP start after either 2 or 27 hours of cold ischemia time (CIT).The shorter duration (2 h) was based on the time needed from clamping the renal arteries in the donor to starting of the first perfusion . The second duration of ice storage was to allow to complete 1 perfusion and set up the second (there was only 1 perfusion system available). The allocation of kidneys into groups (UR and URC) alternated to ensure equality between the groups with respect to CIT.co2 level of 5; or (3) sudden cessation of urine production.The perfusion circuit was assembled and primed with 500 mL autologous whole blood from the same donor animal as the kidney, treated first with a leukocyte filter (HAEMONETICS RS1). Before connecting the kidney, while waiting for the system to reach 38°C, the perfusate was supplemented with bolus doses of 5000 units of unfractionated heparin, cefuroxime, and calcium gluconate. For glucose control, blood glucose measurements were carried out within 30 minutes after perfusion start and hourly thereafter. A bolus injection of 2.5 mL of a lipid-free parenteral nutrition solution was administered once blood glucose level dropped below 4 mmol/L. To optimize microperfusion and to overcome the initial vasospasm of porcine renal kidneys, a combination of 3 vasodilators was used; epoprostenol sodium, verapamil, and glyceryl trinitrate. In kidney perfusions without URC, the cannulated ureter was allowed to drain into a graduated measuring cylinder, and Ringer’s lactate was infused to replace the excreted urine as a 1:1 volume replenishment in 20 mL intervals. Reasons for termination of the kidney perfusions were any of (1) arterial flow 50 mL/min or less; (2) pH <7 or >7.7 measured at a P13 maintained in the range of 90–100 mm Hg, representing physiological MAP in a pig. Also as previously described, urine production was continuously monitored using an in-line flow sensor and arterial blood gas analysis comprised an in-line blood gas analyzer.13 An i-STAT  blood analyzer system was used for hourly glucose, sodium, and lactate measurements.Hemodynamic control was based on mean arterial pressure (MAP) and automaticallyPerfusate samples were taken at 3 different time points: (1) after priming of the circuit with blood and using the leukocyte filter before any supplements were added; (2) after 2 hours of normothermic perfusion; and (3) at the end of perfusion.www.fn-test.com) was used with a detection range of 0.156–10 ng/mL and a minimum detectable dose of <0.094 ng/mL KIM-1. For NGAL, an ELISA kit from DLdevelop (www.dldevelop.com) was used with a detection range of 1.56–100 ng/mL and a minimum detectable level of <0.64 ng/mL.Perfusates were analyzed for kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). This was done by performing an ELISA, according to manufacturer’s instructions. An ELISA kit from FineTest  clustered by pig (subject). In these models, the effect of URC and CIT was assessed on each outcome measure. For univariate comparisons, unpaired Perfusion parameters for all kidneys n = 10), with and without URC, are displayed in Table 0, with aPerfusion failure (defined as perfusate flow of <50 mL/min), occurred in 1 URC kidney after 7 hours. In contrast, perfusion failure occurred in 4/5 (80%) in the group without URC, occurring between 6 and 9 hours of NMP.P = 0.001. The difference in mean arterial flow between URC versus UR was more pronounced in the kidneys with 27 hours of CIT which had also better arterial flow; P for interaction 0.005.The duration of CIT before start of NMP (2 or 27 h) had no negative impact on arterial flow, neither in the overall cohort (n = 10) nor in the groups with or without URC. Therefore, we did not stratify the results solely for short or long CIT or for URC and UR. The linear mixed-effects models  of 7.43 ± 0.08 at 38°C, prior to kidney connection. There was no significant difference in the pH of the autologous blood stored for 27 hours, compared with blood stored for 2 hours; 7.39 ± 0.08 versus 7.46 ± 0.07, P = 0.22.The course of pH for all perfused kidneys is shown in Figure P = 0.54. Perfusate became more alkalotic in kidneys after CIT of 27 hours; P = 0.005. The difference in the mean pH between URC and UR was more pronounced in kidneys with 27 hours of CIT; P for interaction 0.021. Data displayed in Table Comparison of pH within each individual kidney pair, analyzed by URC/replacement did not show differences. Average pH in kidneys with URC was 7.51 ± 0.1 versus 7.49 ± 0.02 in UR perfusions; 17 To investigate the hypothesis that the citrate metabolism is the substrate leading to an alkalotic perfusion solution, the 63 mL CPDA-1 was removed from the blood bags and the bags reprimed with 10 000 units of unfractionated heparin and 20 mL of saline. Comparison of the pH levels of the first 2 kidney perfusions with citrate and the subsequent 8 kidney perfusions without CPDA-1 showed a significant difference; pH 7.72 ± 0.16 versus 7.46 ± 0.17, respectively, P < 0.0001. Based on this observation, CPDA-1 was removed for all of the remaining kidney perfusions.Notably, in the first kidney pair, a pH > 7.7 was observed at hour 5 in the kidney with URC and 2 hours of CIT, and at hour 4 in the kidney without URC and 27 hours of CIT. Both kidneys started perfusion with a pH > 7.45. We hypothesized that a potential reason for this increasing