SGLT-2 inhibitors demonstrated significantly lower hemoglobin A1c (HbA1c)  and body weight  at end of study compared to baseline level. There were no significant changes in eGFR, serum creatinine, urine protein creatinine ratio, and blood pressure. By subgroup analysis, empagliflozin demonstrated a significant reduction in body mass index (BMI) and body weight. Canagliflozin revealed a significant decrease in HbA1C and systolic blood pressure. In terms of safety profiles, fourteen patients had urinary tract infection. Only one had genital mycosis, one had acute kidney injury, and one had cellulitis. There were no reported cases of euglycemic ketoacidosis or acute rejection during the treatment. Conclusion: Among kidney transplant patients with excellent kidney function, SGLT-2 inhibitors for treatment of DM are effective in lowering HbA1C, reducing body weight, and preserving kidney function without reporting of serious adverse events, including euglycemic ketoacidosis and acute rejection.Background: The objective of this systematic review was to evaluate the efficacy and safety profiles of sodium-glucose co-transporter 2 (SGLT-2) inhibitors for treatment of diabetes mellitus (DM) among kidney transplant patients. Methods: We conducted electronic searches in Medline, Embase, Scopus, and Cochrane databases from inception through April 2020 to identify studies that investigated the efficacy and safety of SGLT-2 inhibitors in kidney transplant patients with DM. Study results were pooled and analyzed utilizing random-effects model. Results: Eight studies with 132 patients (baseline estimated glomerular filtration rate (eGFR) of 64.5 ± 19.9 mL/min/1.73 m Diabetes mellitus (DM) is the leading cause of end-stage kidney disease (ESKD) worldwide . In the Sodium-glucose co-transporter 2 (SGLT-2) inhibitors are novel oral glucose-lowering medications that inhibit glucose reabsorption in the proximal tubule and promote renal excretion of glucose . GlycemiTherefore, we conducted the present systematic review and meta-analysis to assess the efficacy and safety of SGLT-2 inhibitors for treatment of DM among kidney transplant recipients.A comprehensive search of several databases from each database’s inception to 30 April 2020, any language, was conducted. The databases included Ovid MEDLINE(R) and Epub Ahead of Print, In-Process and Other Non-Indexed Citations, Daily, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, and Scopus. The search strategy was designed and conducted by an experienced librarian with input from the study’s principal investigator. Controlled vocabulary supplemented with keywords was used to search for studies of SGLT-2 inhibitors in kidney transplant patients. The actual strategy listing all search terms used is available in the Given limited data, our study included any studies, including case series and randomized controlled trials, examining the efficacy and safety of any SGLT-2 inhibitors among kidney transplant patients. We excluded case reports or animal studies. Case series were defined as a study that included multiple cases receiving SGLT-2 inhibitors without a control group. For cohort studies or randomized controlled trials, we focused only on treatment group with SGLT-2 inhibitors in order to be comparable and consistent with case series. The quality assessment tool for case series studies proposed by the National Heart, Lung, and Blood Institute (NHLBI) was used to rate the quality . StudiesRetrieved articles were individually reviewed for eligibility by two investigators (A.C. and N.P.) utilizing a systematic review management program called Covidence, which has been selected by Cochrane to become a standard production platform for their reviews. Disagreements were addressed and resolved by third investigator (W.C.).The following data were extracted: first author name, year of publication, number of participants in SGLT-2 inhibitors group, duration of follow-up, types of SGLT-2 inhibitors, mean age, sex, and duration of transplantation. Efficacy outcomes included estimated glomerular filtration rate (eGFR), serum creatinine, urine protein-creatinine ratio, HbA1C, systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), and body weight. Safety profile outcomes included incidence of urinary tract infection, genital mycosis, euglycemic ketoacidosis, acute kidney injury, acute rejection, ulcer, and cellulitis.2 statistic and the chi-square test. A value of I2 of 0% to 25% represents insignificant heterogeneity, 26% to 50% low heterogeneity, 51% to 75% moderate heterogeneity, and 76 to 100% high heterogeneity ; p = 0.06, I2 = 0%) (Seven studies with 124 patients treated with SGLT-2 inhibitors did not show a significant decline in eGFR between baseline and the end of the study (WMD = −2.51 mL/min/1.73 mI2 = 0%) .p = 0.21, I2 = 0%). Three studies with 38 participants treated with SGLT-2 inhibitors reported no significant difference in urine protein creatinine ratio between levels at baseline and end of study   .2 ; p = 0.09, I2 = 0%) or between levels at baseline and at 12 months among 4 studies with 68 participants . In terms of urine protein creatinine ratio, there was no significant difference between levels at baseline and at 6 months . For serum creatinine, there was no significant difference between levels at baseline and at 12 months  (There was no significant decline in eGFR comparing between levels at baseline and at either 6 months among 5 studies with 72 participants (WMD = −2.38 mL/min/1.73 mI2 = 0%) .p = 0.006, I2 = 85.2%)  .p = 0.03, I2 = 79.8%). However, comparing HbA1C levels at 6 months and baseline, 5 studies with 72 participants showed no significant difference   .p = 0.18, I2 = 21.3%) and DBP . Eight studies with 132 participants treated with SGLT-2 inhibitors demonstrated a significant decrease in body weight between baseline and the end of study . However, there was no significant change in BMI between at baseline and end of study among 3 studies with 38 participants treated with SGLT-2 inhibitors   .2 ; p = 0.007, I2 = 0%) and body weight  than baseline, while at 12 months, only body weight was significantly lower than baseline  but not BMI   .In total of 8 studies, 14 out of 132 participants had urinary tract infection. One participant out of 72 participants from 5 studies had genital mycosis. No euglycemic ketoacidosis were reported in four studies. Based on 4 studies, 1 out of 28 participants had acute kidney injury. One study reported one small ulcer in lower extremity and another study reported one case of cellulitis. Based on five reported studies, no rejections were found during follow-up period .2 ; p = 0.006, I2 = 0%). Four studies with 48 participants treated with empagliflozin showed a lower body weight at end of study compared to baseline . There were no significant changes in kidney function, HbA1C, SBP, and DBP between baseline and at end of study among participants treated with empagliflozin. On the other hand, 2 studies with 34 participants treated with canagliflozin reported a significant decline in HbA1C  and SBP . There were no significant changes in kidney functions, BMI, and body weight between baseline and at end of study among participants treated with canagliflozin . Moreover, a funnel plot for eGFR of the included studies did not suggest any asymmetry. Therefore, publication bias was less likely to occur in our meta-analysis , small ulcers in lower extremities (10%), cellulitis (10%), AKI (3.6%), and genital fungal infection (1.4%), respectively. No euglycemic ketoacidosis and acute rejection events were reported.SGLT-2 inhibitors inhibit sodium-glucose reabsorption at proximal renal tubules, causing osmotic diuresis and natriuresis . As a reIn terms of blood pressure, theoretically, a decrease in body weight combined with osmotic diuresis and reduction of total body sodium can result in lowering blood pressure around 4–6 mmHg in SBP and 1–2 mmHg in DBP, as shown in non-kidney transplant patients with DM . HoweverIn addition to the effect on diminishing intraglomerular hyperfiltration and hypertension, SGLT-2 inhibitors also demonstrate anti-inflammatory, antifibrotic, and protective