Tramadol is an active analgesic drug that is commonly used to treat moderate to severe pain. The present study aimed to assess the arterial blood gas (ABG) analysis of patients with tramadol-induced seizure (TIS).  This prospective cross-sectional study was performed on 50 TIS cases that were referred to emergency department within a maximum of one hour after their last episode of seizure. The results of ABG analysis on admission were collected and their association with dosage and time interval between ingestion and admission was assessed.2 , and PaO2 . The dose of ingestion was negatively associated with respiratory rate , arterial pH , and PaO2 , but was positively associated with PaCO2 . Higher doses of tramadol led to more severe hypercapnia and need for intubation . 5 (10.0%) cases needed mechanical ventilation. All patients improved after supportive care with no in-hospital death. 50 cases with the mean age of 35.10 ± 9.62 years were studied . The mean dosage of ingestion was 1122.00 ± 613.88 (400 to 3000) mg and the mean time interval between ingestion and admission was 7.16 ± 2.18 hours. ABG analysis on admission showed that 49 (98.0%) patients had pH < 7.35 and PaCO2 > 45 mmHg (respiratory acidosis). There was a significant association between ingestion to admission time interval and both PaCOBased on the findings, 98% of TIS cases had respiratory acidosis. Higher doses of ingested drug and longer time interval between ingestion and admission were associated with severity of ABG disturbances.  Tramadol is an active analgesic drug, which is commonly used to treat moderate to severe pain with different sources. According to the literature, this analgesic agent is one of the most prescribed opioids worldwide , 2. The The exact mechanisms of tramadol-induced seizure (TIS) remains unexplained; however, it seems that its inhibitory effects on gamma-aminobutyric acid (GABA) receptors along with its opioid receptor agonist activity play pivotal roles . TIS mayThere are two important points about TIS. First, the minimum stimulant dose of drug that causes seizures and also its blood concentrations remain unknown. In addition, there are some evidence for effects of tramadol on arterial blood gas (ABG) disturbances, especially rise in carbon dioxide pressure (PCO2), which leads to respiratory depression . In thisStudy design and settingThis prospective cross-sectional study was performed on 50 cases that were referred to emergency department of Payambaran Hospital, Tehran, Iran, within a maximum of one hour after TIS , from July to December 2019. The results of ABG analysis on admission were collected and their association with dosage and time interval between ingestion and admission was assessed. The protocol of study was approved by Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (Ethics Code: IR.SBMU.RETECH.REC.1398.355).ParticipantsPatients with a history of tramadol use with subsequent generalized seizure (diagnosed as tramadol-induced seizure) that were referred to emergency department of our hospital within a maximum of one hour after last episode of seizure were included in this study. Patients with history of head trauma, multi drug ingestion, positive amphetamine test, history of methamphetamine, morphine, or methadone abuse, the use of psychological medications such as tricyclic antidepressants, phenothiazine, or selective serotonin reuptake inhibitors, presenting to the hospital more than one hour after the last seizure episode, or any other reason for the seizure, were excluded.Data gathering  On admission, the patients’ baseline characteristics , the level of consciousness , vital signs , need for mechanical ventilation, and at the time of discharge, duration of hospital stay were collected using a predesigned checklist. Also, the results of ABG analysis were collected on admission and then 12 hours after the initial assessment. An expert toxicologist was responsible for data gathering.Statistical analysisThe results were presented as mean ± standard deviation (SD) for quantitative variables and were summarized by absolute frequencies and percentages for categorical variables. Normality of data was analyzed using the Kolmogorov-Smirnoff test. Categorical variables were compared using chi-square test or Fisher's exact test. Quantitative variables were also compared using t test, or Mann-Whitney U test. The association between the quantitative variables was tested via Pearson’s correlation test. To assess the relationship of time and dose of tramadol used with the change in ABG parameters with the presence of other variables as the confounders, the multivariable regression model was employed. For the statistical analysis, the statistical software SPSS version 16.0 for windows  was used. P values of 0.05 or less were considered statistically significant. Baseline characteristics of studied cases50 tramadol-induced seizure cases with the mean age of 35.10 ± 9.62 (range: 25-45) years were studied . The mean dosage of tramadol used was 1122.00 ± 613.88 (400 to 3000) mg that led to one, two and three episodes of Pre-hospital seizures in 72.0%, 26.0%, and 2.0% of patients, respectively. The patients’ characteristics on admission are summarized in Blood gas analysisArterial blood gas analysis on admission showed that 49 (98.0%) patients had pH < 7.35 and PCO2 > 45 mmHg (respiratory acidosis). There was a significant association between tramadol use to seizure time interval and blood pressure , heart rate , body temperature , PCO2 , and PO2 . The dose of medication used was negatively associated with blood pressure , respiratory rate , arterial pH , and PaO2 , but was positively associated with PCO2 . Higher doses of tramadol led to more severe hypercapnia and need for tracheal intubation . OutcomesIn most patients, the blood gas indices had significantly improved within 12 hours via supportive approaches . 5 , which led to severe respiratory acidosis leading to emergency intubation and mechanical ventilation. In another experiment by Ismail et al. , about oTherefore, summing our findings and the results of previous studies shows that in patients suffering from TIS, higher doses of tramadol and also longer time interval between tramadol ingestion and hospital admission may result in more severe acid-base disturbances such as respiratory acidosis. The association between the occurrence of seizure and the change in acid-base balance in TIS has not been previously examined. Respiratory changes with hypercapnia and hypoxemia, which can occur with seizures, have been exclusively studied in previous experiments. However, in our study and due to the lack of designing a case-control study with both seizure and non-seizure subgroups, assessing the relationship between the likelihood of seizure and respiratory acidosis following the use of high dose of tramadol was impossible, which should be considered as a major target in future studies. Based on the findings, 98% of TIS cases had respiratory acidosis. Higher doses of ingested drug and also longer time interval between ingestion and admission were associated with higher severity of ABG disturbances.


The epithet of acute decompensated heart failure (ADHF) is volume overload. ADHF is associated with a rising number of hospital admission for volume overload. Medication non-compliance, excessive salt intake, comorbidities, and/or disease progression can attribute to volume overload. Heart failure (HF) therapy has innovated during the past few decades, but diuretics have been the mainstay of treatment. Diuretics are vital even though these drugs stimulate the renin-angiotensin-aldosterone system (RAAS) and lead to adaptive responses like diuretic resistance, neurohormonal activation, and worsening renal function that may be inimical. There has been a thriving interest in cutting-edge strategies to manage volume overload in ADHF. The American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) guidelines advocate pharmacological and non-pharmacological interventions to treat volume overload in ADHF patients. Ultrafiltration (UF) is, therefore, an emerging stand-in therapy of interest for treating volume overload in ADHF patients. This review article epitomizes available clinical data on the use of diuretics and UF in ADHF patients and identifies challenges for each approach. HF is a complex clinical syndrome due to inability of the heart to adequately fill or eject blood . It is cADHF has been defined as “gradual or rapid change in HF signs and symptoms resulting in a need for urgent therapy.” It is a complex and heterogeneous syndrome characterized by a variable degree of end-organ hypoperfusion and congestion, requiring urgent treatment. It can present as new-onset HF, worsening chronic HF, or advanced HF. Congestion is defined by an abnormally elevated cardiac filling pressure resulting in pulmonary or systemic back up of blood flow, which becomes a major driving force for the symptoms of ADHF. Decongestion is therefore considered a primary goal of acute therapy and is usually achieved in the inpatient setting . TreatmeCirculating natriuretic peptide levels are commonly measured in patients with a suspected ADHF episode. Elevated natriuretic peptides levels support the diagnosis of ADHF in patients suffering from dyspnea and many other comorbidities such as valvular heart disease, arrhythmias, pulmonary hypertension, and renal insufficiency . It has Current guidelines advocate the use of intravenous loop diuretics as a first-line agent for the improvement of congestion and symptoms of dyspnea . It is aCommon challenges in achieving and maintaining decongestion include inadequate diuretic dosing, diuretic resistance, the “breaking phenomena”, and post diuretic sodium retention also known as the “rebound effect” . Felker Prior studies have suggested that high doses of diuretics have a harmful biological effect associated with renal injury. This is thought to occur secondary to electrolyte disturbances or over reactivation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous systems . Loop diDespite preclinical and clinical data supporting the physiological benefits of torsemide, furosemide is the most commonly used loop diuretic . Mentz eDiuretic resistanceDiuretic resistance is defined as the inability to reach the desired therapeutic reduction in edema despite the administration of a proper dose of diuretic . With reMultiple mechanisms contribute to oral diuretic resistance resulting from bioavailability limitations or altered absorption when taken with food. In the setting of hypoalbuminemia, bioavailability is decreased as charged loop diuretics become protein-bound and are more transported to the kidney while bound to albumin. States of venous congestion and renal vein compression that occur in cardiorenal syndrome may also contribute to diuretic resistance. Similarly, albuminuria is another contributing mechanism of resistance; albumin binds to loop diuretics in the lumen and limits the potential of the free drug to act on the site of action .Currently, there are no specific guideline designs on dealing with diuretic resistance . The HeaAronson and Burger concluded in their study that loop diuretic dosage is not the sole predictor of urine output. Other factors such as renal function, blood pressure, and fluid intake are also independent predictors of urine output. An elevated right atrial pressure is a marker of fluid overload; which happens to also be an independent predictor of urine output, while the reduced cardiac output is neither. However, net fluid loss is a better predictor of mortality than urine output .UltrafiltrationUltrafiltration (UF) is a mechanical modality of sodium and fluid removal, exhibiting adjustable volumes and filtration rates that allow optimal preservation of systemic volume homeostasis. It has re-emerged as an effective strategy for decongestion in ADHF . PreviouUltrafiltration versus diureticThe first randomized study that compared intravenous loop diuretics to UF in ADHF was known as RAPID-CHF (Relief for Acutely Fluid-Overloaded Patients with Decompensated Congestive Heart Failure) trial, compared the UF group to the usual diuretic care group. At 24 hours of observation, greater weight loss and fluid removal in the UF group was noted even though an aggressive diuretic dose was given to the usual care group. UF was well-tolerated, no serious complications or significant electrolytes disturbances occurred, neither did any acute renal failure . This result simulated the design of the UNLOAD  trial. Costanzo et al. demonstrated in their study the benefit of early UF in reducing congestion in ADHF with diuretic resistance . In theiThe first clinical trial to evaluate the biohumoral and hemodynamic effect of UF compared to standard intravenous diuretic therapy in ADHF was The ULTRADISCO  study. This prospective, randomized study dealt with 15 patients in each group. In the UF group, N-terminal pro-B-type natriuretic peptide levels decreased significantly from 5063+3811 pg/mL at baseline to 1797+1327 pg/mL at post 36 hours (p 0.001). Whereas levels remained essentially unchanged in the diuretic group (6707+3597 pg/mL at baseline to 5271+3251 pg/mL at post 36 hr). Aldosterone levels showed a similar trend; 0.86+1.04 nmol/l at baseline, 0.25+0.23 nmol/l at post 36 in the ultrafiltration group (p 0.001), and 0.73+0.87 to 0.80+0.46 nmol/l at post 36 (p ¼ ns) in the diuretic group. Their findings also showed that the fluid removal obtained with UF in the acute phase had more beneficial effects on hemodynamic variables compared to the diuretic infusion group [There is a conjecture that UF may also remove harmful myocardial depressant factors, perhaps certain cytokines which are known to be deleterious to cardiac function. Investigators have suggested that the removal of cytokines that have predisposing myocardial depressant effects; including tumor necrosis factor-alpha and other inflammatory cytokines, may improve myocardial contractility. In the ULTRADISCO study, It was theorized that the mechanism underlying the improvement of contractility was induced by UF because of lowered cytokine levels .The most common pathophysiological mechanism underlying both ADHF and the progression of the HF syndrome is volume overload. HF remains the most common cause of hospitalization and the key manifestation is volume overload. Complete decongestion via diuretic strategy is the most important prognosticator of good long-term outcomes and preventing hospitalization. Although there has been clinical data on the effects of diuretic therapy related to reduced intra-arterial volume with RAAS activation, however, no relevance has been rooted between diuretics and cardiovascular mortality. The same is accurate for UF - until substantial clinical evidence is available, its use will be limited to selected cases per current guidelines. New research designed to offset the primary determinants of volume overload could improve the management of patients affected by HF. Until then, diuretic therapy will remain the mainstay of therapy for volume overload in ADHF patients.