pH over time was the metabolism of citrate, which is part of the anticoagulant citrate phosphate dextrose adenine (CPDA-1) in the COMPOFLEX blood bag; citrate is used to prevent coagulation by chelating calcium. Each blood bag contains 63 mL CPDA-1 including 1.7 g sodium citrate dihydrate (26.3 g/1000 mL). Each citrate molecule metabolized in a functioning kidney generates 3 molecules of bicarbonate causing metabolic alkalosis of the perfusate.When lactate levels are compared (mean ± SD) within each kidney pair, the perfusates with URC consistently showed lower lactate levels.Listing the URC kidney first in each case, the numerical data are as follows: first pair: 4.35 ± 1.5 versus 5.99 ± 4.12 mmol/L; second pair: 1.13 ± 0.11 versus 11.64 ± 9 mmol/L; third pair: 2.55 ± 1.28 versus 6.9 ± 1.6 mmol/L; fourth pair: 1.87 ± 0.64 versus 9.07 ± 3.98 mmol/L; and fifth pair: 1.39 ± 0.74 versus 6.49 ± 3.2 mmol/L.P < 0.0001. CITs did not interfere with lactate levels. The relationship of URC/UR and CIT length is significant; P for interaction 0.018.According to the mixed-effects models, Table All kidneys produced urine. The median hourly urine production of all the kidneys with URC was 143 mL (123–175 mL), compared with 350 mL (293–1350 mL) in kidneys without URC.The initial sodium levels in the perfusate were higher (164.6 ± 0.6 mmol/L sodium in blood) in the first 2 perfusions (with CPDA-1 anticoagulant) than in all subsequent perfusions (143.6 ± 3.5 mmol/L sodium in blood). There were no differences in the initial sodium levels between the blood cold stored for 2 versus 27 hours (before NMP was initiated). The course of sodium serum levels for each single kidney is displayed in Figure P < 0.0001. Throughout the perfusion period, perfusate sodium was not significantly influenced by duration of CIT; P = 0.51. URC/UR and CIT are significantly linked regarding perfusate sodium; P for interaction 0.001.Perfusate sodium levels of kidneys with URC were lower throughout the perfusion compared with those in kidneys without URC 129.6 ± 0.7 versus 170.3 ± 2.8 mmol/L; P = 0.008]). As in the perfusate, sodium levels in urine were significantly lower in the group with URC; 94.9 ± 8 versus 117.6 ± 17.6 mmol/L, 95% confidence interval, 10.12-35.15 (P < 0.001).CIT had an impact on initial urine sodium levels. Urine samples taken within 30 minutes after perfusion start revealed higher sodium levels in the 27 hours CIT group (138.2 ± 9.2 mmol/L), compared with the short CIT group  compared with the long CIT group (3467 ± 338.3 mg/L), Table Figure The fourth pair of kidneys did not perfuse as well as the other pairs, and it was not possible to perfuse either kidney for longer than 8 hours either without or with URC.http://links.lww.com/TXD/A245.Time 0 biopsies of both kidneys showed a severe interstitial nephritis, more marked in kidney UR 4 than in kidney URC 4, with patchy multifocal plasma cell-rich mononuclear infiltrates. In UR 4, the interstitial inflammation intensified during NMP , whereas it showed improvement in kidney URC 4; Figure S1A–D, SDC, http://links.lww.com/TXD/A245. KIM-1 positivity could not be detected in any other biopsy-samples at any time  were comparable between the 2 CIT groups; 109.1 ± 3.8 ng/mL after 2 hours versus 111.9 ± 5.1 ng/mL after 27 hours CIT, P = 0.01. The same applies to the difference between start- and 2-hour values; delta 44.5 ± 25.4 ng/mL in the URC group versus delta 3.2 ± 6.4 ng/mL without; P = 0.02.In all but kidney UR 4, KIM-1 levels decreased over time of NMP. Two-hour KIM-1 levels were lower in the group with URC compared to the perfusions without; 65.7 ± 27.7 versus 107.6±5 ng/mL, respectively, P = 0.22.The perfusion-end levels of KIM-1 did not differ significantly between the URC group and the group with UR. The difference between start- and end-perfusion value of KIM-1 was bigger in the URC group, but not significantly; delta 33.8 ± 44.5 with URC compared to delta 18.9 ± 33.6 ng/mL without, P = 0.7. The end-perfusion KIM-1 level was comparable between the short and long CIT group; 65.6 ± 47 ng/mL after 2 versus 103.1 ± 7.8 ng/mL after 27 hours CIT, P = 0.15.The length of CIT did not impact the 2-hour KIM-1 levels; 88.8 ± 23 ng/mL after 2 hours CIT versus 84.5 ± 36.8 ng/mL after 27 hours CIT, NGAL was only detectable in perfusates of kidney pair 4 with the histological diagnosis of interstitial nephritis. In kidney UR 4, the NGAL level 2 hours after perfusion was 62 and 60 ng/mL at the end of perfusion. In kidney URC 4, the NGAL level was 71 ng/mL 2 hours after perfusion and 43 ng/mL at the end of perfusion.13 However, this study was performed using perfusions of human kidneys which had been discarded as unsuitable for clinical transplantation: these were damaged to a substantial but variable degree and, therefore, a heterogeneous cohort. The current study attempts to study the effect of urine circulation in a more consistent model.In our previous publication, we presented encouraging evidence to suggest that the recirculation of urine in kidney NMP was beneficial in respect of maintaining stable perfusion and electrolyte homeostasis.19 the present study demonstrated for the first time that URC makes it possible to extracorporeally preserve undamaged porcine kidneys normothermically for up to 24 hours. A further finding of the study is that the kidneys