effects against deregulation of extracellular matrix evidenced by a reduction in serum levels of TNF receptor 1, IL-6, matrix metalloproteinase 7, and fibronectin 1 . Moreovep = 0.13). This was supported by previous meta-analysis of non-kidney transplant diabetic population, which revealed an increased risk of genital infection but found no significant increased risk of urinary tract infection [2) during the first ~3–6 weeks of treatment SGLT-2 inhibitors has been observed among non-kidney transplant patients, and data has supported this [In light of safety profiles of SGLT-2 inhibitors among kidney transplant recipients with DM, the incidence of urinary tract infection was 43.8% in our study vs. 38.0% among general kidney transplant recipients , which wnfection . In our nfection ,69. Neveted this ,70. Loweted this ,70. Neveted this . While oThere are some limitations in our meta-analysis. Firstly, included studies were mostly case series without a control group, and the sample size was fairly small. Secondly, the median time of follow-up was 12 months. Hence, some complications might not be have been observed during 1–2 years of follow-up. Moreover, we did not have a control group to compare to, so given the design of each study and insufficient data from each study, we could not adjust for potential confounders such as concomitant antidiabetic medications and duration of diabetes. Furthermore, the data on cardiovascular outcomes were lacking. Therefore, large randomized controlled trials with long-term follow-up are required to investigate the effects of SGLT-2 inhibitors on outcomes after kidney transplantation, including cardiovascular events, delayed graft rejection and mortality risk, as well as safety profiles such as hypovolemia . ConversIn conclusion, our study suggested that SGLT-2 inhibitors were effective in lowering HbA1C when treated at least 12 months, reducing body weight when treated at least 6 months, and preserving kidney function among kidney transplant recipients with DM without reported serious adverse events, including euglycemic ketoacidosis and acute rejection. Based on our findings, future randomized controlled trials are required to further investigate the efficacy and safety of SGLT-2 inhibitors, including long-term allograft and patient survival.


In kidney transplant recipients (KTRs), uraemia-induced central nervous system damage partly subsides, while the long-lasting exposure to tacrolimus may cause pathologic visual evoked potentials (VEP) findings, which have not been investigated yet. Thus, the aim of the present study was to assess the effect of tacrolimus maintenance treatment on bioelectrical function of optic nerves in stable KTRs. Sixty-five stable KTRs were enrolled, including 30 patients treated with twice-daily (Prograf) and 35 patients treated with prolonged once-daily (Advagraf) tacrolimus formulation. In all patients, pattern and flash VEP measurements were performed. Tacrolimus dosing and exposure were also analyzed. Overall, 129 eyes were analyzed. In pattern VEP, both (1°) and (15′) latencies of P100 waves were significantly longer, whereas (1°) and (15′) amplitudes were lower in the Advagraf group as compared with the Prograf group. Multivariate regression analyses revealed that the use of Advagraf (vs. Prograf) was independently associated with longer (1°) and (15′) P100 latencies and lower corresponding amplitudes, whereas log tacrolimus daily dose was only related to amplitudes in a whole study group. In flash VEP, log tacrolimus trough level was associated with negative changes in P2 wave amplitude irrespective of tacrolimus formulation, whereas its association with P2 latency was observed only in the Prograf group. Both the type of tacrolimus formulation and drug exposure influenced the VEP parameters in stable KTRs. Nowadays, tacrolimus is the main immunosuppressive agent used in kidney transplantation . The intVisual-evoked potentials (VEP) is a recorded occipital lobe brain wave potential in response to visual stimulation . It has Sixty-five adult recipients of their first kidney graft, who had undergone a successful kidney transplantation at least 24 months previously, were enrolled to this cross-sectional study. The study was conducted in accordance with the Declaration of Helsinki. The Bioethics Committee of the Medical University of Silesia granted permission for the study protocol (No KNW/0022/KB1/93/13) and informed consent was obtained from all patients.At the start, based on the prospectively operated center database, we identified those patients who were treated with tacrolimus since transplantation. Patients with pre-transplant diabetes and those with diabetes diagnosed at any time post-transplantation were excluded. During the routine ambulatory visit, patients were examined by an ophthalmologist. At this stage, we further excluded patients with photosensitive epilepsy; a history of strokes; neurosurgery episodes; serious head injury; a history of optic nerve and/or retina diseases; cataract worsening visual acuity; nystagmus; photophobia or other unpleasant sensations due to flashes of light; and concomitant therapy with potentially neurotoxic medications other than tacrolimus. Furthermore, we excluded patients with unstable kidney graft function over the last 6 months, current infections, a diagnosis of malignancy or liver cirrhosis.In all study patients, intima-media thickness (IMT) was measured and the occurrence of any plaque in both carotid arteries were noted as surrogate markers of atherosclerosis. Additional blood samples were withdrawn for the several biochemical parameters determination and for the calculation of tacrolimus exposure using the area under the curve (AUC). Both pattern and flash VEP measurements were also performed.Carotid ultrasound was performed using a Siemens machine , equipped with a 4.0–9.0 MHz linear transducer. Carotid arteries were examined with a patient in the supine position with the neck extended. The evaluation included the common, internal, and external carotid arteries, and a carotid bifurcation on each side. The common carotid artery IMT was measured within 2 cm proximal to the carotid bulb, omitting any visible plaques, and the higher value from both sides was taken for further analyses. The results from three separate measurements on each side were averaged. At each examined location, the vessels were carefully evaluated in terms of the presence of plaque, which was classified based on the simplified scale: 0—no lesions; 1—non-calcified lesions; 2—at least one calcified lesion; 3—few calcified lesions; 4—carotid bulb heavily covered by calcified lesions.The plasma concentrations of high-sensitivity C-reactive protein (hsCRP) were assessed with the use of an enzyme-linked immunosorbent assay (ELISA) , with the limit of quantification (LoQ) of 0.09 mg/l, intra-assay variation <6%, inter-assay variation <11.6%. Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and brain derived neurotrophic factor (BDNF) were assessed with an ELISA  with an LoQ of 0.7 pg/mL, 0.191 pg/mL and 20 pg/mL, intra-assay variation <4.2%, <8.7% and <6.3%, and inter-assay variation <6.4%, <10.4%, and <11.4% respectively. Plasma concentrations of asymmetric dimethylarginine (ADMA) were measured using ELISA , with an LoQ of 0.16 µmol/l, intra-assay variation <7.6% and inter-assay variation <4%. Plasma concentrations of endothelin-1 (ET-1) were measured using ELISA , with an LoQ of 2.71 pg/mL, an intra-assay variation <10%, inter-assay variation <12%.Kidney graft function was measured using an estimated glomerular filtration rate (eGFR) calculated according to the Modification of Diet in Renal Disease (MDRD) formula.Blood samples for tacrolimus trough level assessments were routinely withdrawn 12 h after the evening dose in patients receiving twice-daily formulation (Prograf) and 24 h after the morning dose in patients treated with once-daily, prolonged-release formulation (Advagraf), immediately prior to the next morning dose. Additional blood samples for AUC calculation were withdrawn 3 h after the morning tacrolimus dose ingestion. Tacrolimus concentrations