We examined standard clinical and laboratory biochemical parameters, as well as the levels of aminothiols in the blood and urine (homocysteine (Hcy), cysteine (Cys), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)) via capillary electrophoresis in patients with CKD at stages II–V. Patient outcomes were assessed after five years. To complete forecasting, correlation and ROC analysis were performed. It was found that the levels of Cys and Hcy in blood plasma were earlier markers of CKD starting from stage II, while the levels of SAM and SAM/SAH in urine made it possible to differentiate between CKD at stages II and III. Blood plasma Hcy and urinary SAM and SAM/SAH correlated with mortality, but plasma Hcy concentrations were more significant. Thus, plasma Hcy, urine SAM, and SAM/SAH can be considered to be potential diagnostic and prognostic markers in patients with CKD. Chronic kidney disease (CKD) is a condition characterized by progressive loss of kidney function over several months or years. The pathogenesis of kidney damage includes a complex set of mechanisms, in which circulatory , coronary heart disease (CHD), etc.) and dysmetabolic disorders (diabetes mellitus (DM), gout, etc.) occur, which, in turn, are the most common causes of renal dysfunction ,2,3,4. CThanks to the development of proteomic diagnostic methods, it has become possible to determine protein metabolites in the blood and urine, which not only reflect the dynamics of the development of renal dysfunction, but are also directly involved in the pathogenesis of these disorders, including metabolites of the methionine cycle. Methionine can exogenously enter the body through consumption of food or be formed as a result of the breakdown of endogenous protein. Methionine is then converted into S-adenosylmethionine (SAM) by the enzyme SAM synthase, which is, in turn, converted into S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed by SAH hydrolase to form Hcy and adenosine. Hcy can be utilized in two ways—remethylation (RM) or trans-sulfonation (TS)—followed by regeneration into methionine or conversion into cysteine, respectively ,19,20. IAll procedures performed in human studies were carried out in accordance with the ethical standards of the institutional and/or national research committee, as well as the Declaration of Helsinki (1964) and its later amendments or comparable ethical standards. This study’s protocol was approved by the Ethics Committee of the Clinical Hospital No. 24 of the Moscow Health Department, where all patients included in this study were hospitalized and treated .The study was conducted from 18 September 2017 to 30 November 2017. The inclusion criteria for the study were as follows: being aged over 18 years old, a history of chronic kidney disease at stages II–V for more than one year, and obtaining written informed consent to participate in this study from the patient or their relatives. The exclusion criteria of this study were as follows: pregnancy, lactation, HIV infection, viral hepatitis, non-vascular diseases of the central nervous system, severe concomitant somatic pathology , and severe purulent-septic diseases. The termination criteria of the study were as follows: the occurrence of any of the conditions included in the exclusion criteria, violation of the protocol, the request of the patient or their relatives to be withdrawn from this study, and the occurrence of a phenomenon forcing the termination of this study.2)). In the presence of arterial hypertension (AH), special attention was paid to the gradation of such parameters, such as the level of blood pressure , as well as the duration and stages of hypertension. Also, to assess the state of the cardiovascular system, an ECG , ECHO-KG  was used. Ultrasound was performed on the abdominal organs using the B-K Medical Pro Focus 2202 ultrasound imaging system .Upon admission, patients anamnestic, measured pulse, respiratory rate, blood pressure (BP), height, and weight data were collected, followed by the calculation of body mass index (BMI) (weight (kg)/height (mn = 50), who were comparable in terms of sex and age to patients in the CKD group, which was confirmed through a ultrasound study of the kidneys, biochemical blood test, and urine test.In our study, we classified CKD stages according to GFR, which was calculated using the CKD–EPI–creatinine formula (Chronic Kidney Disease Epidemiology Collaboration Formula), and divided them into three groups: 1—patients with CKD at stage II (moderate decrease in GFR); 2—patients with CKD at stages IIIa–IIIb (pronounced decrease in GFR), 3: patients with CKD at stages IV–V (a severe decrease in GFR and pronounced decrease in kidney function). To assess the clinical condition of patients in relation to the underlying disease, standard methods of instrumental and laboratory examination were used, which consisted of identifying markers of damage, structural, and functional changes in the kidneys . A standard clinical and laboratory examination was supplemented by a study of aminothiol levels in urine and blood. The comparison group consisted of healthy people , homocysteine (Hcy), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were determined using a 3D capillary electrophoresis (CE) system , along with a quartz capillary with a total length of 32 cm, an effective length of 23.5 cm, and an inner diameter of 50 µm, as well as with UV detection.g. To perform liquid–liquid extraction, 0.1 mL of chloroform and 20 μL of 1-molarity HCl were added to 0.2 mL of the sample, and the mixture was centrifuged for 1 min at 3000× g. The upper phase was discarded, and the lower phase was concentrated in vacuo at 30 °C for 30 min. Then, 50 μL of water was added to the samples and incubated for 5 min, the samples were centrifuged for 1 min at 3000× g, and 30 μL of the solution was taken for analysis. Electrophoretic separation was performed using 0.1-molarity phosphate with 30 mM of triethanolamine containing 25 μM of Stabilite, 2.5 of μM sodium dodecyl sulfonate, and 2.5% (v/v) polyethylene glycol-600 at pH = 2. Samples were introduced into the capillary at 50 mbar for 45 s, and 0.5-molarity KOH at −17 kV was injected over a period of 30 s. The absorption wavelength was 285 nm [Aliquots of blood plasma and urine (100 µL) were first derivatized using 50 mM of 1,1-thiocarbonyldiimidazole (10 µL), and 250 µM of penicillamine (10 µL) was added as an internal standard, followed by incubation at 37 °C for 15 min. The samples were then purified via extraction using 50 mM of 1,1-thiocarbonildiimidazole in acetonitrile (300 µL) and incubated for 30 min at 4 °C, followed by centrifugation for 5 min at 15,000× g after thawing. Na2HPO4  was added to 3.0 mL of urine sample and passed through a solid-phase extraction cartridge grafted using phenylboronic acid . Next, 10 mg of the Bond Elute-PBA phase  was washed with 30% acetonitrile containing 1% (v/v) formic acid (0.2 mL), water (0.4 mL), and 50 mM of Na-phosphate buffer . A sample (0.4 mL) was loaded, and the phase was washed with 50 mM of Na-phosphate buffer  and 10 mM of Na-phosphate buffer . The analytes were desorbed using HCl . Before the start of the experiment, the capillary was washed daily with 1-molarity sodium hydroxide (2 min), water (1 min), and a supporting electrolyte solution  isobutanol, 10% (v/v) polyethylene glycol-300) (3 min). A urine sample was injected for 120 s at 50 mbar, followed by a sample of micellar solution  polyethylene glycol-300), which was injected for 45 s at 50 mbar at −11 kV for the first 2.5 min, followed by −16 kV for the remaining 4.5 min. The capillary was then flushed with BGE for 0.5 min. Analysis was performed at a pH of 2.2. The absorption was 254 nm [Samples were centrifuged for 1 min at 1000× s 254 nm ,39. The To make a prediction, we collected follow-up data 5 years after hospitalization by interviewing patients and/or their relatives , as well as examining and analyzing the data of 61% of patients. The catamnesis data were collected from 19 September 2022 to 31 October 2022.Primary processing of electropherograms was carried out using Data Analysis ChemStation software, version B.01.03 (Agilent Technologies).p < 0.05.When planning the study, the following setting characteristics were used to calculate the minimum sample size: a type I error of no more than 5% (reliability a = 0.05) and power of 80% (β = 1 − 0.8 = 0.2). Data were collected and analyzed using the statistical program Statistical Package for the Social Sciences SPSS 23.0 software, . The normality of data distribution was preliminarily checked (Kolmogorov–Smirnov test) and, since not all data were normally distributed, it was expressed as a median (between the 1st and 3rd quartiles). Correlations were performed using Spearman’s (ρ) rank testing. Differences between groups were tested for significance using the non-parametric Mann–Whitney U test for comparison of two groups and the Kruskal–Wallis test (taking into account the Bonferroni correction for multiple comparisons) for three or more groups. Qualitative indicators  were analyzed according to the contingency table and the Chi-squared test. The receiver operating characteristic (ROC) curve was used to estimate predictors of CKD at various stages, as well as to assess the predictive value of the CKD outcome. Also, to determine the prognostic significance of the indicators that we studied, we used the Cox regression method . The observed difference was considered to be significant at 2, and the mean plasma creatinine concentration was 118.0  µmol/L. The average duration of the underlying disease was two years . Edema was observed in 40.9% of patients. Also, increases in the concentrations of markers of kidney damage, such as urea and uric acid, and decreases in the level of blood plasma albumin and hemoglobin were observed in the blood, while the amount of daily protein present in the urine increased ; 41.8% of these patients were men. Moreover, 33 patients had stage II CKD, 46 patients had CKD at stages IIIa–IIIb, and 31 patients had CKD at stages IV–V. The mean GFR in the study group was 44.1  mL/min/1.73 mncreased .p < 0.001), 2.5 (p < 0.001), 2.3 (p < 0.001), and 1.8 (p < 0.001) times lower, respectively, than in the control group, while the concentrations of Cys and Hcy of blood plasma were 1.6 (p < 0.001) and 1.9 (p < 0.001) times higher, respectively. In contrast, SAH did not show statistically significant differences between these groups.When comparing healthy volunteers and patients with CKD, the studied values of Hcy, Cys in blood and SAM, SAM/SAH, Cys, and Hcy in urine were significantly statistically different from each