Objective: Previous studies have shown that patients with hereditary fructose intolerance (HFI) are characterized by a greater intrahepatic triglyceride content, despite a fructose-restricted diet. The present study aimed to examine the long-term consequences of HFI on other aldolase-B-expressing organs, i.e. the kidney and vascular endothelium.Methods: Fifteen adult HFI patients were compared to healthy control individuals matched for age, sex and body mass index. Aortic stiffness was assessed by carotid-femoral pulse wave velocity (cf-PWV) and endothelial function by peripheral arterial tonometry, skin laser doppler flowmetry and the endothelial function biomarkers soluble E-selectin [sE-selectin] and von Willebrand factor. Serum creatinine and cystatin C were measured to estimate the glomerular filtration rate (eGFR). Urinary glucose and amino acid excretion and the ratio of tubular maximum reabsorption of phosphate to GFR (TmP/GFR) were determined as measures of proximal tubular function. Results: Median systolic blood pressure was significantly higher in HFI patients . Pulse pressure and cf-PWV did not differ between the groups . Of all endothelial function markers, only sE-selectin was significantly higher in HFI patients (p = .004). eGFR was significantly higher in HFI patients than healthy controls . All measurements of proximal tubular function did not differ significantly between the groups.Conclusions: Adult HFI patients treated with a fructose-restricted diet are characterized by a higher sE-selectin level and slightly higher systolic blood pressure, which in time could contribute to a greater cardiovascular risk. The exact cause and, hence, clinical consequences of the higher eGFR in HFI patients, deserves further study. The weight of each consumed product was either provided by the participant or estimated using average quantities per portion. Macro- and micronutrient composition was determined based on the Dutch food composition table . FructosHeight was determined standing upright against a stadiometer and weight was assessed in solely underwear using an electronic scale. BMI was calculated as weight (kg) divided by height (m) squared. Waist was measured with a measuring tape at the level of the umbilicus.Blood was drawn for measurement of whole blood glucose , plasma insulin , and serum lipids .Urinary sodium concentration  was determined in the twenty-four-hour urine samples.2.4U test.Data are expressed as median (interquartile range) and analyzed with a Mann-Whitney cr-cys and urinary total protein concentration) and proximal tubular function  were considered as the primary outcome measurements.Blood pressure, cf-PWV, endothelial function , glomerular function  for the difference (Δ) between medians of HFI patients versus healthy controls as part of the Mann-Whitney test was calculated according to the Hodges-Lehmann method.n = 30), linear regression was conducted with only one potential mediator per analysis.Linear regression analyses were conducted to explore the contribution of potential mediators of any difference in the primary outcome measures. A ≥ 25% reduction in the unstandardized beta of the primary outcome measurement after inclusion of the potential mediator was considered relevant. Due to the low number of study participants  version 23 for Windows .Results were considered statistically significant at 33.12) and predominantly male (73.3%). Three out of 15 HFI patients were smokers, as opposed to none of the healthy controls. Alcohol intake was higher among healthy controls . As expected, patients consumed almost no fructose (median intake: 1.0 g/day) and, as a consequence, protein and saturated fat intake were higher compared to healthy controls . Serum lipids, whole blood glucose and plasma insulin levels did not differ significantly between both groups. None of the participants used blood pressure-, lipid- or glucose-lowering medication.Fifteen HFI patients and fifteen age-, sex- and BMI-matched healthy controls were included in this study. General characteristics are displayed in 3.2p = .54, Δ: 2 bpm, 95%CI: -5;12). Pulse pressure and cf-PWV were not different either , it did not reach statistical significance (p = .10). None of the participants had an eGFR <60 ml/min/1.73m2 (compatible with chronic kidney disease stage 3a). Linear regression analyses were conducted to explore the role of systolic blood pressure and dietary protein intake, which are known risk factors for glomerular hyperfiltration [cr-cys. The unstandardized beta of the HFI affected state did not substantially decline after addition of these variables to the model (data not shown). Urinary total protein concentration was not statistically different between both groups . No statistical differences in urinary amino acid concentrations were found between HFI patients and healthy controls .TmP/GFR and urinary glucose were not statistically different between both groups, as shown in controls , panel E4HFI is an inborn error of metabolism that can be treated with a fructose-restricted diet. Up to now, mostly case reports on HFI at the time of diagnosis have been published . We and p = .056). Five of 15 patients had systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. Since blood pressure was determined only twice during the study visit, we cannot exclude white coat hypertension as a potential explanation for the observed difference. Ideally, this would require 24-h blood pressure monitoring [and diastolic blood pressure as an alternative explanation [HFI patients displayed a higher systolic blood pressure than healthy controls. Diastolic blood pressure tended to be higher as well  mass or diet – which affect serum creatinine levels [cys .Previous studies have shown that ingestion of fructose by HFI patients leads to proximal tubular dysfunction (i.e. Fanconi syndrome) and eventually to renal failure ,40. In te levels ,43 – sinA higher eGFR could reflect glomerular hyperfiltration, as seen in other disease states such as diabetes mellitus , which cand a higher eGFR in the general population [p = .13) [On the other hand, a higher eGFR in HFI might also reflect a truly better glomerular function. Although speculative, a hepatorenal axis in HFI patients may account for the current observations. We and others previously showed that HFI patients are also characterized by greater intrahepatic triglyceride content than controls . Detailepulation ,51. The p = .13) . These sOf all measures of endothelial function, only plasma sE-selectin levels, an endothelium-specific biomarker, were significantly higher in HFI patients compared to healthy controls. The fact that plasma vWF, LDF and RH-PAT were not statistically different between HFI patients and healthy controls, might be due to a lack of statistical power, or alternatively, a difference in the type of blood vessel under investigation, i.e. small arteries (RH-PAT) versus skin arterioles (LDF) versus the microcirculation in general (plasma sE-selectin and vWF). Of note, previous epidemiological studies have shown that sE-selectin levels predict future type 2 diabetes and cardiovascular disease ,54. We pThis study has strengths and limitations. As hitherto mentioned, the small patient population makes it difficult to draw conclusions on non-significant results. Therefore, these results should be interpreted with caution. In addition, formal mediation analyses are required to explore the contribution of potential mediators to the difference in primary outcomes measures between HFI patients and healthy controls. Due to the low number of study participants, however, this was not possible. Last, the healthy control subjects, who were matched for age, sex and BMI, do not necessarily represent the general population.This is the first report of HFI patients treated with a fructose-restricted diet using state of the art methods. Since only case reports on HFI at time of diagnosis have been published , this stE-selectin level compared to age-, sex- and BMI-matched healthy controls. Further studies are required to elucidate the exact causes of these observations, either the direct consequence of the genetic defect or secondary to its dietary treatment. These results do emphasize the need for long-term follow-up of HFI patients, in particular with regard to blood pressure and kidney function.In conclusion, following our previous report on the presence of fatty liver, liver fibrosis and glucose intolerance , we now N.S. performed the measurements, conducted the analyses, researched the data and wrote the manuscript. A.J.H.M.H. conducted the LDF analyses, reviewed the manuscript and provided revisions to the manuscript. J.B. conducted the urinary amino acid analyses, reviewed the manuscript and provided revisions to the manuscript. E.J.F., J.A.P.B. and C.G.S. contributed to the measurements, reviewed the manuscript and provided revisions to the manuscript. C.E.M.H., F.G.D. and D.C. contributed to the inclusion of study participants, reviewed the manuscript and provided revisions to the manuscript. N.C.S., C.D.A.S., F.G.D., D.C. and M.C.G.J.B. conceived the study, supervised the analyses, researched the data, reviewed the manuscript and provided substantial revisions to the manuscript. M.C.G.J.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.