were assessed using the microparticle enzyme immunoassay for Abbott Architect . AUC for tacrolimus was calculated using the formula:ngestion . The timIn all eligible patients, VEP were tested using the Reti-Port electrophysiological apparatus from Roland Consult , in accordance with the standards of the International Society for Clinical Electrophysiology of Vision (ISCEV) . The patA black and white checkerboard pattern with alternating phase change  was used to elicit a visual cortical response. The reversal rate was of 2 reversals per second. Each patient’s eye was stimulated with small  and large (1° per arc) checks. For flash stimulation , standard flashes with a frequency of 1.4 Hz were used in the Ganzfeld stimulator. Each VEP test was preceded by checking the visual acuity of the distance in the patient’s correction with glasses using Snellen charts, an intraocular pressure test using a Goldmann applanation tonometer  and was finished with anterior and posterior examination of the eyeball in a slit lamp (Haag-Streit) with an indirect method using a Volka lens (Super Field) to monitor possible pathological changes in the eye that may affect the result of the VEP test or the result of neurotoxicity . Before the fundus examination, short-acting (4–6 h) mydriatics were given: parasympatholytic  or sympathomimetic  drops. In case of a lack of patient consent for mydriasis, the fundus was checked through the narrow pupil. Refusal of consent for pupil dilation did not affect the patient’s further participation in the study and did not change the patient’s treatment.The amplitude and the latency of the P100 (PVEP) and P2 (FVEP) waves were analyzed. Amplitudes were measured from the top of the negative peak (N) preceding the positive (P) wave (N75-P100 and N2-P2) and the latency from the beginning of the stimulation to the occurrence of the highest value of the marked wave.2 test  or the Mann–Whitney U test . Correlation coefficients were calculated according to Pearson. Data of variables with a not-normal distribution were logarithmized for the correlation analyses. Multivariate regression analyses were performed for the (1°) and (15′) latencies of the P100 wave as dependent variables, including age, log time post transplantation, 12-month tacrolimus exposure, log tacrolimus trough level and the tacrolimus formulation (Advagraf vs. Prograf) as potential independent variables. Analogic regression analyses were performed for the (1°) and (15′) amplitudes of the P100 wave as dependent variables, including eGFR, log time post transplantation, 12-month tacrolimus exposure, log tacrolimus daily dose and the tacrolimus formulation (Advagraf vs. Prograf) as potential independent variables. Lastly, we also performed the backward multivariate regression analysis for P2 wave latency , 12-month tacrolimus exposure, age and the tacrolimus formulation as potential independent variables) and P2 wave amplitude . In all statistical tests, p values less than 0.05 were considered as statistically significant.Statistical analyses were performed using the STATISTICA 13.3 PL for Windows software package  and MedCalc v19.2.1 . Values are presented as means and 95% confidence intervals or frequencies. Variables with skewed distribution were presented as medians and interquartile ranges (IQRs). The main comparison was performed for groups with different tacrolimus formulations: Prograf or Advagraf. The subgroups were compared using the Student t test or χAll study patients were treated with tacrolimus since the transplantation. Thirty patients received a twice-daily formulation (Prograf) and 35 study participants received a once-daily tacrolimus formulation (Advagraf) at least during the 12 months preceding the study measurements . The additional immunosuppressive regimen consists of mycophenolate mofetil or sodium and steroids. The mycophenolate daily doses and the percentage of patients receiving steroids at the time of the study were comparable between subgroups.Both study subgroups did not differ in terms of age, sex, BMI, dialysis vintage prior to transplantation and kidney graft function at the time of the study. Both subgroups were also comparable as regard to the maximum IMT value and the percentage of calcified plaques in carotid arteries. However, the post-transplant follow-up period was significantly longer in the Advagraf-treated patients . ImportaAt the time of the study, there were no differences between the daily tacrolimus dose and the tacrolimus dose calculated per kg of body weight between the study subgroups . In contp < 0.001) and inversely with (1°) and (15′) amplitudes . Except for the above associations, only (1°) and (15′) amplitudes were positively related .In the pattern VEP examinations, both (1°) and (15′) latencies were significantly longer in the Advagraf group as compared with the Prograf group . Accordip < 0.05), whereas log P2 wave amplitude was positively associated with both (1°) and (15′) P100 amplitudes (r = 0.361 and r = 0.327) and inversely with P2 latency (r = −0.327); all p < 0.001.In the flash VEP examinations, there were no significant differences between the groups with regard to the latency and amplitude of the P2 wave. P2 wave latency weakly correlated only with (15′) P100 latency , but not amplitudes were significantly associated with recipient age. On the other hand, P100 wave (1°) and (15′) amplitudes, but not corresponding latencies, were significantly positively associated with log tacrolimus daily dose  (p < 0.001), whereas both were inversely related to eGFR . Notably, there were no associations between P100 wave (1°) and (15′) latencies and amplitudes and log tacrolimus trough concentration and tacrolimus AUC.In univariate analyses, P100 wave (1°) and (15′) latencies  A and 2B p < 0.001) (p < 0.05), BMI , 12-month tacrolimus exposure  (p < 0.05), but not with tacrolimus daily dose or eGFR. On the other hand, P2 wave amplitude inversely correlated with log tacrolimus trough level  (p < 0.01), but not with log tacrolimus daily dose, 12-month tacrolimus exposure or eGFR.In contrast, P2 wave latency presents its most pronounced associations with log tacrolimus trough level  A, log ta < 0.01) B and age < 0.01) C and logp < 0.001) (p < 0.001) were observed only in the Prograf subgroup, whereas no analogical associations were observed in the Advagraf subgroup  and (15′) amplitudes with log tacrolimus daily dose  A,B and lsubgroup C,D.p < 0.001, r = 0.254; p < 0.05, r = 0.307; p < 0.01 and r = 0.281; p < 0.05, respectively), but not the Advagraf, subgroups, except the correlation with BMI, which was universal. Out of the previously described P2 wave amplitude correlations, only the one with tacrolimus trough level was universal, although it was of borderline significance in both subgroups, whereas others were present only in the Prograf subgroup.Similarly, all aforementioned associations between P2 wave latency and other parameters were observed in the Prograf , but not corresponding latencies. Interestingly, plasma ADMA and BDNF concentrations were not associated with P100 (1°) and (15′) measures in the whole group, but both were positively correlated with P100 (15′) latency only in the Advagraf subgroup .In the whole study group, ET-1 concentration was positively associated with P100 wave (1°) and (15′) amplitudes . There was no significant association between those biochemical parameters and P100 latencies.The analysis of inflammatory status parameters revealed that IL-6 and TNF-α, but not hsCRP concentrations showed positive associations with P100 (1°) and (15′) amplitudes  = 0.309; p < 0.001) and the use of Advagraf formulation , but not log time post transplantation or tacrolimus 12-month exposure, independently influenced the P100 wave (1°) latency (R2 = 0.18). In an analogic analysis, age , log time post transplantation  and the use of Advagraf formulation , but not tacrolimus exposure, independently influenced the P100 wave (15′) latency (R2 = 0.22).Stepwise backward multivariate regression analysis revealed that age , log tacrolimus daily dose  and eGFR , but not log time post transplantation or tacrolimus