other . The aveWhen comparing the concentrations of aminothiols, depending on the stage of CKD, the severity of changes in their concentrations progressed in proportion to the increase in renal dysfunction .p < 0.001) times lower in patients with stages IIIa–IIIb of CKD and 2.4 (p < 0.001) times lower in patients with IV–V stages of CKD compared to the control group. No other intergroup differences were found. The urine SAM level was lower than that of the control by an average of 2.9 (p < 0.001) times in patients with stages IIIa–IIIb of CKD and 4.5 (p < 0.001) times in patients at stages IV–V of CKD. Statistically significant differences were also found between stages II and IIIa–IIIb of CKD and stages II and IV–V of CKD: urinary SAM concentrations in patients at stages IIIa–IIIb and stages IV–V of CKD (p < 0.001) were 2.1 (p = 0.019) and 3.2 (p < 0.001) times lower, respectively, than in patients at stage II of CKD. The level of urine ratio of SAM/SAH also significantly differed from the control starting from stage III of CKD. Urine SAM/SAH was lower, on average, by 2.9 times (p < 0.001) and 4.3 times (p < 0.001) in patients at stages IIIa–IIIb and IV–V of CKD, respectively. Statistically significant differences were also found between stages II and IIIa–IIIb and II and IV–V: the urinary SAH/SAM ratio at stages IIIa–IIIb was 2.4 (p = 0.002) times lower and 3.6 (p < 0.001) times lower than in stage II patients. Urinary Cys and Hcy levels showed differences to those of controls starting from stage III CKD. Urine Cys concentrations were 3.2 times (p < 0.001) lower than those in the control at stages IIIa–IIIb and 2.6 times (p < 0.001) lower at stages IV–V, i.e., urine Cys concentrations slightly increased at stages IV–V and showed differences between the second and third stages of CKD (urine Cys of the third stage was 2.7 times lower (p < 0.001) than that of the second stage). Urine Hcy was 2.2 times (p < 0.001) lower than that of the control at stages IIIa–IIIb and 1.9 times (p < 0.001) lower at stages IV–V, though they did not reflect other intergroup differences. The Cys and Hcy of blood plasma were the most sensitive, being significantly different to the control of patients at stage II of CKD, while the level of plasma creatinine significantly differed from the control starting from stage III of CKD (p < 0.001) times higher than in the control group at stages II, IIIa–IIIb, and IV–V. Hcy concentrations were 1.5 (p < 0.001), 1.8 (p < 0.001), and 2.6 (p < 0.001) times higher than in the control group at the second, third, and fourth–fifth stages of CKD, respectively, with At the same time. Hcy concentrations significantly differed between both the II and IV–V stages (p = 0.004) and IIIa–IIIb and IV–V stages (p = 0.031).Urine Cr levels significantly differed from those of the control, starting from stage III CKD; on average, its level was 1.7 ; Cys, blood plasma, µmol/L ; SAM/SAH, urine ; Cys, urine, µmol/L ; and SAM, urine, µmol/L . It is also worth noting that when using the framework of the non-invasive determination of the studied metabolites in urine, SAM had the greatest potential, while urinary creatinine reflected this potential to a lesser extent . Urine aminothiols showed the greatest diagnostic significance between stages II and IIIa–IIIb of CKD, i.e., transition from a moderate decrease in GFR to a more pronounced decrease: SAM, urine ; Cys, urine ; and SAM/SAH, urine . The Hcy  and Cys  parameters of blood plasma most optimally demonstrated their diagnostic significance at stage II of CKD, and Hcy reflected the differences between stages IIIa–IIIb and IV–V .According to the ROC analysis , when asAs a result of the collection of follow-up data five years after hospitalization, it was found that 31 patients out of 110 died. Indeed, 24 patients died from acute renal failure that developed against the background of CKD, 1 patient died from first-time left ventricular myocardial infarction, 4 patients died from repeat LV infarction, and 2 patients died from stroke. Moreover, 1 patient was initially diagnosed with stage II CKD, 9 patients were diagnosed with stage III CKD, and 20 patients were diagnosed with stage IV or V CKD. In order to assess the predictive ability of aminothiol correlation, ROC analyses were performed with the inclusion of 76 variables , which had relationships with the outcome of CKD. For the selected variables, the area under the curve (AUC) scores were calculated. Regarding the set of variables discussed above, those with good predictive quality (AUC above 0.7 or below 0.3) were selected .p < 0.001), plasma creatinine, µmol/L ; age, full years ; CKD stage ; urinary protein, g/L ; urine protein/creatinine ratio (PCR), mg/mmol ; ESR, mm/h ; daily urine protein, g/L ; plasma homocysteine, µmol/L ; granulocytes, % ; adapted systolic blood pressure, mmHg ; leukocytes *109/L ; segmented neutrophils, % ; SAM urine, µmol/L ; total bilirubin, µmol/L ; SAM/SAH urine ; lymphocytes, % ; iron, µmol/L ; hematocrit, % ; daily diuresis, L/day ; hemoglobin, g/L ; erythrocytes *1012/L ; and the glomerular filtration rate, mL/min/1.73 m2 . The average quality of the prognostic model is set for indicators that have AUCs of 0.6–0.7 or 0.3–0.4. These indicators included plasma cysteine, µmol/L , and urine creatinine, µmol/L .Thus, the following 23 indicators reflected a high level of predictive potential: blood urea, mmol/L (AUC = 0.845, We also performed Cox regression to perform survival analysis for two, three, four, and five years. Of the indicators of aminothiols studied in blood plasma and urine, as well as creatinine and urea in blood and urine, at the 10th step of iterations, statistical significance was determined for only one indicator–blood plasma homocysteine. The final calculation formula is presented in As shown in a number of studies, the kidneys play a leading role in the regulation of the methionine cycle and have a direct effect on changes in the concentrations of Hcy, Cys, SAM, SAH, and the SAH/SAM ratio ,41,42. AIt is known that disturbances in the methionine cycle are associated not only with kidney damage , but alsPlasma Cys and Hcy levels were earlier markers of CKD and made it possible to identify kidney damage from stage II CKD onward, demonstrating higher sensitivity than plasma creatinine. In contrast, when performing non-invasive diagnostics in the urine, the levels of SAM and SAM/SAH made it possible to distinguish stage II CKD from stage III CKD. When assessing survival, these markers showed a good prognostic result; however, high plasma homocysteine concentrations were more significant.


However, obesity neither exaggerated the degree of IRI nor attenuated the conditioning effects on the measured outcomes. In conclusion, a single bout of RIPC is an effective means of suppressing subsequent IRI and obesity, at least in Asian young adult men, does not significantly attenuate the efficacy of RIPC.Remote ischemic preconditioning (RIPC) has been shown to minimize subsequent ischemia-reperfusion injury (IRI), whereas obesity has been suggested to attenuate the efficacy of RIPC in animal models. The primary objective of this study was to investigate the effect of a single bout of RIPC on the vascular and autonomic response after IRI in young obese men. A total of 16 healthy young men  underwent two experimental trials: RIPC (three cycles of 5 min ischemia at 180 mmHg + 5 min reperfusion on the left thigh) and SHAM (the same RIPC cycles at resting diastolic pressure) following IRI (20 min ischemia at 180 mmHg + 20 min reperfusion on the right thigh). Heart rate variability (HRV), blood pressure (SBP/DBP), and cutaneous blood flow (CBF) were measured between baseline, post-RIPC/SHAM, and post-IRI. The results showed that RIPC significantly improved the LF/HF ratio ( Remote ischemic preconditioning (RIPC), defined as a set of brief events of ischemia-reperfusion applied in distant tissues or organs from the heart, has been known to protect the cardiovascular systems from subsequent ischemic events in animal ,2 and huHowever, the effectiveness of an acute bout of RIPC is still controversial. For example, a single bout of RIPC was shown to attenuate myocardial ischemic stress through the modification of autonomic nervous system activity in an animal model . In humaObesity is prevalent worldwide and a strong predictor of ischemic stroke  and myocFor the abovementioned reasons, the purpose of the present study was to investigate whether a single bout of RIPC preserves and/or mitigates vascular function and sympathetic reactivity after induced IRI and to compare the possible differences between obese and normal-weight individuals. We hypothesized that (1) a single bout of RIPC would reduce the degree of IRI and (2) obesity would reduce the degree of RIPC-induced preservation of vascular function after IRI.The current study was conducted after the approval from the Institutional Review Board at Kyung-Hee University (KHGIRB-21-531) and conformed to the standard set by the Declaration of Helsinki. All participants provided written informed consent before their study participation.A total of sixteen male participants  were recruited for the present study . Becausem2 . All parThe schematic view of the study procedure is presented in During the remaining two visits, RIPC and SHAM were performed in a counterbalanced order. For the experimental trials, the participants attired medical scrubs upon their arrival at the laboratory and remained in the supine position on a bed for instrumentation. A contoured inflation cuff (18 × 108 cm) was placed on the left proximal thigh for the implementation of RIPC and another cuff (24 × 122.5 cm) was placed on the right proximal thigh to induce IRI. Electrocardiogram standard limb leads  were placed onto the torso to monitor heart rate variability (HRV). Cutaneous blood flow was measured (perfusion unit: PU), using laser Doppler flowmetry  throughout the experiment. The Doppler probe was attached 2 cm from the medial side of the great saphenous vein in the middle of the right leg tibia.After the completion of the instrumentation, participants rested quietly in a supine position for 20 min followed by the experimental measurements, which were carried out three times throughout the test . The measurement started with cutaneous blood flow that was continuously monitored throughout the protocol but analyzed at a specific time point followed by HRV measurement for 5 min. Subsequently, blood pressure and pulse rate were measured using a digital sphygmomanometer  together with the cold pressor test (CPT) to determine sympathetic reactivity. CPT was carried out by the