To investigate the relationships of plasma renin, angiotensin, and aldosterone levels to blood pressure variability and target organ damage in children with essential hypertension.A case-control study was conducted on 132 children diagnosed with essential hypertension . The plasma RAAS levels were measured using the enhanced chemiluminescence method, the ambulatory blood pressure was monitored for 24 h, and then the average real variability (ARV) was calculated. Data on indicators were used for assessing cardiac and renal damages. The correlations of plasma renin, angiotensin, and aldosterone (RAAS) levels to blood pressure variability (BPV) and target organ damage (TOD) were studied. A comparison between the groups was conducted using SPSS 20.P = 0.031; t = 2.672, P = 0.009). Plasma AT II and aldosterone levels were significantly associated with the left ventricular mass index . Linear regression analysis showed that AT II  and aldosterone  were risk factors for LVH.Among the 132 children, 55 cases had target organ damage. The 24-h ARV and the daytime ARV of the systolic blood pressure of the high angiotensin II (AT II) group was significantly higher than that of the normal AT II group (t = 2.175, The AT II level in children with essential hypertension affected the variability of the 24-h and the daytime SBP. Plasma AT II and aldosterone levels were associated with cardiac damage. Results from this study indicated that AT II and aldosterone are risk factors for LVH in childhood hypertension and are of great significance for improving the clinical prognosis of pediatric patients with hypertension. Hypertension is a disease that seriously threatens human health by causing damage to multiple target organs, such as the cardiovascular system, kidneys, central nervous system (CNS), and fundus. Many epidemiological studies have focused on identifying factors that increase the risk of hypertension. Essential hypertension is a multi-gene disease in which the genetic environment interacts with humoral factors. The renin-angiotensin-aldosterone system (RAAS) has a pivotal role in regulating blood pressure and water-electrolyte balance. Over-activation of RAAS plays an important role in the pathogenesis of hypertension . An earlHowever, the relationships of RAAS levels to BPV and target organ damage (TOD) in hypertensive children remain unclear. Hence, we conducted a correlation analysis between RAAS and BPV as well as TOD in children with essential hypertension from August 2017 to June 2019.A total of 132 children, comprising 103 males (78.0%) and 29 females (22.0%) diagnosed with essential hypertension and admitted to the Affiliated Children’s Hospital of Capital Institute of Pediatrics  from August 2017 to June 2019 were included in this case-control study. The research protocol of this study was approved by the Ethics Committee of the Children’s Hospital Affiliated to Capital Institute of Pediatrics. The parents or guardians of the patients received information about the relevant examinations and signed the written informed consent before the study.the Fourth Report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents, and the results were used for hypertension diagnosis and stage classification  and aldosterone  were risk factors for LVH (Table β ± s.e. < 0.001).Correlation analysis between plasma renin/angiotensin/aldosterone levels and target organ damage in 132 pediatric patients. As shown in Table Essential hypertension is a multi-gene disease in which the genetic components interact with physiological factors. RAAS plays an important role in the pathogenesis of hypertension. Classical RAAS refers to the production and function of angiotensin and aldosterone. Angiotensinogen is secreted by the liver into the blood circulation. A decapeptide, angiotensin I, is generated by the action of renin produced by renal mesangial cells. Angiotensin I is then further converted into an octapeptide, angiotensin II, by the action of angiotensin-converting enzyme (ACE) in the pulmonary circulation. Angiotensin II promotes vasoconstriction. Aldosterone is secreted by the adrenal cortex and is essential for sodium retention and potassium excretion to promote water and sodium reabsorption, thereby causing water and sodium retention and elevated blood pressure . RAAS plUnder normal physiological conditions, blood pressure fluctuations are not too extreme (too high or too low). Under pathological conditions, BPV is affected by increased blood volume and neurological and humoral factors, which leads to greater fluctuation in the blood pressure. Among humoral factors, the plasma renin, angiotensin, and aldosterone levels have important effects on BPV. Baltatu et al.  studied Myocardial remodeling in hypertension refers to cardiac hypertrophy and fibrosis. Sustained blood pressure load first and mainly affects the left heart, resulting in LVH. However, the increase in LVM is not always related to blood pressure values . MyocardThe progression of kidney disease is closely related to the regulation of local RAAS. The action of angiotensin II results in increased glomerular capillary pressure in hypertension with renal damage, resulting in increased glomerular permeability and excessive proteins being filtered through the glomeruli; cells in the proximal convoluted tubule absorb the excessive proteins by pinocytosis, which causes tubular damage, interstitial inflammation and fibrosis, as well as the loss of kidney unit function . HoweverConsidering that this was a small-scale study from a single center, the generalizability of our data might be limited. The small patient population and low number of females might also limit the statistical power. In addition, CIMT and PWV are also considered markers of TOD but were not assessed in the study; this is also a limitation of the study. We think that additional prospective research is needed to more fully discern the effect of RAAS activity on BPV and TOD.In conclusion, RAAS plays an important role in the pathogenesis of hypertension. Our results showed that plasma angiotensin II levels affected 24-h and daytime SBP fluctuations and affected the regulation of the systolic BPV. Angiotensin II and aldosterone levels were associated with hypertensive cardiac damage. They were not only the predisposing factors for cardiac damage in hypertensive patients but were also involved in the disease progression of cardiac damage. Elevated angiotensin II activity was closely related to hypertensive cardiac damage. The results from this study indicated that angiotensin II and aldosterone are risk factors for LVH in childhood hypertension and are of great significance for improving the clinical prognosis of pediatric patients with hypertension.


There is an increasing burden of non-communicable disease globally. Tenofovir disoproxil fumarate (TDF) is the most commonly prescribed antiretroviral drug globally. Studies show that patients receiving TDF are more prone to renal dysfunction at some point in time during treatment. Evaluation of kidney function is not routinely done in most HIV public clinics. Identification of renal dysfunction is key in resource constrained settings because managing patients with end stage renal disease is costly.2,or proteinuria of ≥2+ on urine dipstick, glycosuria with normal blood glucose. Electrolyte abnormalities were also documented.This was a cross-sectional study conducted at an outpatient clinic in 2018 involving patients on TDF for at least 6 months who were 18 years or older. Patients with documented kidney disease and pregnancy were excluded. Estimated glomerular filtration rate (eGFR) was calculated using the CKD-Epi formula. Renal dysfunction was defined as any of the following; either eGFR< 60 mL/min/1.73mWe enrolled 278 participants. One hundred sixty nine (60.8%) were females, majority 234(84.2%) were < 50 years old, 205 (73.74%) were in WHO stage 1, most participants 271(97.5%) in addition to TDF were receiving lamivudine/efavirenz. The median age was 37(IQR 29–45) years; median duration on ART was 36 (IQR 24–60) months. The prevalence of renal dysfunction was 2.52% (7/278). Most noted electrolyte abnormality was hypocalcaemia (15.44%).The prevalence of renal dysfunction was low though some participants had hypocalcaemia. Screening for kidney disease should be done in symptomatic HIV infected patients on TDF. People living with HIV (PLHIV) are living longer and as a result face a challenge of increasing morbidity from non-communicable diseases-NCDS , 2. KidnThe 90–90-90 UNAIDS strategy by 2020  is not fThe study was carried out at the Mulago Communicable Disease Clinic under the directorate of medicine situated in Kampala, Uganda. Mulago-Kiruddu hospital is home to the directorate of medicine since May 2016; running different medical outpatient and inpatient clinics. The communicable disease clinic is supported by Makerere University Joint AIDS Program (MJAP), runs on Fridays as an outpatient clinic, and mainly attends to HIV infected patients. All services are provided free of charge to the public.We conducted a cross sectional study with the sample size calculated based on the Kish and Leslie formula for prevalence studies. The sample size was calculated as 396   at a prePotassium 3.5–5.0 mmol/lCalcium 2.2–2.7 mmol/lPhosphate 0.97–1.45 mmol/lPatients, who fulfilled the study criteria were enrolled, interviewed for demographic and social data. Medical history was collected and physical examination performed. Blood pressure measurement was done in standard way . Patients with a value greater than 140/90 mmHg were considered hypertensive. Blood pressure > 140 mmHg was considered systolic hypertension and a value of > 90 mmHg considered diastolic hypertension. Nutritional assessment was done using body mass index (BMI), obtained using the formula body weight in kilograms divided by height squared in meters. Venous blood was collected for renal function test and electrolytes . Reference laboratory ranges for electrolytes;Results for the electrolytes were reported as hypo or hyper depending on these reference ranges.Following aseptic technique, 10 mL of venous blood was drawn from each participant into a syringe. Seven mills (7mls) (of the 10mls) of blood for renal function tests & electrolytes  was placed in a red top vacutainer and centrifuged. Four mls of serum were then put in crial vials. The remaining 3 mls (of the 10 mL) were placed in a grey vacutainer bottle (to determine blood sugar). All these were adequately packaged in Ziploc bags and carrier for processing in old Mulago clinical laboratory. Renal function tests, electrolytes and blood sugar were determined using an automated Abott Machine, Architect plus-Ci Chemistry and immunochemistry analyzer.2or proteinuria of ≥2+ with glycosuria but normal blood sugar. As per Kidney foundation’s kidney Disease outcome quality Initiative (K/DOQI),eGFR< 60 for 3 months implies chronic kidney disease [Each participant was provided with a sterile urine container and educated briefly about provision of 20 mL of a mid-stream urine which was analyzed using a dipstick on the same day to determine presence of leucocytes, nitrites, hematuria or glucose. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic kidney Disease Epidemiology Collaboration (CKD-EPI) formula which is more accurate than the Cockcroft gault formula . For thi disease .X2) test for categorical variables and independent sample t test for continuous variables. The outcome variable was renal dysfunction.Data was analyzed using STATA 14.0 software package. Continuous variables were summarized as means and standard deviation for normally distributed data, medians and interquartile ranges for not normally distributed data. Categorical variables were summarized as frequencies, percentages and also in tables and figures. Tests for the significance of association were made using the Pearson chi-square (From November 2017 through March 2018 (for 4 months), eight hundred eight (808) patients were consecutively screened in Mulago Communicable Disease Clinic for study enrolment. Three hundred five (305) patients met the eligibility criteria and were enrolled. Of these, 278 were entered into the final analysis and 27 patients were excluded due to incomplete data. One hundred sixty nine (60.8%) were female, majority 234 (84.2%) were < 50 years old. Median age was 37 (IQR 29–45) years. One hundred nineteen (42.81%) were married, 141(50.72%) had a primary education, 250(89.93%) had formal employment, 252(83.45%) resided in Kampala and 81 (29.14%) consumed alcohol.  tested positive for nitrites, 7(2.52%) had hematuria and 2(0.72%) had glycosuria.The median duration on ART was 36 IQR (24–60) months, 271 (97.48%) were on TDF/3TC/EFV, 152(72.38%) had CD4 ≥ 200 cells/ml, 109 (86.51%) had viral load of < 1000 copies/ml, 205(73.74%) were in WHO clinical one,155(55.76%) had a normal BMI, 38(13.67%) had systolic hypertension, 42(15.11%) had diastolic hypertension and 270(97.12%) had no concurrent opportunistic infections. Three (1.08%) had random blood sugar ≥11.1 mmol/l, 10 (3.6%) had proteinuria of ≥2Ten (3.59%) had hypophosphatemia, 11(4.44%) had hyperphosphatemia, 15(5.26%) had hyperkalemia, 3(1.32%) had hypokalemia, 43 (15.44%) had hypocalcaemia and 2 (0.67%) had hypercalcemia.7 were found to have eGFR < 60 ml/min/1.73m2, ten [2 patients had glycosuria (but no proteinuria). Therefore prevalence of renal dysfunction was 2.52% (7/278). . In this study contributing factors included WHO region, hypertension and Diabetes Mellitus. Reid et al 2008 in a study  done in Uganda and Zimbabwe, noted a prevalence of 7% among patients on ART, followed up for 96 weeks. Majority (74%) of the patients were receiving TDF based therapy. In this study Glomerular filtration rate was assessed using the Cockcroft gault formula which has its limitations.Udeme et al The variability of different prevalence rates in the studies is attributable to; the different definitions of renal dysfunction, different methods used to estimate GFR and patient characteristics  , 10, 19.The low prevalence in our study could be explained by the small sample size, being an outpatient clinic receiving not very critically sick patients, the possible effect of ART improving kidney function  and use We assessed three serum electrolytes in this study namely; Potassium, Calcium and Phosphate. Albumin and vitamin D levels were not assessed. We noted mainly Hypocalcaemia (15.44%), Hyperkalemia (15%) and hypophosphatemia (3.59%).Hypocalcaemia has been found at a higher prevalence in other studies among HIV infected patients compared to the general population. Vitamin D deficiency is very common among HIV patients and may be the most reasonable explanation , 22. TDFFor patients with hypophosphatemia, data is similar to studies elsewhere that demonstrate that TDF is associated with Proximal tubulopathy manifesting as Fanconi syndrome , 23. RarProteinuria is an important marker of renal dysfunction, however for this study we tried to identify an operational definition that could in cooperate TDF nephrotoxicity. Other studies that have looked at proteinuria have given higher values of renal dysfunction. And this also helps capture the decline in renal dysfunction early to mitigate progress to ESRD.Our study had some limitations; the small sample size , missing information for some patients  which were not done/documented by primary health care giver and no baseline renal function to assess change over time  and we had limited time for the study to check the baseline levels). The results we got will form as baseline for future reference.The prevalence of renal dysfunction among HIV infected patients receiving TDF was low at 2.52% and hypocalcaemia was the most common electrolyte abnormality noted. Renal dysfunction occurs at a low rate in our settings however when assessed should include serum electrolytes.Additional file 1. The datasets generated and/or analyzed during the study are available in excel format and uploaded as supplemental information.