exposure, independently influenced the P100 wave (1°) amplitude (R2 = 0.21). In an analogic analysis, eGFR , log tacrolimus daily dose  and the use of Advagraf formulation  independently influenced the P100 wave (15′) latency (R2 = 0.25).Stepwise backward multivariate regression analysis revealed that the use of Advagraf formulation , BMI  and age  independently influenced the P2 wave latency (R2 = 0.24). In a similar analysis, log tacrolimus dose per kg  and log tacrolimus blood trough level  independently influenced the P2 wave amplitude (R2 = 0.09).Stepwise backward multivariate analysis revealed that log tacrolimus blood trough level . The latter may result in vasoconstriction and relative ischemia of macular optic fibers . InteresIn the present study, we have analyzed both pattern and flash VEP results. Of importance, when both latency and amplitude of the P2 component were negatively influenced by the increased blood tacrolimus trough concentration, the observed association between the tacrolimus dose and P100 wave amplitude was positive, i.e., there was a tendency toward a normal P100 spectrum along with an increasing drug dose. This may be partly explained by the unexpectedly positive correlation between ET-1 concentration and P100 amplitude values in our study, as increased ET-1 levels and vasoconstriction were previously assigned to tacrolimus nephrotoxicity . Thus, wIn contrast, the use of once-daily tacrolimus formulation was independently associated with all identified unfavorable tendencies in the P100 wave spectrum in comparison with twice-daily formulation. As the Advagraf subgroup was characterized by a substantially longer observation after transplantation , we cannot exclude the potential bias. However, the negative association between the time after transplantation and P100 (15′) latency as well as the lack of such associations with the rest of PVEP or FVEP parameters in multivariate analyses does not confirm such an explanation. It is also worth noticing that a positive correlation between P100 (15′) latency and BDNF level was seen only in the Advagraf subgroup. BDNF regulates synaptic plasticity and is a critical protein in maintaining neuronal function . On the Interestingly, the prolonged, once-daily formulation was developed to increase the drug adherence and minimize toxicity due to the more flattened blood level slope. In fact, beneficial changes were noted in renal hemodynamics and function after conversion to prolonged-release tacrolimus in healthy volunteers . HoweverNowadays, an issue with tacrolimus neurotoxicity is emerging as the another prolonged-release tacrolimus formulation was introduced with a different mechanism of drug absorption, based on Melt-Dose technology . NotablyThe main drawback of our study is the limited number of participants. This is mostly the consequence of the strict study exclusion criteria, which covered any diabetes or previous neurologic disease. Such a selection aimed to eliminate patients with comorbidities that may have interfered with the VEP results. Nevertheless, this is the first clinical study analyzing the relationship of tacrolimus dosing and exposure with the bioelectrical function of the visual pathway measured by VEP. Another limitation is the lack of a control group of KTRs receiving calcineurin inhibitor-free immunosuppressive regimen. This is because of the unavailability of such patients who met the study inclusion criteria in our out-patient department KTR cohort. Moreover, as we used a limited sampling strategy (LSS) for tacrolimus AUC calculation which was originally developed for liver transplant patients, it may not be ideal for kidney transplant recipients. However, the analysis of several LSS developed based on different solid organ recipients populations  indicateIn conclusion, in stable KTRs treated with tacrolimus, pathologic values in VEP examination are highly prevalent despite no overt tacrolimus neurotoxicity. There is an association between tacrolimus exposure and simultaneously assessed bioelectrical function of the visual pathway in kidney transplant population. The substantial differences in VEP findings between subjects treated with twice-daily or once-daily tacrolimus formulations were also described. Further investigation is warranted to confirm these findings, including the conversion study switching the tacrolimus formulations with concomitant accurate neurotoxicity monitoring.


Statistical analysis was performed by multiple linear regression. After adjustment for eGFR and other important confounders, only cFGF23 but not iFGF23 was significantly associated with hemoglobin levels and this association was largely accounted for by iron metabolism parameters. cFGF23 but not iFGF23 was also associated with mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), again in dependence on iron metabolism parameters. Similarly, EPO concentrations were associated with cFGF23 but not iFGF23, but their contribution to the association of cFGF23 with hemoglobin levels was marginal. In pre-dialysis CKD patients, the observed association of FGF23 with hemoglobin seems to be restricted to cFGF23 and largely explained by the iron status.Anemia in chronic kidney disease (CKD) is an almost universal complication of this condition. Fibroblast growth factor 23 (FGF23), a key-player in mineral metabolism, is reportedly associated with anemia and hemoglobin levels in non-dialysis CKD patients. Here, we sought to further characterize this association while taking into account the biologically active, intact fraction of FGF23, iron metabolism, and erythropoietin (EPO). Hemoglobin, EPO, iron, and mineral metabolism parameters, including both intact and c-terminal-FGF23  were measured cross-sectionally in 225 non-dialysis CKD patients (stage 1–5, median eGFR: 30 mL/min./1.73m Anemia in chronic kidney disease (CKD) is a major complication of this condition. It is associated with increased morbidity and mortality and its prevalence increases as renal function declines, affecting the great majority of patients at late stages . Among tIron deficiency in non-dialysis CKD is thought to mainly result from elevated hepcidin levels and increased gastrointestinal blood loss ,4. The hThe erythropoiesis-stimulating glycoprotein hormone erythropoietin (EPO) is mainly produced by peritubular renal fibroblasts. Renal tissue hypoxia leads to secretion of EPO via the induction of hypoxia-inducible factors (HIF) . Anemia The osteocyte-secreted polypeptide fibroblast growth factor 23 (FGF23) is a key regulator of phosphate homeostasis . FGF23 iRecently, FGF23 has been linked to physiological processes beyond phosphate homeostasis, including iron metabolism, EPO secretion, and erythropoiesis. Iron deficiency and acute administration of recombinant EPO increased cFGF23, but not iFGF23 serum concentrations in humans ,14. In mIn CKD patients, associations of FGF23 serum concentrations with hemoglobin levels and the presence of anemia have been described ,18,19. HWe included patients that were enrolled in a CKD cohort established at the Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, comprising patients of all six eGFR categories The exclusion criteria for this cohort were age less than 18 years, pregnancy, and history of kidney transplantation. Serum and plasma samples were collected in a biobank repository. Clinical and demographic data were retrieved from the participating patients and from medical charts at the time of inclusion. The study protocol was approved by the ethics committee of the Medical University of Vienna (project number EK2012/2016), adhered to the Declaration of Helsinki, and all of the patients provided written informed consent.We used laboratory tests that are certified for in vitro diagnostics, as follows: Red blood count, hemoglobin, erythrocyte indices , total calcium, inorganic phosphate (iP), albumin, protein, creatinine, intact PTH, iron, transferrin, ferritin, IL-6 , 25-hydroxy-vitamin D, (25(OH)D), 1,25-dihydroxy-vitamin D 2D), intact FGF23 (iFGF23) , erythropoietin (EPO) , and soluble transferrin-receptor (sTFR) . For c-terminal FGF23 (cFGF23), the Immutopics Human FGF-23 (C-Term) ELISA , for Klotho the Soluble alpha-Klotho ELISA , for Hepcidin the Human Hepcidin Quantikine ELISA  were used according to the manufacturers’ instructions. The estimated glomerular filtration rate (eGFR) was determined according to the CKD-EPI formula. All of the analyses were performed in ISO 15189 accredited clinical laboratories of the Department of Laboratory Medicine at the Medical University of Vienna.2 values are reported to quantify the proportion of variability in the dependent variable that is explained by an independent variable or a set of variables, i.e., for comparing their prognostic importance. Multiple regression models were used to adjust investigated influences for the potential confounders albumin, gender, eGFR, and presence of diabetes mellitus type II. 95% confidence intervals are provided for all reported estimates. Residual distributions were graphically checked. None of the potentially influential observations exhibited a relevant impact on the results upon deletion in the adjusted models.The categorical variables are described by counts and percentages. Continuous variables are described by means and standard deviations if approximately normally distributed and by medians and interquartile ranges otherwise. Linear regression models were used to quantify the effect of continuous markers on continuous outcome parameters. Variables exhibiting right-skewed distributions were log-transformed before used in a model. For a log-transformed independent variable, the estimated regression coefficient thus quantifies the effect of doubling this variable. For a log-transformed dependent variable, the regression coefficient was back-transformed to quantify a multiplicative effect on the original variable’s scale. The RThe amount of blood that was drawn at study inclusion was not completely uniform because of variable tube filling at the venipuncture procedure leading to insufficient sample material for some analyses. Therefore, we assume that missing data are missing at random and are not associated with the unmeasured values. Accordingly, each model is based on complete cases with respect to the variables involved. The total numbers of values used for calculation are reported for each variable in p-values that were below 0.05 were regarded as statistically significant. No correction for multiple testing was done due to the exploratory character of this study. All of the calculations were done using SAS version 9.4 .2, eGFR range 2.5–152 mL/min./1.73m2). Native kidney disease was diabetic nephropathy: 26; vascular nephropathy: 41; polycystic kidney disease: 13; glomerulonephritis: 67; interstitial nephritis: 6; other : 35; unknown: 37. We included 225 patients with CKD stage G1 to G5 in this study . Multiple parameters of mineral metabolism, iron metabolism, EPO, and inflammatory markers show a statistically significant effect on hemoglobin in the unadjusted analysis. However, in the adjusted base model, only the influences of calcium, phosphate, serum iron, transferrin saturation, hepcidin, and EPO remain significant beside cFGF23 . Adding EPO to the base model (EPO model) only has a modest impact on the effect of cFGF23 on hemoglobin, which remains statistically significant . Finally, adding the parameters of inflammation  does not influence this association at all. No significant association of iFGF23 with hemoglobin is found in any of these additional models.We added selected sets of parameters to account for mediation via iron, EPO, and inflammation pathways to assess differential pathways implicated in erythropoiesis that might be involved in a proposed cFGF23 hemoglobin axis . Adding The associations of phosphate and calcium with hemoglobin are largely independent of iron status, EPO, and inflammation biomarkers .For further investigation of mediation, we tested the influence of cFGF23, iFGF23, calcium, and phosphate on the potentially mediating parameters discussed above . ConsistNo other statistically significant relationships of phosphate or calcium with any of the potential mediators under assessment are apparent, except for an association of phosphate with EPO .We next looked at the erythrocyte indices mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), given the strong involvement of iron parameters in the association of cFGF23 with hemoglobin. Consistently, we found that cFGF23, but not iFGF23, phosphate, or calcium significantly influences MCH and this association is abolished in the iron, but not in the EPO or inflammation adjusted model . Similar2). Our main findings are as follows:In this cross-sectional study, we aimed to further characterize the role of FGF23 in anemia in CKD. For this purpose, we analyzed both total cFGF23 and bioactive iFGF23, biomarkers of iron metabolism, including hepcidin, inflammation, and EPO, in a cohort of non-dialysis CKD patients with predominantly moderate to severe renal function impairment (median eGFR 30 mL/min./1.73m(1) We confirm the previously described association of total cFGF23 with hemoglobin in non-dialysis CKD patients, however (2) such an association does not hold true for the bioactive iFGF23 in an adjusted model. (3) Biomarkers of iron metabolism and, to a lesser extent, EPO appear to largely explain the association of cFGF23 with hemoglobin.The association of FGF23 with anemia or hemoglobin has been previously described in three independent studies of non-dialysis CKD patients ,18,19. HWe show that the reported associations of cFGF23 with hemoglobin do not extend to the intact form of FGF23 in the adjusted analysis. Similar divergences have been previously described in several settings. In iron deficient renal transplant recipients cFGF23, but not iFGF23, appeared to mediate the association of iron deficiency with mortality . Red celOne of the specific triggers of FGF23 cleavage by proteolysis appears to be iron deficiency, which contributes to the pathophysiology of anemia in CKD. Wolf et al.  reportedWe see a robust correlation of iron indices with cFGF23 . Total cWe observed a significant influence of cFGF23, but not iFGF23 on EPO serum levels . FurtherThe associations of phosphate and calcium with hemoglobin remain robust in adjusted analyses in agreement with previous reports ,29 Supp. HoweverWe note a significant direct association of cFGF23 with IL-6, which is consistent with previous data that inflammatory markers associate with FGF23 levels in CKD ,31. We dJointly, our data render a quite consistent picture of FGF23 in the clinical context of anemia in CKD, in which its interaction with iron metabolism holds a prominent rank. The invariant divergence between cFGF23 and iFGF23 in terms of associations with all studied indices of hematopoiesis and iron metabolism is striking. Interestingly, it was shown that higher doses of iron supplementation in hemodialysis patients improve cardiovascular outcomes . It is tThe main limitation of our study is its cross-sectional design, which precludes definitive causal or mechanistic conclusions. This needs to be addressed in future studies.However, our study has several strengths: The study was conducted in humans, in a representative patient cohort of a large outpatient clinic comprising the full range of renal function impairment not on dialysis. We excluded patients on ESA or intravenous iron therapy, as these interventions could have confounded the FGF23 measurements. Moreover, we wanted to avoid potential cross-reactivity with the EPO assay for the various ESA preparations. We measured FGF23 using both intact and c-terminal assays and performed a detailed analysis of a wide range of parameters that are involved in iron metabolism and inflammation as well as endogenous EPO, which, to the best of our knowledge, has never been analyzed before in the context of FGF23′s role in anemia in human CKD.We demonstrate that cFGF23 levels, which comprise both c-terminal fragments and the intact form, rather than the hormonally active iFGF23, are associated with hemoglobin levels in CKD patients. Iron metabolism and, to a lesser extent, EPO seem to account for this association, in agreement to previous experimental data.