immersion of participants’ right hand in cold water (at 4–5 °C) for 3 min during which blood pressure and pulse rate were measured at the end of the first and third minutes.Following the completion of the baseline measurement, RIPC or SHAM was carried out using a rapid cuff inflation system . The RIPC protocol consisted of 3 cycles of ischemia at 180 mmHg for 5 min and reperfusion for 5 min on the left leg. SHAM was performed in the same manner as RIPC; however, the compression intensity was set at each participant’s diastolic blood pressure measured at baseline. Post-RIPC measurement was started immediately after RIPC application followed by induction of the IRI on the right leg for 20 min of ischemia at 180 mmHg and 20 min of reperfusion. We judged that the ischemic stress threshold was reached when the cutaneous blood flow value fell below 20% compared with the baseline [Power spectral analysis of HRV (1600 Hz sampling frequency) was conducted using the fast Fourier transform and expressed as the ratio of the low frequency (0.04–0.15 Hz) to high frequency (0.15–0.4 Hz) (LF/HF ratio) to determine an overall balance between the sympathetic and parasympathetic activities. In addition, the lower limb cutaneous vascular conductance (CVC) and cutaneous vascular resistance (CVR) were calculated based on cutaneous blood flow and mean arterial pressure as shown below.CVC (PU/mmHg) = Cutaneous blood flow ÷ Mean arterial pressure × 100CVR (mmHg/PU) = Mean arterial pressure ÷ Cutaneous blood flowAll data in this study were analyzed using SPSS  and presented as mean and standard deviation. A two-way repeated measures ANOVA (2 conditions × 3 time points) with obesity as a between-subject factor was used to compare dependent variables between RIPC and SHAM. When a significant F-value was detected with Greenhouse–Geisser correction for sphericity, a post hoc pairwise comparison with Bonferroni correction was carried out to compare conditions at each time point. The significance level of all statistical analyses was set at α = 0.05.p = 0.027) (p = 0.047) and MAP  after IRI were significantly lower in RIPC compared with SHAM, but no difference was found for DBP  and HR  .There was a significant interaction for the LF/HF after IRI in the RIPC compared with the SHAM  . Consist= 0.550) . Howeverp = 0.039); however, no significant difference was found in MAP  or DBP . Similarly, the existence of obesity did not alter the sympathetic reactivity .A significant interaction was found for sympathetic reactivity, where there was an attenuated SBP response to cold in RIPC compared with SHAM after IRI . Consequently, a significantly increased CVC and decreased CVR were found after IRI in the RIPC . However, RHI did not differ between conditions  .A significant interaction was found for CBF, where there was an alleviated reduction in CBF after IRI in the RIPC compared with SHAM  , and theTo our knowledge, this is the first study investigating the effectiveness of RIPC in obese humans. It was found that a single bout of RIPC significantly suppressed the IRI-induced aggravation of vascular function and sympathetic reactivity compared with SHAM. However, there was no difference in any outcome measures between obese and normal-weight individuals. These results suggest that a single bout of RIPC is an effective means of mitigating injuries resulting from a subsequent ischemic event and such modifications were neither blunted nor magnified by obesity, at least in young adult males, in the present study.The present results showed the significant inhibitory effects of RIPC on the reduction in CBF; in agreement with previous findings, these effects mitigated the IRI-related impairment in CVC and CVR. Kraemer et al. 2011) reported that a single bout of RIPC significantly increased blood flow and tissue oxygen saturation during the reperfusion phase in healthy young men . Moreove1 reporteWhen considering IRI-induced aggravation in vascular function is attributed to a decline in nitric oxide bioavailability and sympathetic-overactivation-induced vasoconstriction ,28, a siBoth obesity and IRI are hypoxic and share similar inflammatory profiles including excessive production of reactive oxygen species and inflammatory cytokines ,33. TherThis might be due to the characteristics of the obese subjects participating in this study. Activation of phosphatase, known to limit the efficacy of both preconditioning and postconditioning with aging, was more pronounced in obese rats ,35, but This study has several limitations. First, the present results and interpretation regarding obesity are limited to Asian men. Secondly, we also excluded female participants to rule out the effect of hormonal changes on measurements such as HRV. Finally, any blood parameters that may have been responsible for explaining the mechanisms and/or effects of RIPC on the outcomes were not included. Therefore, the potential roles of some important markers such as inflammatory cytokines and various vasodilators were limited in the present interpretation.A single bout of RIPC was found to be an effective means of reducing impairment in vascular function and hyper-sympathetic nerve activity resulting from an acute ischemic event. Further, based on the present outcome measures, obesity neither significantly aggravated the degree of IRI nor abolished the favorable effects of RIPC in Asian obese young men. Future studies are warranted to investigate how repeated bouts of RIPC could further influence the functioning of the vascular systems in diverse obese populations.


Background and Objectives: Hypertension and vascular damage can begin in adolescents affected by Autosomal Dominant Polycystic Kidney Disease (ADPKD). This study aimed to evaluate markers of vascular damage and left ventricular geometry in a sample of children with ADPKD. Materials and Methods: Several vascular measurements were obtained: ambulatory blood pressure monitoring (ABPM), carotid intima-media thickness (cIMT), carotid distensibility coefficient (cDC), pulse wave velocity (PWV), and echocardiographic measurements  and left ventricular mass index (LVMI)). Results: Eleven ADPKD children were recruited . Four children were hypertensive at the ABPM, five were normotensive, and for two ABPM was not available. RWT was tendentially high (mean 0.47 ± 0.39). Eight patients had concentric cardiac remodeling, while one patient had cardiac hypertrophy. cIMT was above the 95° percentile for sex and height in 80% of the children (0.5 ± 0.005 mm). The average PWV and cDC were between the normal range . We observed a positive correlation between the PWV and RWT  and a negative correlation between cDC and RWT . Cardiovascular damages (cIMT > 95° percentile) were found in normotensive patients. Conclusions: Increased RWT and high cIMT, indicating subclinical organ damage, are already present in ADPKD children. RWT was significantly correlated to that of cDC and PWV, implying that vascular stiffening is associated with cardiac remodeling. None of the children had an alteration in renal function. Subclinical cardiovascular damage preceded the decline in glomerular filtration rate. Autosomal dominant polycystic kidney disease (ADPKD) is a disorder occurring in about 1 in every 400 to 1000 live births . Most paPatients with ADPKD were recruited from October to December 2018. These patients were referred to the Unit of Pediatric Nephrology of the University Hospital of Verona. The study was approved by the Ethical Committee of the University Hospital of Verona (CESC n.9427), and written informed consent was obtained from each participant’s parents.Several vascular measurements were obtained. Specifically, ambulatory blood pressure monitoring (ABPM), office blood pressure (OBP), carotid intima-media thickness (cIMT), carotid distensibility (DC), pulse wave velocity (PWV), and echocardiographic measurements  and left ventricular mass index (LVMI)).An oscillometric device recorded ABPM (Intermed A&D TM-2430). It was placed on the non-dominant arm and was set such that measurements were taken every 15 min during the day and every 30 min throughout the night, adapting “day” and “night” according to the diary form completed by the child or parents. All the values derived from the blood pressure (BP) measurements were also Z-score transformed according to normative values ,29.cIMT was assessed with an ultrasound of the carotid arteries . The cIMT was estimated by tracking the artery wall in the last centimeter of the common carotid artery and calculated using dedicated hardware . The relative z-scores and percentiles were calculated using reference values .The cDC was calculated as cDC = ΔA/(A × ΔP), where A is the diastolic lumen area, ΔA is the stroke change in lumen area, and ΔP is pulse pressure (PP). Changes in diameters were detected using ultrasound B-mode image sequences of the right and left common carotid arteries acquired at different steps. These changes were analyzed using the above-mentioned automatic system . The relPWV was measured with SphygmoCor XCEL . To conduct a carotid–femoral PWV measurement, a cuff was placed around the femoral artery of the child to capture the femoral waveform, and a tonometer was used to capture the carotid waveform. The distance between the carotid and femoral arteries was measured, and the velocity was automatically determined by dividing the distance by the pulse transit time. The subtraction method calculated the distance between the carotid measurement site and the cuffed site. The distance was calculated from the sternal notch to the top edge of the femoral cuff  and from the carotid artery to the sternal notch . To assess the above, the proximal distance was subtracted from the distal distance to determine the aortic lative z-score, and the percentile was calculated according to reference values .3 − tele-diastolic diameter3] + 0.6 gr). LVM was indexed (LVMI) to height (m2.7) [A single sonographer performed transthoracic echocardiography  in all participants. In the parasternal long-axis view, the 2D method was used to measure interventricular septum thickness end-diastole (IVSd), left ventricular end-diastolic diameter (LVEDd), and left ventricular posterior wall thickness at end-diastole. The Relative Wall Thickness (RWT) was calculated through the following formula: (IVSd + LVPWd/LVEDd). The Devereux equation was used to obtain left ventricular mass (LVM = 0.80 * 1.04 [(tele-diastolic diameter + PW + IVS)t (m2.7) . Left vet (m2.7) . The thrt (m2.7) . Normal t (m2.7) .Venous blood and urinary samples were collected after an overnight fast. Biochemical parameters such as triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol, non-HDL