Eight potential biomarkers have been identified using 1H NMR metabolomics and nine mass spectral features which differed significantly (p < 0.05) between kidney cancer patients and healthy volunteers, as observed by LDI MS. A partial least squares discriminant analysis (OPLS-DA) model generated from metabolic profiles obtained by both analytical approaches could robustly discriminate normal from cancerous samples (Q2 > 0.7), area under the receiver operative characteristic curve (ROC) AUC > 0.96. Compared with healthy human serum, kidney cancer serum had higher levels of glucose and lower levels of choline, glycerol, glycine, lactate, leucine, myo-inositol, and 1-methylhistidine. Analysis of differences between these metabolite levels in patients with different types and grades of kidney cancer was undertaken. Our results, derived from the combination of LDI MS and 1H NMR methods, suggest that serum biomarkers identified herein appeared to have great potential for use in clinical prognosis and/or diagnosis of kidney cancer.Kidney cancer is one of the most frequently diagnosed and the most lethal urinary cancer. Despite all the efforts made, no serum-specific biomarker is currently used in the clinical management of patients with this tumor. In this study, comprehensive high-resolution proton nuclear magnetic resonance spectroscopy (The online version of this article (10.1007/s00216-020-02807-1) contains supplementary material, which is available to authorized users. Kidney cancer is the third most frequently diagnosed cancer of the urinary tract in the world. In 2018, this disease affected over 400,000 individuals worldwide and is responsible for nearly 180,000 deaths annually . NowadayThe survival of advanced kidney cancer patients, for whom standard chemotherapy is not very effective, has improved with the advent of targeted therapy. The targeted drugs act against vascular endothelial growth factor (VEGF) and other important proteins (tyrosine kinases) that enable cancer cell growth and survival, but treatment response is varied; and at best, these targeted drugs can only slow the growth of the cancer for a time but do not actually cure kidney cancer. Specific targeting molecules, most of which are proteins, have been proposed (C-reactive protein (CRP), PTEN, carbonic anhydrase IX (CAIX), hypoxia-inducible factors (HIF-1α and HIF-1β), vascular endothelial growth factor , and monitoring their activity might generate a timely prognosis of the metastatic potential of RCC. However, these biomarkers suffer from low abundance and are not particularly specific to metastatic RCC, and using them as diagnosis of metastasized RCC is very limited .Unfortunately, though great efforts have been undertaken in the past decades, there are still no acknowledged robust biomarkers available for diagnosis or prognosis of RCC cancers. In order to create more effective therapies, additional kidney cancer biomarker research is needed, and has important clinical significance especially for early detection, diagnosis, as well as guiding treatment interventions, monitoring treatment effectiveness, identifying relapse, and elucidating the molecular processes underlying RCC disease states.1H nuclear magnetic resonance (NMR) [Over the past decade, sensitive analytical methods have been developed, enabling to better understand the metabolic changes underlying kidney cancer phenotypic changes. These analytical platforms include ce (NMR) , liquid ce (NMR) , gas chrce (NMR) , matrix-ce (NMR) , desorptce (NMR) , and surce (NMR) .NMR and MS combined with multivariate statistical data analysis are the two most powerful and commonly used analytical methods for the rapid, noninvasive, and simultaneous measurements of endogenous metabolites present in biological fluids and tissues . These tMetabolic profiling of biofluids, especially serum, generates a biochemical fingerprint of small molecule metabolites, and allows for identification and characterization of potential biomarkers associated with cancer. Since genetic studies has shown that RCC is a metabolic disease, a growing number of studies is focusing on profiling of tissue, serum, plasma, and urine samples of kidney cancer patients. However, relatively low number of NMR metabolomics studies have been undertaken to characterize the serum metabolomes of kidney cancer patients in recent years .1H NMR) spectroscopy and found 18 metabolites significantly differentiating between three groups: (i) RCC patients and controls; (ii) RCC patients with metastases and without metastases; and (iii) RCC patients before and after nephrectomy [1H NMR spectroscopy to analyze serum metabolome of plasma in 32 RCC patients and 13 controls and found 17 metabolites that can distinguish RCC patients form controls [1H NMR [1H NMR-derived metabolomics profile of serum samples from 104 participants was studied by Zheng et al. in order to prepare RCC predictions. They revealed that cluster of 7 metabolites  can be used for the early diagnosis of RCC [Lin and co-workers are pioneers in the analysis of the serum of patients with kidney cancer using mass spectrometry. In 2010, they employed separately two MS-based techniques to analyze serum samples from 31 kidney cancer patients and 20 healthy volunteers. The authors found a total of 71 variables useable as potential markers but the identification of chemical compounds was only successful for only a few of them [hrectomy . Zira etcontrols . In anots of RCC . Moreoves of RCC , 22, 23 s of RCC , 25 froms of RCC .1H NMR and laser desorption/ionization MS using a 109-silver nanoparticle-enhanced steel target platform (109AgNPET LDI MS) [109AgNPET LDI MS approach for metabolomics has been demonstrated in the analysis of metabolites in plant, animal, and human tissues [1H NMR untargeted metabolomics.In the current work, the metabolomics analysis of 50 serum samples from patients with kidney cancer and 49 healthy subjects serving as controls was undertaken using two orthogonal analytical methods: high-resolution  LDI MS) . The val tissues . Both mu109Ag) isotope was purchased from BuyIsotope (Sweden) and transformed to trifluoroacetate salt using commonly known methods  and recrystallized from tetrahydrofuran/hexane system. 2,5-Dihydroxybenzoic acid (DHB) was purchased from Aldrich. Steel targets were locally machined from H17 stainless steel. All solvents were of HPLC quality, except for water and methanol . The silver-109 nanoparticles were synthesized on the surface of steel targets as described in our publication [Silver-109  had stage T1 disease, four patients had stage T2, ten patients had stage T3, and one patient had stage T4. In three patients, the stage of the disease could not be determined. Among tumors diagnosed, there were 33 clear cell renal cell carcinomas (ccRCC), 4 oncocytomas, 4 angiomyolipomas (AML), 2 chromophobe renal cell carcinomas (chRCC), 2 papillary renal cell carcinomas (pRCC), 1 collecting duct carcinoma (CDC), 1 simple renal cyst (SRC), and 1 tubulocystic renal cell carcinoma (TCRC) according to the 2016 WHO Classification of Tumors of the Urinary System and Male Genital Organs. Most of the diagnosed cancers were malignant (n = 41), but few patients (n = 7) had benign (non-cancerous) kidney tumors. In this study, benign tumors of the kidney include oncocytoma and angiomyolipoma, while other types of tumors are considered malignant. In addition, one patient had a lung adenocarcinoma metastasis. The majority of patients  had stage T1 disease, four patients had stage T2, ten patients had stage T3, and one patient have stage T4. In three patients, the stage of the disease could not be determined. The pathological and clinical characteristics of the patients are presented in the Electronic Supplementary Material  (permission no. 2018/04/10). We confirm that all research was performed in accordance with relevant guidelines and regulations. Specimens and clinical data from patients involved in the study were collected with informed consent. Blood samples were obtained from fifty patients with kidney cancer and 49 age- and sex-matched healthy control subjects, following detailed clinical questioning at John Paul II Hospital in Kolbuszowa (Poland). All laboratory test results  were within normal ranges. Serum samples from 50 patients  with kidney cancer and 49 healthy control subjects were collected. The majority of patients  and vortex vigorously. The samples were subjected to centrifugation at 12000×g for 10 min at 4 °C. The supernatants from the pellet wash were combined with the supernatants from the first spin. Finally, from 990 μL of resulting samples, 50 μL was taken and used for 109AgNPET LDI MS analysis. The rest of the sample was dried to complete dryness in a SpeedVac vacuum concentrator, with no heat. Dried extracts were re-suspended in 600 μL of NMR buffer consisting of 25 mM NaH2PO4/Na2HPO4, 0.4 mM imidazole, 0.25 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 90% H2O/10% D2O, pH 7.0. Following re-suspension, samples were centrifuged at 21,000 rpm for 1 min to pellet insoluble debris, and then transferred to 5 mm NMR tubes for NMR metabolomics analysis.Prior to NMR analysis, serum samples were thawed at 4 °C, then centrifuged at 12000×1H NMR spectra were collected at 298 K (25 °C) using a Bruker 600 MHz (1H Larmor frequency) AVANCE III solution NMR spectrometer, equipped with a SampleJet automatic sample loading system, a 5 mm triple resonance , liquid-helium-cooled TCI NMR cryoprobe, and Topspin software (Bruker version 3.6). 1D 1H NMR spectra acquisition was performed using the Bruker-supplied excitation sculpting (ES)-based “zgesgp” pulse sequence, and NMR spectra were recorded with 256 scans and a 1H spectral window of 7211.538 Hz. Free induction decays (FIDs) were collected with 64 K data points and a dwell time interval of 69.33 μsec, amounting to a data acquisition time of 4.54 s. Relaxation delay (D1) times between acquisitions were set to 2 s, resulting in an overall 6.5-s delay between scans. DSS chemical shift referencing and phase correction of 1D 1H NMR spectra were conducted using Topspin software (Bruker version 3.6).1D 1H-1H total correlation spectroscopy (TOCSY) spectra were acquired for representative samples using the Bruker-supplied “mlevphpr.2/mlevgpph19” pulse sequences . 2D 1H-1H TOCSY spectra were processed using Topspin software (Bruker version 3.6).For verification of Chenomx-annotated metabolites, 2D 1H NMR spectra and metabolite profiling analyses were conducted using the Chenomx NMR Suite software . Baseline correction of NMR spectra following import of preprocessed “1r” NMR spectral files into Chenomx software was performed using the automatic cubic spline function in Chenomx, and subsequent manual breakpoint adjustment to obtain a flat, well-defined baseline, following recommendations from Chenomx application notes and previously reported methods [1H chemical shifts were referenced to the 0.0 ppm DSS signal, and the 1H NMR signals arising from imidazole were used to correct for small chemical shift changes due to slight variations in sample pH. Metabolite identification and quantification were performed by fitting the 1D 1H spectral splitting patterns, chemical shifts, and spectral intensities to reference spectral patterns of small molecules using the Chenomx small molecule spectral database for 600 MHz (1H Larmor frequency) magnetic field strength NMR, and manually peak-based fits, where adjustments were made to achieve optimal spectral pattern fits for compound peak cluster location and intensity. An internal (0.25 mM DSS) standard was used for metabolite quantitation. Although pulse sequences utilizing the “ZGESGP” pulse sequence scheme suppress proton signals around the water region to a greater extent than the NOESYPR pulse sequence , the relative intensities observed for these particular 1H signals whose resonance frequencies are close to that of the water 1H are largely identical to those seen using the noesypr1d sequence. To adjust for minor differences between 1D 1H spectra acquired using “zgesgp” versus “noesypr1d” pulse sequences, we have created our own in-house “zgesgp”-acquired 600 MHz metabolite library using pure standards and the “Compound Builder” module of Chenomx NMR Suite program (version 8.1), as described previously in the operational manual.Further processing of 1D  methods . 