Hypertension is the leading risk factor for disability and mortality globally. It is estimated that in 2019, high systolic blood pressure accounted for 10.8 million deaths  and 235 million disability-adjusted life years  worldwide [th percentile) and 12.7% (95% CI: 2.1-30.4) for slightly elevated blood pressure (systolic or diastolic blood pressure ≥ 90th percentile and < 95th percentile) [Hypertension has long been a neglected issue in children. Until recently, it was rarely searched for or diagnosed in children. For long, the diagnosis of hypertension was made only in the presence of very elevated blood pressure levels. In this context, only the most severe cases of secondary hypertension were identified . With thcentile) . Moreovecentile) .Hypertension in children is increasingly recognized as a serious public health problem, not only because of its rising prevalence, but also because growing evidence indicates that elevated blood pressure early in life has detrimental lifelong cardiovascular effects . Indeed,th percentile) was found in 8.5% of children, and slightly elevated blood pressure (systolic or diastolic blood pressure ≥ 90th percentile and < 95th percentile) in 9.6% of them. Factors associated with elevated blood pressure included age ≥ 10 years , female sex , overweight , obesity , eating fried food , drinking sugary soft drinks  and not eating fruits  [In this volume of the Pan African Medical Journal, Edson Elias Sungwa and colleagues report on a cross-sectional study that investigated the blood pressure profile and factors associated with elevated blood pressure in 742 school children aged 6 to 16 years from Mwanza, Tanzania . Elevate.1-65.8) . BesidesIndeed, the substantial contribution of modifiable risk factors to the occurrence of hypertension in children suggests that it can be prevented to a large extent. The study by Sungwa and colleagues corroborates that interventions for the promotion of physical activity, avoidance of energy-dense foods and sugar-sweetened beverages, increased consumption of fruits and vegetables are crucial for primordial prevention of hypertension . These iSystematic screening and pharmacological management of elevated blood pressure in children remain grey areas. According to a recent report of the US Preventive Services Task Force, there is inadequate evidence about the accuracy of screening for elevated blood pressure in children and adolescents, and it is unknown whether this screening would delay or lessen adverse health outcomes . AdditioDisclosures: Dr Noubiap is supported by a Postgraduate Scholarship from the University of Adelaide.


PP) but different levels of systolic blood pressure (SBP), although the underlying mechanisms have not been described. We hypothesized that, for a given level of PP, differences in SBP relate to peripheral vascular resistance (PVR); and we tested this hypothesis in a large cohort of healthy young adults.Two individuals can have a similar pulse pressure . Moreover, stratifying individuals by quartiles of PP and PVR revealed a stepwise increase in SBP from the lowest to highest quartile for each variable, with the highest SBP occurring in those in the highest quartile of both PP and PVR . PVR was also increased with increasing tertile of minimum forearm vascular resistance, in both men (P=0.002) and women (P=0.03).Demographic, biochemical, and hemodynamic data from 3103 subjects were available for the current analyses. In both men and women, for a given level of PVR, mediated in part through altered resistance vessel structure, strongly associates with the elevation of SBP for a given level of PP in young adults. An impaired ability to adapt PVR appropriately to a given level of PP may be an important mechanism underlying elevated SBP in young adults.Increased  AIxaugmentation indexaPWVaortic pulse wave velocityBMIbody mass indexMAPmean arterial pressureMFVRminimum forearm vascular resistancePPpulse pressurePPApulse pressure amplificationPVRperipheral vascular resistanceSBPsystolic blood pressureTACtotal arterial complianceElevated peripheral vascular resistance is the major mechanism explaining why differences in systolic blood pressure occur between young adults presenting with similar levels of pulse pressure and stroke volume.The differences in peripheral vascular resistance are mediated, in part, through altered resistance vessel structure and are also associated with greater body weight and body mass index.High systolic blood pressure in young adults suggests that the vascular adaptation to a given level of stroke volume is impaired, and this impairment is associated with alterations in the underlying vascular structure.The most common form of hypertension in young adults is isolated systolic hypertension (ISH).The key hemodynamic determinants of PP are stroke volume and aortic stiffness, although in young adults, it is stroke volume rather than aortic stiffness that is predominantly associated with increased PP, particularly in men.Systolic and diastolic BP values oscillate around mean arterial pressure (MAP), which is determined, physiologically, by cardiac output and peripheral vascular resistance (PVR). Therefore, we hypothesized that for a given level of PP, variations in SBP between individuals are related to PVR, and the aim of this study was to test this hypothesis in a large cohort of healthy young adults. In addition, we measured minimum forearm vascular resistance (MFVR) in a random subset of subjects to determine whether structural differences in the resistance vasculature were associated with variations in PVR.The data that support the findings of this study are available from the corresponding author on reasonable request.Subjects were drawn from The Enigma Study population, which consists of 3145 individuals aged between 18 and 40 years, selected at random from 2 university populations in the United Kingdom .Each participant completed a detailed lifestyle and medical history questionnaire. Height and weight were measured; body surface area and body mass index (BMI) were calculated. After 20 minutes of supine rest, brachial BP and radial artery waveforms were recorded, as well as aortic pulse wave velocity (aPWV). Stroke volume and cardiac output were assessed, and PVR was calculated. Blood samples were collected from the antecubital vein under fasting conditions. Total cholesterol, low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, triglycerides, and serum glucose were assessed using standard laboratory methods in an accredited laboratory. MFVR was assessed in a random subset of individuals.Brachial BP was measured in the dominant arm, using a validated oscillometric sphygmomanometer and an appropriately sized cuff , while participants rested in a supine position. Three readings were obtained, and the mean value was used for analysis. A high‐fidelity micromanometer , interfaced with a computer using SphygmoCor software , was applied to evaluate central BP, augmentation index (AIx), and aPWV. In brief, radial artery waveforms were recorded, and pulse wave analysis was used to generate a corresponding central (ascending aortic) waveform. From this, central BP, MAP, AIx, and heart rate were derived. PP amplification (PPA) was calculated as the ratio between brachial and central PP. Pulse wave recordings were then