cholesterol, uric acid, creatinine, cystatin C, microalbuminuria (spot urine sample), and proteinuria (24 h urine collection) were analyzed in a single reference centralized laboratory with standard methods on routine clinical chemistry instrumentation . The estimated glomerular filtration rate (eGFR) was calculated using the Schwartz equation.Anthropometric parameters were also assessed. Body weight was measured using a calibrated balance, and height was measured using a calibrated stadiometer. The body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Overweight and obesity were defined as BMI ≥ 85th and 95th percentile for sex and age, respectively . The WHOwww.graphpad.com).For the statistical analysis, data were expressed as mean ± standard deviation for continuous variables or percentages for categorical ones. The Spearman correlation coefficient (rS) was used to quantify the linear relationship between variables. Statistical analyses were performed using SPSS software . Graphs were created with GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla, CA, USA (Patients’ characteristics are reported in 2. Three children were overweight (>85th), and eight were normal weight. None of the subjects had altered glomerular filtration rate (GFR), and the mean GFR was 109.6 ± 13.5 mL/min/m2. The mean microalbuminuria measured by the albumin mg/mmol ratio was 1.1 ± 1.3. The cardiovascular parameters according to the percentile reference values are reported in Eleven patients, seven males and four females, were included in the study. The mean age of the population was 9.5 ± 3.2 years. The median BMI was 18.9 ± 3.4 kg/m−3/KPa, respectively). We observed a positive correlation between the PWV and RWT  according to ABPM, five were normotensive, and only two patients were hypertensive at office blood pressure (OBP) measurement, consistent with the possible diagnosis of masked hypertension. One child was already under chronic therapy with an ACE inhibitor. RWT was, on average, higher concerning the cut-off for remodeling (mean 0.39 ± 0.05). In particular, eight patients had concentric cardiac remodeling, while one had cardiac concentric hypertrophy. cIMT was above the 95th percentile for sex and height in 80% of children (0.5 ± 0.005 mm), while average PWV and cDC were within the normal range  ,50,51,52According to the results of our study, the role of blood pressure/hypertension is not pivotal since the markers of early vascular and cardiac damage are not different in children with or without high blood pressure , and carAnyhow, it is conceivable to speculate that early antihypertensive treatment, especially with drugs that modulate renin-angiotensin-aldosterone, may be important for preventing renal worsening or cardiovascular protection. Both these speculations are supported by the notion that cardiovascular disease prevention occurs by reducing blood pressure and LVH, even in ADPKD patients ,61. So fThe cross-sectional design of the present study does not allow us to see if cardiac and vascular damages develop contemporarily or are, in part, causes or consequences of each other. Another limitation of the present study is that our population consisted of only 11 patients. However, the rarity of the disease can, at least partially, justify the restricted number of included patients, which is consistent with other studies ,27,41. WIn conclusion, in this study, we showed a high prevalence of organ damage even in children (with an average age < 10 years old) affected by ADPKD. In these children, we report tendentially increased RWT and high cIMT, with likely masked hypertension in almost half of patients despite normal renal function. Although this observation is clinically relevant, the present study should be read cautiously due to several limitations. First of all, because of the small sample size, this remains a pilot study, and firm conclusions cannot be drawn. Second, a control group is missing, and thus differences between ADPKD patients and healthy children cannot be determined, but only interpreted in the light of references values. Anyhow, our data should be a warning and prompt towards early cardiovascular screening and BP measurements using ABPM in children affected by ADPKD. Future studies using a higher sample size and adequate control groups may help to address the unmet points raised by our pilot study.


Recently, the VALENF instrument, Nursing Assessment by its acronym in Spanish, was developed as a meta-tool composed of only seven items with a more parsimonious approach for nursing assessment in adult hospitalization units. This meta-tool integrates the assessment of functional capacity, the risk of pressure injuries and the risk of falls. The general objective of this project is to validate the VALENF instrument by studying its diagnostic accuracy against the instruments commonly used in nursing to assess functional capacity, the risk of pressure injuries and the risk of falls. An observational, longitudinal, prospective study is presented, with recruitment and random selection based on admissions to six adult hospitalization units of the Hospital Universitario de La Plana. The study population will be made up of patients hospitalized in these units. The inclusion criteria will be patients over 18 years of age with a nursing assessment within the first 24 h of admission and an expected length of stay greater than 48 h and who sign the informed consent form. The exclusion criteria will be transfers from other units or centers. A sample of 521 participants is estimated as necessary. The evaluation test will be the VALENF instrument, and the reference tests will be the Barthel, Braden and Downton indices. Sociodemographic variables related to the care process and results such as functional loss, falls or pressure injuries will be collected. The evolution of functional capacity, the risk of falls and the risk of pressure injuries will be analyzed. The sensitivity, specificity and positive predictive values of the VALENF instrument will be calculated and compared to those of the usual instruments. A survival analysis will be performed for pressure injuries, falls and patients with functional loss. The VALENF instrument is expected to have at least the same diagnostic validity as the original instruments.Trial registration The study will be retrospectively registered . Nurses who work in hospitalization units are responsible for evaluating, planning, executing and reassessing the care that patients require throughout the care process and documenting all of this in the clinical history. However, this group perceives health documentation as an administrative burden due to the increase in the amount of data and the duplication of elements . MoreoveNursing assessment is defined as a planned, systematic, continuous, and deliberate process of collecting, classifying, and categorizing individualized information to recognize individuals' responses to their actual or potential health problems and needs . These aSome factors that could justify nurses perceiving nursing assessment as an administrative burden  include Different studies have shown that nursing assessments do not meet adequate standards of quantity or quality of information –15, incl2adj = 0.938), Braden (R2adj = 0.926) and Downton (R2adj = 0.921) indices, with high interobserver reliability (ICC > 0.9) and good construct validity  and internal consistency (Ω = 0.864), although its sensitivity, specificity, and predictive values for detecting patients at risk of functional loss, pressure injuries, or falls remain to be determined. Consequently, the main objective of this project is to study the diagnostic accuracy of the VALENF instrument compared to instruments commonly used in nursing in adult hospitalization units to assess functional capacity, the risk of pressure injuries, and the risk of falls.Recently, the “VALoración ENFermera” (VALENF) instrument, Nursing Assessment by its acronym in Spanish, was developed as a meta-tool composed of only 7 items with a more parsimonious approach for nursing assessment in adult hospitalization units , 24. ThiAn observational, longitudinal, prospective study with recruitment and random selection of hospitalized patients is presented to estimate the diagnostic accuracy of a meta-tool that collapses other instruments for assessing functional capacity and the risk of pressure injuries and falls in a public hospital in the province of Castellón. The study began in January 2023 and will end in December 2024. Figure The study population will be made up of patients admitted to the adult hospitalization units of the Hospital Universitario de La Plana. Special service units , home hospitalization units and maternal-infant and obstetric-gynecological hospitalization units will not be part of the study. Patients over 18 years of age who are assessed in the first 24 h after admission to the hospitalization unit using the Barthel Index, Braden Index, Downton scale and VALENF instrument, are expected to stay longer than 48 h, and who agree to participate in the study and sign the informed consent form will be included in the study. Patients who are transferred from other units of the same hospital and other hospitals will be excluded since their care process is in progress and their assessment on admission does not correspond to the initial assessment after admission.The sample size was calculated with Epidata v4 and was based on a comparison of proportions for paired data, since the condition of the patient will be unknown at the time of the evaluations, and the reference tests will be used for all subjects. A confidence level of 95%, an accuracy of 80% and a replacement rate of 10% were considered. Thus, the sample size includes 521 participants who will be stratified considering the monthly mean number of discharges from each hospitalization unit (Table The study includes sociodemographic variables (age and sex); variables related to the care process, such as hospitalization unit, type of process , type of admission , main diagnosis, Charlson Comorbidity Index , 26, preBefore starting data collection, the nurses responsible for carrying out measurements will receive training to guarantee homogeneity in data collection. This training program aims to equip them with the necessary skills to use the software used in data collection and standardize patient measurements. To achieve this, several meetings will be held with nurses who voluntarily choose to participate in data collection. During these meetings, the project will be explained in detail, presenting the objectives, participant selection criteria