1H chem109AgNPET and allowed to dry at room temperature, then target was inserted into a MALDI ToF/ToF mass spectrometer.Volume of 0.3 μL of each sample was placed on a m/z lower than 80, with a m/z range of 80–2000 Da. Spectrum for each extract contained data from 20k laser shots with a default random walk applied (random points with 50 laser shots). All spectra were calibrated with the use of silver ions (109Ag+ to 109Ag10+). The first accelerating voltage was held at 19 kV, and the second ion source voltage was held at 16.7 kV. Reflector voltages used were 21 kV (the first) and 9.55 kV (the second). FlexAnalysis 4.0 software was used for data processing and analysis.Laser desorption ionization mass spectrometry imaging (LDI-MSI) experiments were performed using a Bruker Autoflex Speed time-of-flight mass spectrometer in positive-ion reflectron mode. The apparatus was equipped with a SmartBeam II 1000 Hz 355 nm laser, with a laser impulse energy of approximately 100–190 μJ, laser repetition rate of 1000 Hz, and deflection set on 1H NMR spectra were recorded, corresponding to serum samples of 50 patients with kidney cancer and 49 healthy controls, and 100 LDI MS spectra were recorded for the same 50 patients with kidney cancer and 49 healthy controls, and resulting data were subjected to multivariate data analysis. Both NMR and MS data sets were analyzed using multivariate data analysis to reveal metabolic changes in the serum of patients with kidney cancer with respect to healthy controls.A total of 99 Q2), and goodness of fit (R2). Variable Importance in the Projection (VIP) plots and S-plots were generated to identify metabolites whose level changes were most significantly responsible for group separation. Metabolites with VIP scores > 1, |p| > 0.05 (magnitude), and |p(corr)| > 0.5 (reliability) were considered to be potential biomarkers that distinguish kidney cancer patients from healthy controls. In this work, 10-fold cross validations were used to define the number of latent variables (PLS components) in the model. To test the accuracy of multivariate models and minimize the possibility that the observed separation in the PLS-DA and OPLS-DA is due to chance (p < 0.05), permutation tests were performed with 2000-fold repetition. Statistical significance of metabolite level differences was assessed using unpaired parametric t test with Mann-Whitney and Bonferroni correction. p values and false discovery rates  less than 0.05 were considered statistically significant. Moreover, receiver operating curve (ROC) analysis was done to evaluate the diagnostic value of selected metabolites. Standard chemometrics tools such as 2D PCA and PLS-DA analysis were also used to assess metabolic profile similarities and differences between cancer types  and grades . An approximation was used whereby all malignant renal tumors like ccRR, chRCC, pRCC, CDC, SRC, and benign kidney tumors (oncocytoma and AML) were grouped together, expecting that changes in serum metabolite levels in tumor types would follow similar trends.Metabolite concentrations normalized by sum were further log-transformed to ensure a Gaussian distribution of the data and auto-scaled , prior to statistical analysis, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA), which was accomplished using the MetaboAnalyst software 4.0 . The ove1H NMR spectra on 99 total (50 RCC and 49 controls) metabolite extracts from serum samples.In this study, we characterized the metabolic profiles of patients suffering from kidney cancer, in an effort to develop serum-specific metabolomics signatures for early and specific detection of kidney cancer. For this purpose, we recorded high-resolution 1D 1H NMR spectral patterns resulted in the resonance assignment and quantitation of 43 metabolites in each serum sample. Representative 1H NMR spectra of serum samples from healthy subjects and kidney cancer patients are shown in Fig. Analysis of Significant differences were observed in levels of individual metabolites when the metabolic profiles of serum samples from patients with kidney cancer were compared with those of healthy controls. Multivariate and univariate statistical analyses of these metabolite patterns were performed to assess similarities and differences in serum metabolomes from the two different groups. This analysis also enabled us to identify a panel of polar small molecules whose levels are altered significantly in kidney cancer.Multivariate analysis was conducted based on metabolite concentration data obtained by NMR. Unsupervised principal component analysis (PCA) was performed to search for trends in group separation and to identify potential outliers. The results are presented as 2D PCA scores plots, where each colored dot represents a serum sample from a cancer patient (red) or healthy control (green) in Fig. The 2D PCA score plot shown in Fig. R2 and Q2 values and data permutation tests. This analysis resulted in an R2 value of 0.92, a Q2 of 0.86, an accuracy of 0.99, and p values < 5E−4 for the data shown in Fig. R2Y and Q2 were respectively 0.92 ) and 0.865  giving an indication of good fitness and predictability of the two-class model | > 0.5). Mean metabolite concentrations with all relevant statistical parameters are summarized in Table myo-inositol, and pi-methylhistidine , followed by glucose , choline , and lactate . The distribution of concentration values of individual metabolites in control and cancer serum samples is shown in Fig. Among these metabolites, best results with highest significant were achieved for the metabolite Ten metabolites exhibiting the highest AUC > 0.8 in the ROC curve generated Fig. , ESM wer1H NMR metabolomics can significantly differentiate between kidney cancer tumor types  and healthy control subjects, we assessed whether the 8 most significant metabolites identified in our PCA and PLS-DA analyses can also serve to distinguish between the different grades of kidney cancer tumors (grades 1–3) and the healthy control groups. A 2D-PCA score plot highlighting the extent of the separation three groups  based of distinct metabolic serum is shown in Fig. To demonstrate whether metabolite fingerprinting of serum extracts using 1H NMR. This suggests that metabolic patterns for those groups are not easily separable by just simply assessing differences in polar metabolite levels in serum samples. Similarly, 2D-PLS-DA analysis could not distinguish clearly between tumor types and grades, as illustrated in Fig. Both 2D PCA score plots shown in Figs. p < 0.05)  that contributed most to group separation were considered significant with p and q values < 0.05. As a result, 62 m/z variables were detected as potential serum discriminators of kidney cancer versus controls, using 109AgNPET LDI MS  were removed from this analysis. Next, OPLS-DA was applied to the MS dataset to reveal the features that had the greatest contribution to the discrimination between serum samples from kidney cancer and controls ) and 0.708 , giving an indication of good fitness and predictability of the two-class model  [m/z values could be used as diagnostic variables enabling the distinction between kidney cancer and the control groups with high specificity and sensitivity.Selected e (HMDB) . All of p < 0.05) metabolite level changes, cancer patient versus control groups could be clearly differentiated.Kidney cancer is one of the most studied neoplasms characterized by metabolic reprogramming. Changes in cellular metabolism are reflected in the concentration changes of detected metabolites. The best material for biomarker and metabolomics research is cell cultures and tissues. However, tissue samples must be obtained in invasive way . Therefore, body fluids like urine and serum, which can be obtained with minimal discomfort, are the most preferable materials for biomarker discovery in cancer research. Metabolite levels are in constant flux between tissue and body fluids; therefore, changes in tissue metabolism are reflected in changes in the metabolomes of biofluids. In this study, we analyzed changes in metabolites, which were extracted from serum samples of kidney cancer patients and healthy controls. These data demonstrated that, based of statistically significant  that are not synthesized de novo by humans. Our results do not coincide with previous NMR-based serum metabolomics studies that described the increased leucine levels from RCC patients , 11. RegRegarding lipid metabolism, choline levels were decreased in the serum samples of RCC patients compared with controls, which may be a consequence of increased choline uptake by cancer cells to fuel phospholipid production for cell membrane formation. Similar results were reported by Gao et al. who observed, in two separate studies, decreased level of serum choline and increased concentration of choline in RCC tissue , 21. MorIn this study, we observed decreased level of glycerol in serum samples of RCC patients. Glycerol is a precursor for the synthesis of triacylglycerols and phospholipids, the latter being major structural components of cellular membranes. Moreover, during prolonged fast or in a nutrient-depleted environment (cancer cachexia), glycerol and other substrates (lactate and α-keto acids) may be transformed in liver and kidney into glucose through gluconeogenesis. This finding is also supported by the research of Catchpole et al. who has shown that glycerol is lower in abundance in RCC tissue .myo-Inositol, a carbocyclic sugar, is a precursor of secondary messengers including inositol triphosphate and phosphatidylinositol, which mediate cell signal transduction in response to hormones, neurotransmitters, and growth factors. In our study, and when comparing the kidney cancer group to controls, myo-inositol levels were found to be decreased in the serum of RCC patients. Similar results concerning the level of myo-inositol were reported by Popławski et al. and Catchpole et al. [e et al. . The resOther metabolites that were found in lower abundance in the sera RCC patients included 1-methylhistidine. Prior studies conducted by Felagan et al. have also confirmed that level of 1-methylhistidine is found to be decreased in the urine of kidney cancer patients . 1-Methy1H NMR and 109AgNPET LDI MS, along with multivariate statistics, are useful approaches to characterize the serum metabolome differences between patients with kidney cancer and healthy people and to separate these two groups based on their distinct serum metabolite profiles. Moreover, 1H NMR enabled to identify trends between tumor grades and to discriminate between different types and grades of kidney cancer and healthy controls. With regard to biomarker discovery, 8 potentially robust metabolic biomarkers in 50 serum samples of kidney cancer patients and 49 controls were identified using 1H NMR spectroscopy, while 9 mass spectral features were obtained by LDI MS. The most important endogenous compounds having bioactive properties and pharmacological applicability were discussed in details. Both methods have a valuable potential for use as diagnostics and analysis of serum metabolic profiles may be a useful, less invasive way to screen patients with kidney cancer.This work has demonstrated that high-resolution ESM 1(PDF 2697 kb).