preserved with URC were comparable whether they had undergone 2 or 27 hours of CIT prior to NMP for up to 24 hours. Remarkably, arterial flow and IRR reached even better, more physiological values, in kidneys cold stored for 27 hours. Perfusate lactate and sodium levels were not negatively impacted either by duration of CIT. This presents the intriguing possibility of overall preservation times in excess of 50 hours; subject to confirmation of safe transplantation of such kidneys in future studies. A limitation of the study is the sample size. The first pair of kidneys developed and experienced severe alkalosis due to the metabolization of citrate in the blood collection bag, as we suspected. The severe alkalosis was the reason for terminating perfusions earlier as planned. However, kidney URC 1 could kept longer on the device as the acid-base balance deteriorated slower. Also, in the fourth pair of kidneys 24 hours could not be reached, neither with UR nor with URC. Pathology results revealed interstitial nephritis in those kidneys and interestingly, NGAL was only elevated in perfusates of URC 4 and UR 4 with decline of NGAL over time only in the URC 4 samples despite the longer CIT of the URC 4 kidney with 27 hours. Besides the unexpected events in kidney pairs 1 and 4, the differences between the URC and the UR group were still clear and we decided, according to The Three Rs and based on the experience we gained with human kidneys before,15 not to increase the number of porcine perfusions.Building on the work of previous researchers and using leukocyte-depleted blood-based perfusate,20 attest to the benefits of shorter durations of NMP with simple urine volume replacement, we believe that this URC may be of value in enabling longer-term perfusions by facilitating electrolyte and acid-base balance. The composition of the perfusate is a fundamental challenge to maintaining a kidney in a functioning state: the kidney excretes the perfusion volume multiple times over a 24-hour perfusion. Whereas accurate volume replacement is relatively straightforward and is easy to envisage as method whereby this could be automated for a clinical device, the same is not true for electrolytes, which are lost in unpredictable proportions. This leads rapidly to electrolyte and acid-base disruption, sufficient to cause cellular dysfunction.Although the work of the groups of Nicholson and Selzner21Ringer’s lactate also includes 130 mmol sodium per liter and has a lactate content of 28 mmol/L. One of the most significant findings in our analysis was that the perfusate sodium levels, and the urine sodium levels, were significantly higher in the perfusion group without URC. High perfusate sodium levels are a possible reason for the decline in arterial blood flow in the group without URC, as vasoconstriction is a natural response of a normal kidney to hypernatremia.http://links.lww.com/TXD/A245); a greater level of glucose administered correlated with a higher lactate level, and the higher the arterial flow the lower the lactate levels. In addition, as described by Bartlett et al,22 hourly urine production correlated with final lactate levels.Changes of lactate levels during perfusion are complicated to interpret in a metabolically active kidney. The kidney is the second most major lactate-consuming organ after the liver and plays a major role in lactate metabolism. A correlation between lactate levels, glucose administration, and arterial flow was shown : KIM-1 levels were lower in the 5 kidneys with URC. These differences in KIM-1 levels were based on a small number of experiments which calls for caution in the interpretation of nonparametric t-tests.If normothermic perfusion is to be clinically useful in viability assessment, it will be necessary to identify biomarkers that predict posttransplant performance. In this study, we studied NGAL and KIM-1. The former was only detectable in the kidney pair suffering from interstitial nephritis, showing lower levels of NGAL in the kidney perfused with URC over time, although the relevance of this remains unclear. KIM-1 is a well-recognized kidney injury marker that has been studied in kidney transplantation, and also in NMP.In summary, we have presented data supporting and going beyond what was published previously. Normothermic organ perfusion has been shown by many investigators to be beneficial in organ preservation and reconditioning, but its application in the field of kidney transplantation has lagged behind that of other organs for unknown reasons. Balanced perfusion as well as the opportunity to operate with a fully automated and transportable kidney perfusion device, represent, in our opinion, 3 main advantages of applying URC in kidney NMP. Further technical development is needed to move long-term kidney NMP from the experimental phase to clinical utility. We believe that resolution of the urine problem may prove to be a useful step in this direction and that this will bring improvements in both function and usability.Cordial thank you to the team of the Oxford University Biomedical Services, University of Oxford, for their support and collaboration. Special thanks to our statistical advisor Professor Dr Hanno Ulmer, executive director of the Department of Medical Statistics, Informatics and Health Economics at the Medical University of Innsbruck.