and measurement instruments, as well as the recruitment flow and monitoring of participants during hospitalization. At the end of the training, nurses will be provided with a notebook with the necessary information to serve as a reference at any time during data collection.Initially, data collection will take place between October 2023 and May 2024 using Research Electronic Data Capture (REDCap) software. Sociodemographic variables and variables related to the care process will be collected only upon admission. Moreover, the test under evaluation (VALENF instrument) and the reference tests  will be employed upon admission, every five days, and upon discharge. Finally, the outcome variables sensitive to nursing care will be collected at the time of discharge.Nurses who work in the hospitalization units that will participate in the study will carry out the recruitment, data collection and follow-up during the hospitalization of the patients included in the study prospectively.The recruitment procedure will begin when a patient is admitted to one of the participating hospitalization units, as long as one of the nurses participating in the study is on their work shift during the first 24 h after entry. To randomize the recruitment of the participants, a list of random numbers will be generated for each unit, and a box will be prepared with opaque envelopes with included/not included cards. The nurse who conducts the recruitment will explain the project and request informed consent (IC). The patients will be assessed upon admission, at five days, and upon discharge , type of admission (urgent or programmed) and the main diagnosis will be evaluated. For this, the normality and homoscedasticity of the sample will be studied with Shapiro‒Wilk and Levene tests, respectively. Depending on the results, Student’s t test or ANOVA will be applied, depending on the number of groups, or the corresponding nonparametric tests. In addition, an analysis of paired measures will be carried out to analyze the evolution of the scores of the Barthel, Braden and Downton indices, what will serve us as a measure of diagnostic accuracy for our instrument, using these measurements as criteria. Categorical variables will be analyzed using the chi-square or Fisher’s exact test, and correlations will be studied with the Pearson or Spearman test, depending on the application conditions.After this initial analysis, the cumulative incidence and period incidence of pressure injuries, falls and patients with functional loss will be estimated by unit and overall. A survival analysis will be carried out with pressure injuries, falls or functional loss as dependent variables and the other study variables as independent variables.Next, the sensitivity, specificity, and positive and negative predictive values of the VALENF instrument will be analyzed to detect patients at risk of pressure injuries, falls, and functional loss using the Barthel, Braden, and Downton indices as reference tests. At this point, it should be considered that the protocolized prevention measures will be applied for those patients who are assessed to be at risk of pressure injury or fall to safeguard the ethical and deontological commitment of nonmaleficence. For this reason, this analysis will be carried out only with the positive cases first and, second, including the true positives with those other patients to whom the corresponding prevention measures were applied. We understand that this situation may bias the diagnostic accuracy results of the VALENF instrument, but we believe that it is a viable solution and that it can also provide information on the effectiveness of the measures used.p < 0.05 will be considered in the comparisons of the hypotheses, and the statistical analysis will be carried out with SPSS v-25 and Jamovi v-2.3.2 software.Finally, the VALENF instrument cutoff points will be established to detect the risk of pressure injuries, falls, and functional loss through simple linear regressions with the original instruments and cluster analysis to categorize patients. Moreover, and analysis of the area under the ROC curve will be carried out, considering acceptable values 0.7 to 0.8, excellent if 0.8 to 0.9, and outstanding if more than 0.9, and the Youden index will be used to calculate the optimal cut-off point . A valueThe project was positively evaluated by the Ethics and Research Committee of the participating hospital in July 2023. This project complies with Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April, 2016, regarding the protection of natural persons and Organic Law 3/2018 of 5 December, on Protection of Personal Data and Guarantee of Digital Rights. The participants will receive adequate and timely information about the objective and methodology of the study, as well as about their rights to access the information and to abandon the study. This information will be provided by the nurses who participate in the study. After being informed, they will provide informed consent with their signature. The database will not include personal data that allow the identification of patients.The limitations of this study mainly involve sample selection bias and measurement bias due to misclassification of the subjects due to the subjectivity of the measurement and underreporting of results sensitive to nursing practice. Another important aspect to highlight is the risk of patient loss during hospitalization follow-up and discharge.To try to control these limitations, a team of nurses who participate voluntarily in the study and who will receive specific training on the objective and methodology of the study will be selected. Adequate training on REDCap will be carried out, and standardized measurements will be used, verifying compliance with the requirements through a CDRe pilot. In addition, during data collection, the researchers are expected to visit the units periodically to resolve doubts and problems related to the recruitment, data collection, and hospitalization follow-up of participants, and three researchers will audit the REDCap database to verify its accuracy and correct completion and reduce the risk of loss.Finally, patients at risk  need preventive measures to avoid these undesirable results. These measures will be applied when necessary and considered in the CDR and data analysis.


There is no consensus strategy for mineralocorticoid (MC) therapy titration in patients with primary adrenal insufficiency (PAI). We aim to measure serum fludrocortisone (sFC) and urine fludrocortisone (uFC) levels and to determine their utility, alongside clinical/biochemical variables and treatment adherence to guide MC replacement dose titration.+, K+), mean arterial blood pressure (MAP), total daily glucocorticoid (dGC) and MC (dMC) dose, and assessment of treatment adherence were incorporated into statistical models.Multi-centre, observational, cross-sectional study on 41 patients with PAI on MC replacement therapy. sFC and uFC levels (measured by liquid chromatography-tandem mass spectrometry), plasma renin concentration (PRC), electrolytes  and between sFC and the time from the last fludrocortisone dose . Total dMC dose was related to dGC dose , K+  as well as sFC  and uFC . PRC was related to Na+  and MAP , but not to MC dose, sFC or uFC. Regression analyses did not support a role for sFC, uFC or PRC measurements and confirmed K+  as the most important variable to guide dMC titration. Of the patients, 32% were non-adherent with replacement therapy. When adherence was inserted into the regression model, it was the only factor affecting dMC.We observed a close relationship between sFC and uFC (sFC and uFC levels are not helpful in guiding dMC titration. Treatment adherence impacts on clinical variables used to assess MC replacement and should be included as part of routine care in patients with PAI. Primary adrenal insufficiency (PAI) is a rare disease consisting of the inability of the adrenal cortex to produce adequate levels of glucocorticoids (GCs), with or without concomitant inadequate production of mineralocorticoids (MCs) and adrenal androgens . The mosFludrocortisone (FC) has been the most commonly prescribed MC since its discovery in 1954 . The phaMuch attention has focused on the optimization of GC replacement in patients with PAI, driven, at least in part, by the increasing awareness of the detrimental adverse effects of circulating GC excess alongside the development of novel GC preparations , 20, 21.Previous studies to examine the potential utility of measuring FC levels in serum and urine  alongside accurate assessments of treatment adherence to guide MC preplacement have not been performed. We performed an observational clinical study to determine if combining sFC and uFC measurements, treatment adherence assessment with other routine clinical parameters  could help guide MC replacement dose titration.We performed a multi-centre, observational, cross-sectional study on patients with PAI treated with GC and MC replacement therapy. Consecutive patients were enrolled from a dedicated endocrine outpatient clinic in two different referral centres  from June 2019 until March 2020.All the adult patients attending the dedicated outpatient endocrine clinics for patients with AD or CAH were asked to participate in the study. Data and samples were collected as part of ‘real-life’ clinic consultations and included: anthropometric parameters , systolic and diastolic blood pressure); details on MC and GC replacement therapy  and other concomitant medications (including drugs interfering with RAA systems such as antihypertensives or diuretics); standard laboratory biochemical analyses including sodium, potassium and plasma renin concentration (PRC); blood and urine (spot) samples were collected for serum and urinary FC assays. Mean arterial blood pressure (MAP) was calculated as diastolic blood pressure + 1/3 of differential blood pressure (systolic blood pressure minus diastolic blood pressure). To ensure uniformity and comparability of data the collected data, all GC doses were expressed as hydrocortisone equivalent (HCeq) using standardised conversions .Patient adherence with both GC and MC replacement was evaluated through specific questions asked to the patients see ; patient125I-labelled monoclonal antibody. After incubation, unbound reagents are removed by washing the tubes. The amount of 125I-labelled anti-renin bound to the tube is directly proportional to the concentration of renin present in the sample. A standard curve is constructed, and PRC is obtained from the curve by interpolation. EDTA plasma samples were collected and processed at room temperature, to