Anorectal malformations (ARM) are a spectrum of anomalies of the rectum and anal canal affecting 1 in 2500 to 5000 live births. Functional problems are common and related to the type of ARM and associated malformations. We aimed to evaluate the results of Three-dimensional High Resolution Anorectal Manometry (3D-HRAM) in long-term follow-up after surgical correction of ARM with special reference to fecal incontinence. Twenty-one patients with anorectal malformations and primary repair at our center consented to participate in the study. Pressures of the anal sphincter muscles and defects were addressed by 3D-HRAM. Fecal incontinence and disease-specific quality of life were evaluated by the Fecal Incontinence Quality of Life score and Wexner incontinence score respectively. The study was approved by the Committee in Health Research Ethics and the Danish Data Protection Agency. Median age was 22(12–31) years and 13(67%) participants were females. Sphincter defect was present in 48% (N = 10) of participants. Participants with sphincter defects had significant higher Wexner score and size of sphincter defects and mean anal squeeze pressure were correlated to Wexner score. Participants with or without sphincter defects did not differ on manometry parameters including resting anal and squeeze pressure or disease-specific quality of life. In a study of the long-term outcome after repair of anorectal malformations we found a higher Wexner incontinence score in the presence of an anal sphincter defect and the size of the defect and mean anal squeeze pressure were correlated to the Wexner incontinence score. Even after proper reconstruction, functional problems are common and related to the type of ARM and associated malformations. Only few studies have addressed the findings on advanced anorectal manometry in relation to the long-term functional outcome3.Anorectal malformations (ARM) are a spectrum of anomalies of the rectum and anal canal affecting 1 in 2500 to 5000 live births4. The results have been contradictory and with dubious clinical value. Some studies have found a correlation between the extent of sphincter damage and the degree of continence and others have not5. Three-Dimensional High Resolution anorectal manometry (3D-HRAM) is a technique providing an image of the pressure profile of the sphincter complex. In patients with fecal incontinence of other causes, 3D-HRAM has shown a high negative predictive value for the finding of an acquired anatomic sphincter defect compared to EUS6. To our best knowledge no previous studies has addressed the value of the 3D-HRAM anal manometry in the long-term follow-up after surgery for ARM.Conventional anorectal manometry has gained widely use and acceptance for the evaluation of the anorectal function in different types of anorectal malformationsThe aim of the present study was to evaluate the results of 3D-HRAM in the long-term follow-up after surgical correction of ARM with special reference to the status of fecal continence.The incidence and severity of soiling and constipation are presented in Table In approximately half of the subjects (N = 10) a sphincter defect was detected by HRAM and in 30% the defect affected more than half of the sphincter circumference. Largest sphincter defect amounted 249 degrees and the smallest 23 degrees. Median circumferential sphincter defect was 90°. Subjects with lower ARM had significant higher anal resting pressure and anal squeeze pressure compared to subjects with intermediate ARM.One participant was unable to distinguish between first sensation, urge and discomfort in rectal sensitivity test and in one patient first sensation and urge appeared at the same volume.Participants with a sphincter defect, was similar to participants without sphincter defect regarding age, gender, resting pressure, squeeze pressure, rectoanal pressure gradient and HPZ as presented in Table In participants with sphincter defect, more suffered from soiling compared to participants without sphincter defect (P = 0.009).Participants with low malformations had lower anal resting pressure and squeeze pressure (p < 0.05). No differences were found regarding age, gender, anorectal pressure gradient, size of HPZ, appearance or size of functional sphincter defects between participants with low and intermediate malformations. One subject without a fistula and one patient with a cloaca malformation were not included in this calculation.Figure In our study approximately half of the participants (48%) had an anal sphincter defects detected by 3D-HRAM after repair of anorectal malformations. We found size of functional sphincter defects and the mean anal squeeze pressure to be correlated to the Wexner incontinence score.7. The extent of the internal anal sphincter was proportionally correlated to a decrease in the mean resting anal pressure. The severity of fecal incontinence was assessed with Wexner score. The study was different from our study in only including women with a mean age of 60 years after vaginal delivery without repair of the anal sphincter. We found the extent of sphincter defect correlated to a decrease in mean anal squeeze pressure. A similar observation was found by Mion et al. in a study comparing 37 female patients with fecal incontinence to 36 asymptomatic volunteers and 40 constipated patients, all subjected to 3D HRAM8. Significantly more patients with fecal incontinence had pressure defects compared to asymptomatic controls. Patients with fecal incontinence with a median age of 62 years were significant older than both asymptomatic and constipated patients. Apart from being older patients were different from our population due to the fact that only 11 out of 37 (29%) patients had previous anorectal surgery compared to 18/21(86%) in our patients ignoring patient treated with dilatations.The extent of anal sphincter defect as detected by anal ultrasonography has been correlated to the severity of fecal incontinence in other populations. In a study of 250 women with a history of vaginal delivery the authors were unable to find any significant correlation between the size of an anal sphincter defect and severity of fecal incontinence9. The problem is that, although the structural defects may be important in the consideration of sphincter repair, the functional defects may explain the relative poor outcome in re-do procedures10.Endosonography has been the gold standard for assessing the anatomical integrity of the anal sphincter after surgery for anorectal malformationset al., internal anal sphincter lesions were found to be common after surgical repair of anorectal malformations. It was detected in 60% of patients with low and intermediate malformations using 3D-endoanal ultrasound (3D-EAUS)11. The study included 17 children after surgical repair with posterior sagittal anorectoplasty. Six out of 10 patients with low or intermediate malformations had a sphincter lesion, which they deemed to be confined to internal anal sphincter (IAS). However, an anatomic distinction between the different elements of the sphincter complex after PSARP may be difficult and the clinical importance of this is unknown especially for the minor defects. The 3D-HRAM examination gives detailed information on the function of the sphincter complex, which was correlated to functional outcome in our study. The correlation between anal defects shown by 3D-HRAM and defects by ultrasonography or MRI in patients operated for ARM has yet to be proven. We were not able to make an anatomic distinction between the different elements of the sphincter complex with 3D-HRAM but nonetheless able to correlate sphincter defects to functional outcome.In a study by Caldaro 9. The mean age at follow-up was 16 years (range 1–22). Gender distribution was not described. Twenty-five of the 40 included patients were characterized with a high malformation. In our study only two participants were registered with high malformations. An average defect of 2.5 and 3 quadrants in the internal (IAS) and the external sphincter (EAS) was found respectively. A four level continence scoring system was applied ranging from one to four. Grade four incontinence was defined as incontinence for gas, loose and solid stool. The extent of muscle defect in the IAS was found to be correlated with the degree of incontinence.In former studies with conventional manometry a correlation between manometric parameters and clinical outcome has been found. Emblem and colleagues assessed postoperative anatomy in 40 patients with 2D - EAUS after repair of anorectal malformations and compared it to 20 healthy participantset al. 39 adult patients with fecal incontinence but with no previous history of ARM underwent EAUS and 3D-HRAM6. Mean age was 65 years. Thirty-one patients were females (79%) and approximately two out of three had had at least one vaginal delivery. Defects were defined as any pressure measurements below 25 mmHg equal to pressure limit for detecting anal sphincter defect in our study. The negative predictive value of HRAM in detecting sphincter defects was found to be 92%. Unfortunately there is no information on any correlation between findings from HRAM and functional outcome. In a similar study where the cut-off value for sphincter defects was a circumferential area with pressure below 10 mmHg the sensitivity and specificity of detecting EAS defects by 3D-HRAM were 65%12. It is generally accepted that the resting anal pressure is maintained by the IAS accounting for about 55% with the EAS and hemorrhoidal cushions adding 30% and 15%, respectively14. The anatomy and function of the pelvic flour play an important role for the size of squeeze pressure15. Thus, a comparison of the results of HRAM between ARM patients with other populations should be interpreted with caution. The fact that low-pressure areas on the 3D cylindrical presentation of pressure contribution as detected by HRAM may be misinterpreted as sphincter defects needs to be taken into account. Normal asymmetrical pattern of pressures in the anal canal is properly a common cause16. In patients with ARM hypo-development of the anal sphincter complex in different degrees is common and may alter the pressure distribution as detected by HRAM17.In a study by Rezaie et al. in a retrospective study of 221 patients18. The 3D probe enhances diagnostic gains by the identification of 29 focal anal sphincter defects not identified by 2D examinations and had a fair inter-reader agreement. It is important to identify the sphincter defects with clinical contribution in order to stratify patients to the proper treatment. Future studies in large populations need to correlate localization and extent of low-pressure areas in anal canal as detected by HRAM and perform HRAM standardized according to consensus recommendations19. The strength of this trial was the standardized and protocolized HRAM investigations performed by one experienced examiner. The study has some imitations. The inclusion rate was low and a possible cause of selection bias and a low statistical power. A reason for non-consenting could be that patients with anorectal malformations have been subjected to several unpleasant examinations of the anorectum during childhood and the fact that the local ethical committee only allowed invitation by letter. We assume that invitation by phone could have increased the participation rate. Although most of the patients included had a “favourable” type of anorectal anomaly, the population was heterogenic concerning age and type of repair.The application of a three-dimensional probe to HRAM equipment as performed in our study has been compared to the use of a two-dimensional probe in a study by Raja The 3D-HRAM is able to provide information on the functional anatomy of the sphincter complex after reconstruction for ARM. The clinical significance of the method has yet to be proven.Participants were recruited from a surgical center at Odense University Hospital (OUH), Denmark with a catchment area of approximately 3.5 million people. A thorough search in local databases using the ICD-9 codes: 725.1, 725.2 (1985–1994) and ICD-10 codes: Q42  and Q438K (1995–2004) was performed. Inclusion criteria were primary surgery for anorectal malformations from 1985 to 2004 at OUH. Exclusion criterion was