performed consecutively at the common carotid and femoral arteries to assess aPWV. Path length was calculated as the superficial distance from the sternal notch to the femoral pulse site minus the sternal notch to the carotid pulse site distance, using a tape measure.6, 5% N2O, and 94% O2) over 20 seconds, with a breathing rate of 15/min. Expired gases were sampled continuously and analyzed by an infrared photoacoustic gas analyzer, for the determination of stroke volume and cardiac output. PVR was calculated using the formula: PVR (dynes·s·cm−5)=MAP (mm Hg)×80/cardiac output (mL/min), and total arterial compliance (TAC) was defined as the ratio of stroke volume/central PP.Stroke volume and cardiac output were measured using a validated noninvasive, inert gas rebreathing technique, which has been validated against thermodilution and direct Fick methods for measurement of pulmonary blood flow and cardiac outputMFVR was assessed in 263 randomly scheduled individuals. Venous occlusion plethysmography  was undertaken in the nondominant arm to measure forearm blood flow before and after 13 minutes of ischemia, as previously described in detail.P<0.05.Continuous variables are presented as means±SD, and categorical variables are presented as absolute numbers (percentages). All analyses were performed separately for men and women because of the significant differences in hemodynamics and mechanisms of BP elevation between sexes in young adults. Three strategies were adopted to examine our hypothesis that for a given level of PP, variations in SBP between individuals are related to PVR. Participants were initially grouped according to quartile of brachial PP, with each PP quartile further stratified by tertile of brachial SBP. This approach ensured adequate separation of hemodynamic data across PP quartiles, while retaining relatively large sample sizes in each SBP group. For the primary analyses, we chose to focus on 4 groups within each sex, comprising the extremes of PP (lowest and highest PP quartiles) and their corresponding lowest and highest SBP tertiles, to allow us to examine, in detail, the hemodynamic mechanisms underlying SBP elevation within a given level of PP. One‐way ANCOVA was used to explore differences in demographic, biochemical, and hemodynamic variables among the 4 groups, with age included as a covariate in all models (except the age model). Height and heart rate were additionally included as covariates in the AIx/PPA models, MAP and heart rate were included in the aPWV model, and BMI and heart rate were included in the TAC model. Post hoc analyses were conducted using the Bonferroni method. Additional analyses, based on all 4 PP quartiles and all corresponding SBP tertiles , were also undertaken. Second, using the entire cohort, men and women were grouped by quartile of PVR and PP and an ANOVA model was constructed with SBP as the dependent variable and PVR/PP quartile as the independent variable for analysis of the overall trend. Third, treating all data as continuous variables, univariable and multivariable linear regression models were constructed to investigate the association of SBP with each hemodynamic determinant of BP. Models were constructed for the whole cohort, and the lowest and highest PP groups. Models for the middle 2 quartiles of PP are shown as a supplementary analysis. Finally, in a separate analysis, men and women were grouped according to tertile of MFVR. One‐way ANOVA was used to explore differences in PVR between MFVR tertiles for each sex, with PVR as the dependent variable and MFVR group (3 groups representing the lowest to highest tertiles of MFVR) as the independent variable, for analysis of the overall trend. Statistical analysis was performed using SAS software, version 9.4 . Statistical significance was defined as Characteristics of the study participants are presented in Table Comparisons of demographic, biochemical, and hemodynamic characteristics between lower and higher SBP groups within the lowest and highest quartiles of PP are shown in Tables In both men Table  and womeP<0.001 for trend in both sexes), indicating that at any given level of PP, SBP increased with an increase in PVR. The highest level of SBP occurred in those individuals in the top quartile of PVR and PP. In multivariable linear regression analyses, independent associations of PVR, TAC, and AIx with SBP were consistently observed in various subgroups stratified by sex and PP  and women (P for trend=0.03) . This work was funded, in part, by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NIHR. The authors appreciate the financial support provided by China Scholarship Council (Grant No. 201806260070) for Dr Yu and acknowledge help from Fondazione per la Ricerca Biomedica Cardiovascolare Foundation.None.Tables S1–S6Click here for additional data file.


Fatty acid products derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω‐1)‐hydroxylase pathways are a superclass of lipid mediators with potent bioactivities. Whether or not the chronic kidney disease (CKD) and hemodialysis treatments performed on end‐stage renal disease (ESRD) patients affect RBC epoxy fatty acids profiles remains unknown. Measuring the products solely in plasma is suboptimal. Since such determinations invariably ignore red blood cells (RBCs) that make up 3 kg of the circulating blood. RBCs are potential reservoirs for epoxy fatty acids that regulate cardiovascular function. We studied 15 healthy persons and 15 ESRD patients undergoing regular hemodialysis treatments. We measured epoxides derived from CYP monooxygenase and metabolites derived from LOX/CYP ω/(ω‐1)‐hydroxylase pathways in RBCs by LC–MS/MS tandem mass spectrometry. Our data demonstrate that various CYP epoxides and LOX/CYP ω/(ω‐1)‐hydroxylase products are increased in RBCs of ESRD patients, compared to control subjects, including dihydroxyeicosatrienoic acids (DHETs), epoxyeicosatetraenoic acids (EEQs), dihydroxydocosapentaenoic acids (DiHDPAs), and hydroxyeicosatetraenoic acids (HETEs). Hemodialysis treatment did not affect the majority of those metabolites. Nevertheless, we detected more pronounced changes in free metabolite levels in RBCs after dialysis, as compared with the total RBC compartment. These findings indicate that free RBC eicosanoids should be considered more dynamic or vulnerable in CKD. Whether or not the chronic kidney disease (CKD) and hemodialysis treatments performed on end‐stage renal disease (ESRD) patients affect RBC epoxy fatty acids profiles remains unknown. Measuring the products solely in plasma is suboptimal since such determinations invariably ignore red blood cells (RBCs) that makeup 3 kg of the circulating blood. RBCs are potential reservoirs for epoxy fatty acids that regulate cardiovascular function. We studied 15 healthy persons and 15 ESRD patients undergoing regular hemodialysis treatments. We measured epoxides derived from CYP monooxygenase and metabolites derived from LOX/CYP ω/(ω‐1)‐hydroxylase pathways in RBCs by LC–MS/MS tandem mass spectrometry. AAarachidonic acid, C20:4CYPcytochrome P450DHAdocosahexaenoic acid, C22:6 n‐3DHETdihydroxyeicosatrienoic acidDiHDHAdihydroxydocosahexaenoic acidDiHDPAdihydroxydocosapentaenoic acidDiHETEdihydroxyeicosatetraenoic acidDiHOMEdihydroxyctadecenoic acidEDHFendothelium‐derived hyperpolarizing