avoid cryoactivation of prorenin, giving falsely high active renin levels. The measurement range  is 0.81–500 pg/mL. The high specificity of the assay was confirmed by extremely low or undetectable cross-reactivity with several molecules .Two different assays were used for PRC measurement. In Rome, PRC was measured by immunoradiometric assay, using Beckman Coulter reagents (ref. DSL25100). Mouse monoclonal antibodies directed against two different epitopes of renin are used (sandwich-type assay). Samples or calibrators are incubated in tubes pre-coated with one monoclonal antibody, and with a second liquid In Oxford, PRC was measured by automated immunoassay, using the IDS-iSYS Direct Renin assay . Two monoclonal antibodies directed against two different epitopes of renin are used in the assay; one labelled with acridinium and the other labelled with biotin.Samples, calibrators and controls are incubated with both antibodies in specific system buffers. Streptavidin-coated magnetic particles are added, and following further incubation, the particles (bound to the renin-antibody complexes) are ‘captured’ using a magnet. The particles then undergo a wash step and a ‘trigger’ reagent is added. The amount of light emitted by the acridinium label is directly proportional to the concentration of renin present in the original sample.The IDS-iSYS assay utilises a two-point calibration, measuring three replicates of each level. A four-parameter logistic curve fit is used to calculate the renin concentrations. The measurement range of the assay is 1.8–550 mIU/L. Typical assay performance characteristics in terms of coefficient of variation are as follows: 6.0% at 15.5 mIU/L, 1.8% at 101.8 mIU/L and 1.9% at 327.5 mIU/L.To ensure comparability across assays, all the PRC levels were expressed as mIU/mL, using 1.67 as conversion factors as previously described .sFC and uFC were measured following liquid–liquid extraction by isotope dilution liquid chromatography-tandem mass spectrometry. Deuterated internal standard  was added to each sample, QC and calibrator followed by diethylether. Samples were mixed and then placed into an ethanol cold bath and the sample was frozen. The top solvent layer was poured off into clean glass tubes which were then evaporated to dryness in a vacuum drier . Samples were reconstituted in 50:50 methanol:water and injected into a Shimadzu HPLC system coupled with an AB Sciex 5000 tandem mass spectrometer. A Hypersil GOLD 1.9 µm, 100 × 2.1 mm (Thermo Fisher) chromatography column and samples eluted using mobile phases ammonium acetate in water (A) and 0.2% formic acid in methanol (B). APCI ionisation and positive mode ionisation were used. FC was monitored using transition 381.2>343.2 and internal standard using 386.2>348.4.The distribution of continuous variables was tested using the Shapiro–Wilk’s test and is reported as mean and 95% confidence interval (95% CI) or median and interquartile range , as appropriate. Categorical variables are expressed as percentage and frequency. Linearity was established by visual inspection of a scatterplot.U test was run to determine possible differences in the outcome variables between groups. A multiple regression analysis was run to test the effects of selected variables on the likelihood of predicting the total daily MC dose. The level of statistical significance was set at a P value < 0.05. All statistical computations were conducted with the IBM SPSS Statistics  and Prism 7.0 software package .As per the distribution of data, a Pearson’s product–moment or Spearman’s rank-order correlation was run to assess the baseline relationship between different variables; an independent sample t-test or a Mann–Whitney This study was conducted under the Declaration of Helsinki and good clinical practice. Informed consent has been obtained from each patient after a full explanation of the purpose and nature of all procedures used. The study was approved by the Trust audit team and conducted and registered as a local quality improvement (QI-8109).n = 27) were treated with hydrocortisone (HC) as GC replacement , whereas 34% (n = 14) were treated with prednisolone. GC treatment regimens were as follows: 37% (n = 15) received GC as a single dose and 44% (n = 18) and 19% (n = 8) were prescribed with two (BID) or three (TID) doses, respectively. All patients were treated with FC: 88% (n = 36) received FC as a single dose and 10% (n = 4) and 2% (n = 1) were prescribed with TID and BID regimens, respectively. Patient characteristics are presented in Forty-one patients  with PAI  receiving stable daily GC  and FC  replacement therapy were consecutively recruited into the study . Of the r = 0.434, P = 0.005)   . Endorsi= 0.023) , with a = 0.023) , 33, 34.r = 0.356, P = 0.022; uFC: r = 0.531, P < 0.001)  and an inverse relationship between MAP and PRC   and between sodium and PRC levels on univariate analysis    and D. T= 0.006)  andB. In= 0.006) , there w< 0.001)  and D. AP = 0.744). Among the 26 patients treated with more than one dose of GC per day, 11 showed non-adherence (P = 0.084). There were no differences in adherence between patients taking HC or MRHC (P = 0.545).Eight patients (20%) missed at least one dose of their GC replacement therapy in the preceding 7 days; Four patients (10%) declared missing at least one dose of both GC and FC whilst one patient missed a single FC dose in isolation. In total, 13 (32%) patients were classified as ‘non-adherent’. MC dose frequency did not affect patients’ adherence (P = 0.009), were mainly affected by SW-CAH , were prescribed higher FC  and higher GC replacement doses  and had lower potassium levels . There were no differences in sodium levels, PRC or MAP (P = 0.622) and uFC (P = 0.127) when comparing FC adherent (n = 36) and non-adherent (n = 5) patients. In patients with CAH (n = 17), biochemical makers of disease control were lower in the GC adherent (n = 7) compared to non-adherent (n = 10) patients : 2.4 ( 1.0–14.9) nmol/L vs13.3 (9.8–34.43), P = 0.04; 17OH-progesterone: 2 (1–90) nmol/L vs 55 (36–119), P = 0.07)  .Table 2r = 0.578, P < 0.001). In addition, the relationship between total daily GC and MC doses persisted , as did the inverse relationship of PRC to sodium levels  and the positive association of PRC with potassium levels   as well as data on patient’s treatment adherence. The model was significant , there was no relationship between sFC or uFC with total daily MC dose, suggesting that they are unhelpful in guiding total daily MC dose. Treatment adherence was the only variable able to predict total daily MC dose . All the computed and relative coefficients generated by the models are summarized in P < 0.001).A multiple regression model was constructed to identify variables that could predict total daily MC dose and included all measured clinical and biochemical variables  and biochemical variables  , 35. HowAchieving electrolyte levels in the normal range, a normal blood pressure without postural hypotension , 37 and In the current study, we have also demonstrated a dissociation between PRC and MC daily dose. Indeed, the total daily MC dose was only inversely related to serum potassium levels. Higher PRC was associated with lower sodium levels and MAP, confirming PRC as a marker of plasma volume and sodium state . These dAdherence to medication is commonly overlooked in ‘real-life’ clinical research, retrospective studies as well as in clinical trials , 29. TheIn patients with chronic diseases requiring long-term treatments, poor medication adherence is particularly prevalent . MedicatIn total, we found 32% of our patients to be ‘non-adherent’ with FC and/or GC treatment. This observation does raise concerns, especially in this patient group who are at significant serious risk of adrenal crisis and the associated morbidity and mortality. We assessed treatment adherence in two different conditions (AD and SW-CAH). Little is known about medication adherence in adults with PAI; however, endorsing our findings, two published studies have shown non-adherence rates in adults with CAH ranging from 26 to 36% , 51. In Through the incorporation of adherence assessment into our models to predict MC dosing, we have demonstrated that its impact is strong affecting the reliability of clinical and biochemical variables. In addition, by refining our analysis and excluding ‘non-adherent’ patients, we found significant associations reflective of underlying physiology including the relationships between PRC and electrolytes levels, and GC with MAP. Current guidance for the treatment of PAI state that ‘…assessment of renin should be done in response to changes in clinical status or if compliance is in question…’. Whilst There are several limitations to the current study and analysis. The sample size was relatively small and as a result, our findings may not be representative. PRC was measured, without a standardised posture protocol, in two different centres and a direct comparison between the two assays was not performed. No data about FC treatment duration and administration in relation to food, nor about salt craving were collected, although such data are difficult to quantify and the assessment of adherence was limited to a small number of specific questions. uFC levels were measured in spot urine samples which, albeit less onerous for participants, might be dependent on hydration status, duration and volume of the collection as well as residual bladder volume. Moreover, no data about urine electrolytes (notably sodium and potassium) were collected. Whilst there are studies demonstrating the reliability of urine spot samples, our results should be confirmed using 24-h urine measurement of FC and electrolytes. The observational nature of our study design is clearly an additional limitation; however, it does reflect real-life clinical practice and is representative of the challenges faced by clinicians managing patients with PAI.In conclusion, our study provides real-life insights into addressing the clinically important challenge of optimizing MC replacement in patients with PAI. Whilst we demonstrate a lack of utility of the measurement of sFC and uFC, this study highlights the crucial role of adherence with medication and the need for this to incorporate as part of the routine clinical care assessment in patients with PAI. Only once the degree of adherence is known, can a meaningful interpretation of other relevant clinical variables including blood pressure and electrolytes and perhaps PRC be made.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.The study was supported by an MRC programme grant awarded to JWT ref. MR/P011462/1.