severe mental disability.Patients were invited by letter. A second invitation was send after 14 days if the subject had not answered. Twenty three subjects fulfilled inclusion criteria and consented to participate Fig. . It was In all included patients primary surgery was performed within the first year of life. There for the HRAM examination was performed at least 11 years after reconstructive anal surgery owing to the fact that the youngest participant was 12 years of age.The following data were retrieved: age, comorbidity, type of malformation and type of primary surgery, other congenital abnormalities or malformations and in women, childbirth.th revision of the Helsinki declaration. Prior to inclusion verbal and written informed consent was obtained. In participants below 18 years of age, the consent was retrieved from parent(s) or guardian(s). Approval was obtained from the National Committee on Health Research Ethics and the Danish Data Protection Agency.The study was performed in accordance with the 7TM anorectal High Resolution Manometry system . A 3D probe was mounted. The probe is composed of 256 pressure sensors circumferentially aligned over the length of 64 mm and a circumference of 10 mm. The probe was calibrated before every examination. A disposable sheath with a rectal balloon was applied to the probe and lubricated before use. Examinations were performed with the patient in the left decubitus position with hips and knees flexed 90 degrees. A rectal examination was done to ensure the rectum was empty before introducing the probe. The examinations were carried out by Author (TBM) with experience in performing anorectal manometries.The manometries were performed with a ManoscanA resting period of one minute was awaited after introducing the probe before measurements. First the resting pressure was measured during a period of 20 seconds three consecutive times. The mean value was obtained. Next, Squeeze and push maneuvers were performed three times with 30 seconds intervals and mean values were used for the calculation. The recto-anal inhibitory reflex was elicited after the rectal balloon was forcefully inflated with increasing volumes of air in 10 ml aliquots. The attempt was ended at 60 ml if the reflex was not elicited. Finally, rectal sensitivity was examined. It was evaluated by inflating 10 ml of air consecutively until the patient expressed first sensation, urge to defecate and discomfort.The procedure was stopped after inflating a total volume of 400 ml.The presence, size and location of a functional sphincter defect were calculated from the 3D presentation of the anal resting pressure profile  if not otherwise indicated. The Fisher’s exact test was used for discrete data and for continuous data the Mann-Whitney U test was applied. A p-value below 0.05 was considered statistical significant and p-values were reported 2-sided. Spearmann’s rank order correlation was used to measure the strength and direction of association between variables and reported with a correlation coefficient rho (ρ).20 of postoperative results  - score22.Soiling and constipation were evaluated with the Krickenbeck Classificationts Table . The sevSubject characteristics are presented in Table


Conclusion: KeGFR computed from the first day after renal transplantation was able to predict SGF with good discrimination, outperforming CKD-EPI estimation. SGF patients had lower renal graft function overall at the one-year follow up.Background: Rapid identification of patients at high risk for slow graft function (SGF) is of major importance in the immediate period following renal graft transplantation, both for early therapeutic decisions and long-term prognosis. Due to the high variability of serum creatinine levels after surgery, glomerular filtration rate (GFR) estimation is challenging. In this situation, kinetic estimated GFR (KeGFR) equations are interesting tools but have never been assessed for the identification of SGF patients. Methods: We conducted a single-center retrospective cohort study, including all consecutive kidney allograft recipients in the University Hospitals of Geneva from 2008 to 2016. GFR was estimated using both CKD-EPI and KeGFR formulae. Their accuracies for SGF prediction were compared. Patients were followed up for one year after transplantation. Results: A total of 326 kidney recipients were analyzed. SGF occurred in 76 (23%) patients. KeGFR estimation stabilized from the day following kidney transplantation, more rapidly than CKD-EPI. Discrimination ability for SGF prediction was better for KeGFR than CKD-EPI (AUC 0.82 and 0.66,  Kidney transplantation is the current best treatment for end-stage renal disease . DelayedIn the post-transplantation setting, KeGFR could be of great interest as the serum creatinine levels are not stable . One stuWe conducted a single-center retrospective cohort study at Geneva University Hospitals, Switzerland. Patients were included from January 2003 to December 2016. All kidney allograft recipients older than 18 years old were enrolled. Patients were secondarily excluded if the recorded serum creatinine measurements were not sufficient to calculate eGFR and define the status “slow graft function”. This study was approved by the ethical committee for human studies of Geneva, Switzerland , and performed according to the Declaration of Helsinki principles.Slow graft function (SGF) was defined as a creatinine reduction rate >20% at postoperative day (POD) 3 with a POD3 serum creatinine level greater than 132 µmol/L . The creThis study was completed using the intensive care unit database from Geneva University Hospitals, updated with all serum creatinine values measured during the 12 months following discharge. All data were anonymized and analyzed in a blinded manner. 2. Renal function during the follow up was estimated using CKD-EPI equation.Serum creatinine concentrations were measured using the Jaffé method with isotope dilution mass spectrometry (ID/MS) standardization. CKD-EPI was calculated as described. KeGFR was calculated using Chen’s formula :KeGFR =  by Chen . KeGFR wContinuous variables for individuals were expressed as the mean ± standard error (SE) or as the median and the 25th–75th percentile for non-normally distributed variables. Comparisons between groups were performed using the Wilcoxon rank-sum test.Categorical variables were expressed as the absolute and relative (%) frequency and compared using Fisher’s exact test.eGFR and c-statistic were expressed as the mean ±95% confidence interval estimated by bootstrapping. ROC curves were compared by the log-rank test.Individual trajectories of eGFR was fitted using a linear model.p-value below 0.05 was considered significant, and all p-values were two tailed. p-values were adjusted for multiple tests using Holm methods when necessary. Statistical analyses were performed using R software.A n = 185, 57%) and donation after cardiac death . Median eGFR at 12 months using CKD-EPI equation was 54 (44–67) mL/min/1.73 m2. SGF occurred in 76 (23%) patients. Comparatively to the kidney recipients who did not experience SGF, patients displaying SGF were older , received an allograft less frequently from a living donor , which donors were also generally older  and needed more frequently dialysis . In patients with SGF, 3 month, 6 month and one-year estimated GFRs after transplantation were lower (42 (32–63) mL/min/1.73 m2 versus 56 (48–68) mL/min/1.73 m2, 46 (31–60) mL/min/1.73 m2 versus 54 (45–69) mL/min/1.73 m2, 47 (38–60) mL/min/1.73 m2 versus 57 (45–69) mL/min/1.73 m2, respectively, p < 0.005). From January 2003 to December 2016, 359 patients older than 18 years old who underwent a kidney allograft in the Geneva University Hospitals were screened. We excluded 21 (6%) patients as KeGFR estimations could not be performed at POD 1. Finally, we excluded 12 (4%) patients as the SGF status could not be assessed due to missing creatinine values. We finally analyzed 326 patients . In this2/day, p < 0.001). CKD-EPI evolved differently. Although it did not increase in SGF patients , patients without SGF displayed a rapid increase in eGFR  following the serum creatinine levels   a,b. 2, p < 0.0001 and 10 (8–15) versus 16 (10–26) mL/min/1.73 m2, p < 0.001, respectively) (p < 0.001). At POD 1, the area under the ROC curve for SGF prediction was better for KeGFR compared to CKD-EPI  and  for GFR estimated using KeGFR or CKD-EPI respectively). Using POD1 KeGFR, the optimal cut-off point giving by the ROC01 index was 15 mL/min/1.73 m2 and predicted SGF with a sensitivity of 0.79, a specificity of 0.74, and a positive predictive value of 0.91. The optimal cut-off point for CKD-EPI was 12 mL/min/1.73 m2 and predicted SGF with a sensitivity of 0.64, a specificity of 0.66 and a positive predictive value of 0.86 (Median POD1 KeGFR and CKD-EPI were lower in patients experiencing slow graft function (10 (4–17) versus 33 (17–49) mL/min/1.73 mctively) a but the of 0.86 b. We then considered the 262 patients who had KeGFR and GFR recorded each in the three first postoperative days following transplantation. During the whole period, KeGFR better discriminated the SGF and non-SGF groups than CKD-EPI eGFR. c.2 LOF  for KeGFR and −37 mL/min/1.73 m2 LOF  for CKD-EPI . By contrast, patients who had initially experienced SGF had lower renal function during the entire follow-up period   c.). Usinp = 0.004)    a. At PODr males) b as wellctively) c.In this retrospective analysis, we compared the estimation of GFR using CKD-EPI and KeGFR formulae in the five days following renal transplantation to predict SGF in kidney allograft patients. We confirmed the high incidence of SGF, occurring in one over five patients, and its association to a mid-term worse renal prognosis. KeGFR estimation was stable from the day following surgery and did not change in a clinically significant manner in the next five days. On the other hand, CKD-EPI increased all over the five first days. KeGFR was more discriminative to predict SGF than CKD-EPI from POD1 to POD3.p < 0.001). This is in line with the incidence of SGF reported in the literature, ranging from 11.2% to 42%, depending on the definition used, and the population studied [In this work, we showed that SGF is a frequent condition occurring in 23% of patients, more in grafts originating from donation after death than in kidneys from living donors (33% versus 9%, respectively,  studied ,22,23.Secondly, we confirmed the greater responsiveness and reliability of KeGFR in the immediate postoperative period. KeGFR has been specifically designed to estimate GFR when serum creatinine levels are not stable, for example during acute kidney injury . Other bWe show here that identification of this high-risk population using KeGFR is possible as early as at POD1, independently of the gender, potentially allowing care optimization of these patients. However, our study has some limitations. First, our analyses were retrospective and limited to the recorded data, missing some variables, such as cold ischemia time, immunosuppressive regimen, HLA mismatch ratio or native kidney disease. Nevertheless, these data were not crucial for our study, and the number of excluded patients because of missing data remains low. Second, as a single-center study, we were not able to validate the predictive value of KeGFR in an independent cohort. However, our other findings were consistent with the literature. Third, as there is not a universally accepted definition of the SGF phenotype, our results are limited to the SGF definition we chose, based on recent literature . We furtOur study has several strengths. First, we included a larger number of patients than most studies published on KeGFR. Second, to our knowledge, this study is the first describing the discriminative value of KeGFR to predict SGF. Third, since we included renal graft recipients from both living and deceased donors, our results are easily transposable to other populations.Altogether, we show that KeGFR may be used from the first day after transplantation to identify patients with SGF with good sensibility and specificity, and much more rapidly than CKD-EPI, which should not be used in this situation. Patients with SGF were further shown to have lower renal graft function at one-year follow up.