To explore the characteristics of retina microvascular changes in patients with diabetic nephropathy (DN) and its risk factors.Retrospective, observational study. 145 patients with type 2 diabetic mellitus (DM) and DN were included in the study. Demographic and clinical parameters were obtained from medical records. Presence of diabetic retinopathy (DR), hard exudates (HEs) and diabetic macular edema (DME) were evaluated according to the color fundus images, optical coherence tomography (OCT) and fluorescence angiography (FFA).p = 0.004), HbA1c (P = 0.037), Urine albumin creatine ratio (ACR) (p < 0.001) and lower level of estimated glomerular filtration rate (eGFR) (P = 0.013). Logistic regression analysis showed DR was significantly associated with ACR stage (p = 0.011). Subjects with ACR stage3 had higher incidence of DR compared with subjects with ACR stage1 . 138 eyes of 138 patients were analyzed for HEs and DME, of which 23.2% had HEs in posterior pole and 9.4% had DME. Visual acuity was worse in HEs group than in non-HEs group. There was significant difference in the LDL-C cholesterol level, total cholesterol (CHOL) level and ACR between HEs group and non-HEs group.DR accounted for 61.4% in type 2 DM patients with DN, of which proliferative diabetic retinopathy (PDR) accounted for 23.6% and sight threatening DR accounted for 35.7%. DR group had significantly higher levels of low-density lipoprotein cholesterol (LDL-C) (A relatively higher prevalence of DR was found in type 2 DM patients with DN. ACR stage could be recognized as a risk factor for DR in DN patients. Patients with DN needs ophthalmic examination more timely and more frequently.The online version contains supplementary material available at 10.1186/s12902-022-01250-w. Diabetes mellitus (DM) is a chronic hyperglycemic state characterized by defects in insulin secretion or/and insulin action  and has Diabetic retinopathy (DR) is one of the most common microvascular complications of DM, which is the leading cause of preventable blindness in working-age people. The prevalence of DR in the general population and diabetic patients in a meta-analysis was 1.7% and 22.4% respectively. The highest prevalence of DR was found in patients aged 50–59 years in China .The retina complications of DM result from damage to small vessels, while the kidney complications share the same mechanism. As all belongs to microvascular complications of DM, several investigations have demonstrated the relationship between DR and diabetic nephropathy (DN) in DM patients . PreviouHard exudates (HEs) are the result of extravasation of fluid, lipids and lipoproteins into the retinal layers from microaneurysms and dilated capillaries , which cTherefore, we hypothesized that DN severity may be a potential risk factor to microvascular changes in retina. The purpose of our study is to test the hypothesis and to explore the characteristics of retina microvascular changes in patients with DN. These analyses might provide useful information for the prevention and management of DM complications.This was a retrospective, observational, single-center study. The medical records of 145 patients diagnosed with type 2 DM and DN in China-Japan Friendship Hospital from January 2019 to December 2019 were retrospectively reviewed , abdominal circumference, blood pressure, history of hypertension, serum lipid profile, history of hyperlipidemia, serum uric acid level, serum homocysteine level, history of peripheral atherosclerosis and history of diabetic peripheral neuropathy. Laboratory indexes associated with DN were collected: serum creatinine (Scr) level, urine microalbumin level and urine creatinine level. Estimated glomerular filtration rate (eGFR) was calculated according to previous guidelines  = 186* Scr-1.154*(age)-0.203; for female: eGFR (ml/min/1.73 m^2) = 186*Scr-1.154* (age)- 0.203*0.742). Urine albumin creatine ratio (ACR) was calculated as the urine microalbumin level divided by urine creatinine level. The stage of kidney function was classified as follows: stage I was defined as eGFR at least 90 ml/min/1.73 m^2 as, stage II was defined as eGFR at least 60 ml/min/1.73 m^2 but less than 90 ml/min/1.73 m^2, stage III was defined as eGFR at least 30 ml/min/1.73 m^2 but less than 60 ml/min/1.73 m^2, stage IV was defined as eGFR at least 15 ml/min/1.73 m^2 but less than 30 ml/min/1.73 m^2, while stage V was defined as eGFR less than 15 ml/min/1.73 m^2. The stage of urine microalbumin was classified as follows: stage I was defined as ACR less than 30 mg/g, stage II was defined as ACR at least 30 mg/g but less than 300 mg/g, while stage III was defined as ACR at least 300 mg/g.All patients had a throughout ophthalmic examination, including best corrected visual acuity (BCVA) using a Snellen chart, intraocular pressure (IOP) measurement, slit lamp examination, color fundus photography, optical coherence tomography  and fluorescein angiography (FFA) if needed. Visual impairment was graded according to BCVA in Snellen chart as: mild, moderate, severe and blind. BCVA ≥ 0.8 was defined as normal vision; 0.6 ≤ BCVA < 0.8 was defined as mild visual impairment; 0.3 ≤ BCVA < 0.6 was defined as moderate visual impairment; 0.1 ≤ BCVA < 0.3 was defined as severe visual impairment; BCVA < 0.1 was defined as blind.The presence of DR was examined by general ophthalmologists at Department of Ophthalmology and was re-examined by one of our authors YYJ, an experienced retinal specialist, according to the color fundus image, OCT and FFA. The stage of DR was graded according to literature report  as: abseThe presence of HEs was made by visible white-yellow exudates in the posterior area in the color fundus images. Confirmation was made by cross-sectional OCT images as hyperreflective round elements in the outer plexiform layer of the retina. Macular edema was defined as hard exudates in the presence of microaneurysms and blot hemorrhage within 1500 um from the foveal center with a central retinal thickness (CRT) ≥ 250 μm due to intraretinal fluid or subretinal fluid . MacularP values less than 0.05 were considered to indicate statistical significance.All statistical analyses were carried out with SPSS software . Data are expressed as mean ± SD or medians  for continuous variable and as numbers (percentage) for categorical variables. The t-test was used to compare continuous variables with normal distribution and Mann–Whitney test was used to compare continuous variables which were not normally distributed. The numbers of categorical variables were compared using chi-square test or Fisher’s exact test. Multivariate logistic regression analysis was performed to assess the independent predictive effects of the variables on the risk for progression of DR. Records of 145 patients with type 2 DM and history of DN were examined. The duration of DM was 15.14 ± 8.56 years (Table p = 0.004), HbA1c (P = 0.037), ACR(p < 0.001) and lower level of eGFR(P = 0.013). , serum uric acid, serum homocysteine (HCY), serum creatinine. However, DR group had significantly higher levels of low-density lipoprotein cholesterol (LDL-C) . Table .Table 2Dp = 0.011). Subjects with ACR stage3 had higher incidence of DR compared with subjects with ACR stage1 (OR = 24.15. 95%CI: 2.06–282.95) . Visual acuity was worse in HEs group than in non-HEs group, however, no significant difference was found between the groups(p = 0.058) . Visual acuity was worse in DME group with significant difference compared with non-DME group (p = 0.003)  had HEs in the area of posterior pole and 13 eyes 9.4%) had DME. There was significant difference in the LDL-C level, total cholesterol level and ACR between HEs group and non-HEs group  (TableMacular HEs are often seen in patients with diabetic macular edema (DME) and they are known to be an independent risk factor for vision loss , 28. Eve11Our study has several limitations. Firstly, the present study was a retrospective cross-sectional study, thus causal relationships cannot be established. Secondly, the study was conducted in hospitalized patients in one center, thus our results may not be representative to other populations. Further prospective study in multi-center is needed in the future to prove the association between urine microalbumin and DR.In conclusion, a relatively higher prevalence of DR was found in type 2 DM patients with DN. DM was significantly associated with ACR stage, thus ACR stage could be recognized as a risk factor for DR in DN patients. Besides routine ophthalmic examination in patients with type 2 DM, patients with DN needs ophthalmic examination more timely and more frequently. These data may provide useful information for the prevention and management of DM complications.Additional file 1.Additional file 2.


Bipolar disorder (BD) is a serious and chronic mental disease of mood. Lithium is used for treatment and studies have demonstrated that it is the most efficient drug, reducing suicide risk in a high percentage of patients. However, this drug has well known side effects, such as kidney damage. Lithium could cause chronic kidney disease, specially with the presence of other risk factors.Observational and retrospective study of creatinine levels and glomerular filtration rates observed in blood analysis (follow-up period of 11 years). Sample size of 263 patients diagnosed of BD I and BD II in treatment with lithium. We used socio-demographic  and clinic variables  and/or diuretics) to generate bivariate and multivariate analysis.Our main objective is to analyze the deterioration of kidney function and the development of chronic kidney disease that chronic treatment with lithium can induce in patients with BD. Our secondary objective is to determine variables which could promote the development of chronic kidney disease, and to assess if these variables could be considered as risk factors during the treatment with lithium.11,3 % of patients in our study developed chronic kidney disease during monitoring. The deterioration of GFR in patients in treatment with lithium was significantly associated with female sex and NSAIDs consumption. A trend towards statistical significance was found regarding the use of diuretics . No statistical significance was found between diabetes mellitus, hypertension or type of BD and the deterioration of kidney function in our sample. An inverse association was found between the GFR decline and the age but no statistical significance was demonstrated.We conclude that female sex and use of NSAIDs are predicting factors of GFR decline in patients with BD in chronic treatment with lithium. We must take into account these drugs or even avoid concomitant treatment (lithium and NSAIDs) in order to prevent chronic kidney disease. In addition to it, we should recommend careful use of diuretics during treatment with lithium because of risk of dehydration. Diabetes mellitus and hypertension have universally been associated to increase risk of development of chronic kidney disease. However, we have not found statistical significance in our study. Therefore, research should be done in order to determine specific risk factors in this group of patients and, consequently, optimize their treatment.None Declared