IntroductionPercutaneous nephrolithotomy (PNL) has replaced open surgery for the treatment of kidney stones due to its less invasive nature. Bleeding still occurs due to renal vascular injuries, dependent upon the access route of the procedure. Several other factors are also related to the increased risk of bleeding. This study aims to find the association between blood transfusion and other factors such as age, gender, body mass index (BMI), size of the stone, operative time, preoperative hemoglobin (Hb) level, stone surface area, hypertension, and diabetes mellitus.Materials and methodsThis was a descriptive cross-sectional study conducted over a period of six months between November 2019 and April 2020 at a tertiary care hospital in Karachi, Pakistan. The sample size of 131 patients was calculated using open-source epidemiological software (Open-Epi). Inclusion criteria included patients from both genders and ages between 26 and 70 years. Patients ≤25 years, having a liver disease or bleeding disorders, or refusing to participate in the study, were excluded. Laboratory data included preoperative routine complete blood count, serum creatinine , platelet count, bleeding and coagulation profile, and urine culture. All patients also underwent renal ultrasound scans. Treatment was postponed until a negative urine culture was obtained from patients with a positive urine culture. ResultsThe mean age of the patients was 42.4 ± 15.65 years. One third (29.8%) of the patients were females. The stone size was 850 ± 121.43 mm², the mean operative time of the procedure was 125.76 ± 53.4 minutes, and the mean number of cell packs transfused was 1.10 ± 0.31 units. Blood transfusion was done in 24 (18.3%) of the patients. Gender, diabetes mellitus, stone size, preoperative Hb level, and operative time were significantly related to blood transfusion. ConclusionsIncreased bleeding risk while performing PNL has been associated with many factors such as operating time, the gender of the patients, and stone size. Therefore, these factors should be controlled for the procedure to decrease the risk of bleeding and the need for blood transfusion. Furthermore, the kidney vasculature should not be compromised while performing the procedure. In percutaneous nephrolithotomy (PNL), the percutaneous technique of insertion of a small-caliber nephrostomy tube is used to access the kidney and drain an obstructed renal unit. Ever since its discovery, the percutaneous removal of kidney stone has replaced open techniques and is now being used with extracorporeal shockwave lithotripsy (SWL) in the clinical setting . These nMany factors affect renal hemorrhages, such as the operative time, upper calyceal access, multiple access sites, method of tract dilatation, number of tracts, size and location of stones, staghorn calculi, diabetes mellitus in patients, and the experience of the surgeon . BleedinGuidelines of the American Association of Urology (AUA) describe the use of PNL as the first-line for large kidney stones . This prStudy design and samplingThis descriptive cross-sectional study was conducted from November 2019 to April 2020 for a duration of six months in the urology department of a tertiary care hospital in Karachi, Pakistan. A sample size (n) of 131 patients was calculated using open-source epidemiological software (Open-Epi). The expected prevalence was set as 14.2% at a 95% level of confidence and a 5% margin of error. The inclusion criteria included kidney stone patients aged 26-70 years of either gender. Patients aged ≤25 years, having a chronic liver disease or bleeding disorders as per their record, and refusing to participate in the study were excluded.Data collectionPatients who fulfilled the inclusion criteria were included in the study and written informed consent was taken. Laboratory data included preoperative routine complete blood count, serum creatinine , platelet count, bleeding and coagulation profile, and urine culture. Renal ultrasound scans were performed on all patients. Treatment was suspended until a negative urine culture was achieved from patients with a positive urine culture. Socioeconomic status was defined as lower class for patients with monthly income <10,000 rupees(Rs)/month, middle class for 10,000 - 30,000 Rs/month, and upper class for >30,000 Rs/month.All PNL procedures were performed with patients in a prone position and under general anesthesia. The experience of the surgeon was at least three years. The entrance into the collecting system was confirmed on the removal of the stylet of the needle. A 0.038-inch J-tip wire was inserted and access was dilated. The tracts were dilated up to 30F in adults. The number of tracts was made according to the stone volume and dimensions. In all cases, pneumatic lithotripsy was used. Either 14F reentry or 8F pigtail nephrostomy tubes were inserted after completion. No hemostatic agent was used and the nephrostomy tube was clamped overnight and subsequently removed. The stone burden was calculated in mm² by the product of the longest dimension and the one perpendicular to it. Postoperative hemoglobin (Hb) and hematocrit were measured. Blood loss was calculated using the following formula: (preoperative blood Hb - postoperative Hb) + (number of units transfused x 1 g/dL per unit transfused) [Data analysisData were entered and analyzed through the Statistical Package for Social Sciences (SPSS) version 23.0 . Mean and the standard deviation was calculated for quantitative variables including age, BMI, serum creatinine level, size of the stone, operative time, number of cell packs, preoperative Hb level, decrease in Hb level, and stone surface area. Frequencies and percentages were calculated for qualitative variables such as hypertension, diabetes mellitus, preoperative urinary tract infection (UTI), gender, staghorn stone, chronic renal failure, socioeconomic status, number of tracts, access calyx, dilation method, side of puncture, and outcome . Effect modifiers such as age, gender, socioeconomic status, BMI, hypertension, diabetes mellitus, size of the stone, stone surface area, preoperative Hb level, operative time, preoperative UTI, chronic renal failure, staghorn stone, number of tracts, access calyx, dilation method, and side of puncture were controlled through stratification. The post-stratification chi-square test was applied by taking p-value ≤0.05 as significant. 2. The serum creatinine level of the patients was 2.20 ± 1.42 mg/dL, the stone size was 850 ± 121.43 mm², the mean operative time of the procedure was 125.76 ± 53.4 minutes, and the mean number of cell packs transfused was 1.10 ± 0.31 units. The mean preoperative Hb level was 12.88 ± 7.5 g/dL, the mean decrease in Hb level was 2.3 ± 1.98 g/dL, and the mean stone surface area was 5.9 ± 4.3 cm2. These descriptive statistics of all quantitative variables with their means and standard deviations are shown in Table The mean age of the patients was 42.4 ± 15.65 years and the mean BMI was 24.7 ± 6.33 kg/mOver 92 (70.2%) of the study subjects were male and 39 (29.8%) were female. Approximately one-third of the patients were female. 75 (57.25%) study subjects had a history of hypertension and 56 (42.75%) were normotensive patients. 78 (59.54%) of the study subjects were diabetic and 53 (40.46%) were non-diabetic patients. 75 (57.25%) study subjects had preoperative UTI and 56 (42.75%) did not have pre-operative UTI. Type of staghorn stone was also recorded; 49 (37.4%) of the patients had partial staghorn stone, 40 (30.5%) had complete staghorn stone, and 42 (32.1%) had multiple stones. 92 (70.2%) study subjects had chronic renal failure and 39 (29.8%) had a normal renal function. In socioeconomic status, it was found that most of the patients were from the middle class, i.e., 52 (39.69%), 31 (23.66%) were from the lower class, and 48 (36.65%) were from the upper class. Socioeconomic status did not affect the outcome. In the distribution of the number of tracts, 67 (51.14%) patients had single and the remaining had multiple tracts. Access calyx was also distributed, 28 (21.37%) patients had access from superior, 51 (38.93%) had access from medium, and the remaining 52 (39.69%) had access from the inferior calyx. 71 (54.2%) patients’ side of puncture was left and 60 (45.8%) patients’ side of puncture was right. This analysis of all qualitative variables is shown in Table The blood transfusion was needed in 24 18.3%) of the patients while 107 (81.7%) of the patients did not need a blood transfusion, as shown in Figure 8.3% of t2), hypertension, diabetes mellitus, preoperative UTI, gender, staghorn stone, chronic renal failure, socioeconomic status, number of tracts, access calyx, dilation method, and side of puncture with blood transfusion was performed. Gender, diabetes mellitus, stone size, preoperative Hb level, and operative time were significantly related to blood transfusion, as shown in Table Stratification of age, BMI, size of the stone, operative time, preoperative Hb level, stone surface area  . In anotTaking a closer look into Table There are some limitations to our study. Univariate and multivariate analysis can be done to further consolidate the comparison of the variables to blood transfusion. Our age group mostly includes older patients. A larger sample size with more variation in age groups is needed. Furthermore, conservative management and embolization groups can be created to further evaluate transfusion necessity as well as hospital stay and drop in hemoglobin after the procedure.PNL is a safe procedure for kidney stone removal, especially in the treatment of large stones. However, certain factors need to be controlled to remove the risk of severe bleeding. Access of the kidney should be appropriate to ensure that the kidney structure is minimally manipulated and the vasculature is not compromised. At the same time, operative time should not be too long. Female gender, stone size, diabetes mellitus, and preoperative Hb level may also predispose patients to more bleeding. Thus, bleeding risk can be reduced by controlling these factors.