TTV load did not relate to mean tacrolimus blood level, CV, TTR, C/D and AUC-MPA. To conclude, although TTV is a useful marker of net immunosuppressive status after KTx, it is not related to exposure to maintenance immunosuppression.Measuring the non-pathogenic Torque Teno Virus (TTV) load allows assessing the net immunosuppressive state after kidney transplantation (KTx). Currently, it is not known how exposure to maintenance immunosuppression affects TTV load. We hypothesized that TTV load is associated with the exposure to mycophenolic acid (MPA) and tacrolimus. We performed a prospective study including 54 consecutive KTx. Blood TTV load was measured by an in-house PCR at months 1 and 3. Together with doses and trough blood levels of tacrolimus and MPA, we calculated the coefficient of variability (CV), time in therapeutic range (TTR) and concentration/dose ratio (C/D) of tacrolimus, and the MPA-area under the curve (AUC-MPA) at the third month. TTV load at the first and third month discriminated those patients at risk of developing opportunistic infections between months 1 and 3 (AUC-ROC 0.723, 95%CI 0.559–0.905,  Kidney transplantation is the best therapy for patients with end-stage renal disease. While in liver transplantation a significant number of patients can be withdrawn immunosuppressive drugs due to the development of variable grades of tolerance, most of the other solid organ transplants need to maintain the immunosuppressive drugs while the graft is functioning to avoid alloimmune response ,2. RoutiTo improve the results of kidney transplantation, it is important to have new, minimally invasive monitoring methods that would make it possible to assess the risk of both infection and rejection. Some of these methods, such as the ability to activate T helper lymphocytes, have already been used in clinical trials for monitoring liver transplantation, although their value has not been sufficiently proven . RecentlSo far, it is not known precisely which immunosuppressive-related variables influence TTV levels. Some studies have linked higher doses of antimetabolite or CNI drugs or higher CNI blood levels to higher blood TTV loads, but this has not been demonstrated in all studies ,24,25,26This prospective study was conducted following the guidelines of the Declaration of Helsinki and approved by the Regional Ethics Committee in our institution . A total of 54 consecutive kidney transplants from deceased donors performed in our center from January 2021 to April 2022 were recruited after giving written consent prior to kidney transplant. Recipients from non-controlled cardiac death donation, with preformed donor specific antibodies and highly hypersensitized with a panel reactive antibody over 98% were excluded.Pneumocystis jirovecii pneumonia, Candida and invasive fungal infections such as Aspergillus) infections. Standard microbiology techniques were used for the detection of viral, bacterial and fungal infections.Relevant information about recipient, donor, and transplant characteristics was collected. All acute rejection episodes were biopsy proven. Indication biopsies were performed when the level of creatinine increased by 25% or more over its previous value or when proteinuria persisted >1 g per day. We considered opportunistic infections those related to cellular and humoral immunosuppression, which included viral , bacterial , and fungal  of tacrolimus were determined by chemiluminescent microparticle immunoassay on the Architect iSystem . This immunoassay is designed to have a precision of ≤10% total coefficient of variation (CV), a mean recovery of 100 ± 10% of the expected value, a limit of detection of ≤1.5 µg/L, and a functional sensitivity of ≤2 µg/L. All levels of tacrolimus up to day 90 were collected. The tacrolimus target trough blood levels up to month 3 were 8 to 12 ng/mL. The variability of tacrolimus blood levels was estimated by means of the coefficient of variation (CV) calculated according to the following equation: samples . The per samples . Tacroli samples .Trough blood concentrations of mycophenolic acid (MPA) in human plasma (mg/L), were quantified by homogenous enzyme immunoassay  at months 1 and 3. This immunoassay is designed to have a precision of ≤10% total CV, and a limit of detection of ≤0.1 mg/L. The dynamic range of this immunoassay is from 0.1 mg/L to 15 mg/L based on the analytical sensitivity of the assay. If the analysis of a patient sample is outside the calibration range, it is diluted, allowing it to determine the concentration. No cross-reactivity with MPAG (mycophenolic acid glucuronide), the main metabolite of MPA, or with cyclosporine or tacrolimus has been observed.0–12h estimation that gave the best performance for this model was: 7.75 + 6.49.C0h + 0.76.C0.5h + 2.43.C2h [The full mycophenolic acid area under the curve (AUC-MPA) was calculated at month 3 using an abbreviated procedure of drawing blood samples at time 0, at 30 min and 2 h after having taken the drug, according to the procedure previously reported by Pawinski et al. The regression equation for AUC2.43.C2h .3 viral genomes per ml of plasma sample. This protocol has recently been compared and validated against the commercial TTV R-GENE® kit  [Samples for measuring TTV load were drawn before transplantation and at days 30 and 90 post-transplantation. Briefly, the blood obtained by venipuncture with a BD Vacutainer K2E 5.4 mg tube, cat #368856  was centrifuged at 1.300 rpm and the plasma was frozen at −80 °C until processing. Free viral DNA was purified from 400 µL of plasma from all specimens using the QIAamp MinElute Virus Spin Kit Cat. # 57704  as specified by the manufacturer. The presence and viral load of TTV in the samples were determined in duplicate using a previously described TaqMan (TM)-PCR assay human TTV APP2XDMP  in a StepOnePlus Real-Time PCR System . This assay is based on the specific amplification of a highly conserved viral segment in the untranslated region of TTV, which has the potential for sensitive and specific detection of all TTV genotypes present in GenBank . The pro France) .12 copies, B = 1.0 × 1010 copies, C = 1.0 × 108 copies, D = 1.0 × 106 copies, E = 1.0 × 104 copies, F = 1.0 × 102 copies, G = 1.0 copies, H = 0 copies.As a positive control, a 143 bp PCR fragment from the same untranslated region of TTV genome (NC_015783.1) was amplified using the primers TTV Sen (5′ GTGCCGTAGGTGAGTTTA 3′) and TTV AntisL (5′ ATGGACCGGCGGTCTCCACGG 3′) and cloned into the pCR™2.1 cloning vector . The resulting plasmid was then purified with QIAprep Spin Miniprep Kit, # 2710  and quantified using Nanodrop 2000C spectrophotometer ThermoFisher Scientific # ND-2000C . The standard curve was established with the points A = 1.0 × 10p-value less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS, version 15.0 .Continuous variables were expressed as mean ± standard deviation if normally distributed or as median and interquartile range (IQR) if non-normally distributed. Categorical variables were described as relative frequencies. The Spearman’s rank correlation coefficient was used to explore the relationship between TTV load and continuous variables. The Wilcoxon rank test was used for comparing TTV loads at different time points. The Mann–Whitney U test was used to compare TTV load differences among dichotomous variables. The ability of TTV load to discriminate infection and rejection was analyzed by constructing receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression analyses were used to analyze the relationship between TTV load and infection and rejection. A The main patient characteristics are shown in p < 0.001) and month 3  (p = 0.200), to donor age  and to time in renal replacement therapy  according to Spearman correlation analysis. Baseline TTV load was significantly higher in male recipients . Neither CMV serostatus  nor preemptive transplant  showed significantly different baseline TTV loads.Median TTV load values increased from pretransplant  to month 1  . Baselinp = 0.890) and at month 3  were not associated with the accumulated thymoglobulin dose. The Mann–Whitney U test did not find significant differences in TTV load at month 1 comparing recipient gender  , diabetic nephropathy , retransplant , preemptive transplantation , induction use , thymoglobulin , any tacrolimus level <5 at month 1 , any tacrolimus level <6 at month 1  and fast tacrolimus metabolizers .Spearman correlations between TTV load at months 1 and 3 and continuous variables are shown in p = 0.333), diabetic nephropathy , retransplant , preemptive transplantation , thymoglobulin , any tacrolimus level < 5 at month 3 , any tacrolimus level < 6 at month 3  and fast tacrolimus metabolizers . Induction use  was the only immunosuppression-related variable associated with the TTV load at month 3.By the Mann–Whitney U test, we did not find significant differences in TTV load at month 3 comparing recipient gender  (p = 0.023) (p = 0.010). After multivariate logistic regression analysis adjusting for other variables related to TTV load , TTV load at month 1 remained independently associated with a higher risk of an opportunistic infection from months 1 to 3 .Patients who developed an opportunistic infection between months 1 and 3 showed higher levels of TTV load at first month  . TTV loa= 0.023) . By univp = 0.026) (p = 0.028) (p = 0.040) and this relationship remained significant  after adjusting by confounders variables such as recipient age, donor age, female recipient gender, estimated GFR and diabetes mellitus.Similarly, patients who developed an opportunistic infection between months 3 and 6 showed higher levels of TTV load at the third month  . TTV loa= 0.028) . TTV loap = 0.177) (p = 0.165).By contrast, TTV load at the first month was not significantly higher in patients who developed acute rejection between months 1 and 3   and was After transplantation, TTV load increased in month 1 and to higher levels in month 3. Similar to us, most authors analyzing kinetic changes in TTV load have reported that TTV load increases after transplantation reaching its maximal value at months 3 to 6 ,17,36,37Due to the low number of rejection episodes in our study, we did not find a relationship with TTV load, although the median differences between patients with (3.89 log) and without rejection (2.57 log) were striking. Most previous studies have found that patients with lower TTV loads are at a higher risk of acute rejection ,20,21,23The only immunosuppressive-drug variable related to TTV load was induction use. Those patients who have received induction showed higher TTV loads. Although the use of thymoglobulin did not relate statistically to TTV load, there was a trend in the median TTV load between those treated and non-treated with lymphocyte-depleting antibodies as induction. Induction with antibodies and/or thymoglobulin is the immunosuppressive treatment most consistently related to higher TTV loads, since it is associated with a stronger immunosuppressive effect, although this relationship has not been confirmed by all authors ,14,19,36The main finding of our study was that higher exposure to maintenance immunosuppressive drugs such as tacrolimus, mycophenolate mofetil, or prednisone was not associated with a higher TTV load. We conducted a detailed study of global tacrolimus exposure. To this end, we collected variables related to sustained exposure to tacrolimus that had not been previously studied in relation to TTV burden. The tacrolimus CV has been related to a worse outcome in solid organ transplantation, not only in relation to under-immunosuppression and increased risk of rejection, but also in relation to over-immunosuppression and increased risk of infections ,29. A shMoreover, different from previous studies, we analyzed the relationship between TTV load and exposure to mycophenolic acid by measuring the gold standard AUC-MPA. Previous authors reported that MMF dose related to TTV load, but neither of them analyzed AUC-MPA . There iIn our study, male recipients showed higher baseline TTV loads. Previous studies reported a similar finding ,19,36. BThe main limitation of our single-center study was the sample size. We demonstrated a relationship between the burden of TTV and opportunistic infections, but not with rejection, mainly due to its low incidence. Another limitation was that we used a non-standardized in-house PCR technique to measure the TTV burden. A recent publication has highlighted the agreement between in-house and standardized techniques and that both methods are useful for measuring TTV . As an aTo conclude, we carried out a prospective study in which it was shown that the TTV loads at one month and the third month after transplantation were independently related to the subsequent risk of infection in renal transplantation. A trend was also detected where patients who experienced an acute rejection had lower levels of TTV. TTV viral load was related to induction use but was not related to overall exposure to maintenance immunosuppression. It is necessary to analyze in greater depth which variables determine the blood values of TTV to use it as a non-invasive biomarker useful to assess the global level of immunosuppression in kidney transplants and other solid organ transplants.