factorEDPepoxydocosapentaenoic acidEEQepoxyeicosatetraenoic acidEETepoxyeicosatrienoic acidEPAeicosapentaenoic acid, C20:5 n‐3EpOMEepoxyoctadecenoic acidHDHAhydroxydocosahexaenoic acidHEPEhydroxyeicosapentaenoic acidHETEhydroxyeicosatetraenoic acidHODEhydroxyoctadecadienoic acidHPETEhydroperoxyeicosatetraenoic acidHpODEhydroperoxylinoleic acidLAlinoleic acid, C18:2LOXlipoxygenasePUFApolyunsaturated fatty acid1Chronic kidney disease (CKD) is a risk factor for the composite outcome of all‐cause mortality and cardiovascular disease xy fatty acids (or oxylipins) are lipid peroxidation products of polyunsaturated fatty acids (PUFA), including C18:2 linoleic (LA), C20:0 arachidonic (AA), C20:5 n‐3 eicosapentaenoic (EPA), and C22:5 n‐3 docosahexaenoic acids (DHA). These products are derived from CYP monooxygenase, cyclooxygenase (COX), and LOX/CYP ω/(ω‐1)‐hydroxylase pathways, which catalyze the production in a highly tissue‐dependent and regioisomer‐specific manner Figure . CYP‐der2via person‐to‐person interview. Prior to participation in the study, 15 healthy volunteers  and 15 CKD patients  undergoing regular hemodialysis treatment signed informed consent forms which outlined the treatments to be taken and the possible risks involved. All healthy control subjects were not taking medications. Venous blood was collected in each healthy subject by subcutaneous arm vein puncture in the sitting position. In the group of dialyzed patients (CKD group), all the blood samples were collected on the fistula arm right before beginning of the dialysis  and at the end of the dialysis . Patients underwent thrice‐weekly dialysis, which lasted from 3 hr 45 min to 5 hr, based on high flux AK 200 dialyzers . All samples were analyzed for RBC lipids. All blood samples were obtained by 4°C precooled EDTA vacuum extraction tube systems. Cells were separated from plasma by centrifugation for 10 min at 1,000–2,000 g using a refrigerated centrifuge RBCs were separated from EDTA blood by centrifugation as previously described . Recruitment was primarily t test or Mann–Whitney test was used to compare the values of CKD versus control groups. Paired t‐test or paired Wilcoxon test were used to compare pre‐HD versus post‐HD values. In order to determine statistical significance between the four classes of epoxy‐metabolites hydrolyzed to appear in the circulation, Friedman's test followed by applying Dunn's multiple comparison test was used. In order to determine statistical significance between the four classes of epoxy‐metabolites hydrolyzed to appear in the circulation, Friedman's test followed by applying Dunn's multiple comparison test was used. The analysis included Mauchly's test of sphericity followed by applying the test of within‐subjects effects with Greenhouse–Geisser correction to ensure sphericity assumption  was chosen. All data are presented as mean ± SD. All statistical analyses were performed using SPSS Statistics software (IBM Corporation) or All‐Therapy statistics beta (AICBT Ltd).Descriptive statistics were calculated and variables were examined for meeting assumptions of normal distribution without skewness and kurtosis. In order to determine statistical significance, 33.1p > .05, n = 15 each). The body mass indices between the two groups were also not different . The patients in the group CKD were diagnosed for the following conditions: diabetes mellitus (n = 4 patients), hypertension (n = 3), membranous glomerulonephritis (n = 2), autosomal dominant polycystic kidney disease (n = 1), other or unknown (n = 5). Major cardiovascular complications in the CKD group included peripheral artery disease (n = 3), cardiovascular (n = 2) and cerebrovascular (n = 1) events. Subjects were Caucasians, with the exception of one Black patient in the CKD group and one Asian subject in the control group.The age between ESRD patients and the healthy subjects was not different ‐hydroxylase products in RBCs of the HD patients (Table ‐hydroxyl3.3p > .05), EpOMEs and EDPs  are preferentially metabolized into their diols. In fact, the following order of ratios was identified: DiHOMEs/EpOMEs=DiHDPA/EDPs>DHETs/EETs=DiHETEs/EEQs . ESRD patients showed increased ratios for DHET/EET and DiHDPA/EDP, which indicates that increased sEH activity preferred for EET and EDP substrate classes in vivo may have caused the observed accumulation of 8,9‐DHET, 14,15‐DHET, 7,8‐DiHDPA, 10,11‐DiHDPA, 13,14‐DiHDPA, and 16,17‐DiHDPA in the RBCs in ESRD. The observed accumulation of EEQs is unlikely to result from changes in sEH activity , and dihydroxydocosapentaenoic acids (DiHDPAs) by the sEH, respectively Figure . To provty Table  or accumty Table   and various HETEs, including 5‐HETE, 8‐HETE, 9‐HETE, 11‐HETE, 12‐HETE, 15‐HETE, and 19‐HETE, compared to control subjects. Furthermore, hemodialysis treatment is insufficient to change the total concentrations of these and other LOX/CYP metabolites in RBCs of ESRD patients. Since the four subclasses of CYP epoxy metabolites increase in plasma after the dialysis treatment  and EETs upon exposure to a low O2 environment  and EDP/DiHDPAs  and HETEs  in RBCs of our ESRD patients. Little is known about the functions of EEQs and EDPs. Both EEQs and EDPs are potent vasodilators ‐hydroxylase metabolites in RBCs in ESRD, which were augmented by hemodialysis. The mechanism by which CKD and hemodialysis raises the levels of those erythro‐metabolites is not known. Since those metabolites cannot be synthesized endogenously in appreciable amounts, accelerated release into and uptake from plasma could be a possible explanation. The more pronounced changes observed in free metabolite levels within the RBCs, as compared with the total RBC compartment, indicate that free erythro‐eicosanoids should be considered more dynamic or vulnerable with respect to metabolite flux. The design of our study does not differentiate between patient groups undergoing long‐term dialysis therapy with regard to the specific underlying renal disease. Nevertheless, the impact of those epoxides and hydroxy metabolites has yet to be integrated into a (patho)physiological context.5Our results show that CKD affects the levels of numerous CYP epoxides and hydroxy metabolites  in circulating RBCs compared to control subjects, which on release may act in a vasoregulatory capacity. Although hemodialysis treatment was insufficient to change the majority of those total metabolites, we detected pronounced changes in free metabolite levels within the ESRD RBCs and in response to hemodialysis, indicating that free erythro‐epoxides could also contribute to the cardiovascular risk, for example, in diabetes or hypertension. More research is needed to determine the contribution of RBC epoxy‐ and hydroxy‐metabolites to cardiac performance and blood pressure regulation in health, cardiovascular, and specific kidney diseases.None.BG, MG, and FCL planned and designed the experimental studies. MR and ID performed the LC–MS/MS spectrometry experiments. All authors contributed to the implementation and analyses of the experiments. BG drafted the article, and all authors, contributed to its completion.


Hypertension is the leading risk factor for global disease burden. Self-management of high blood pressure (BP) through self-monitoring and self-titration of medications, has proved to be one successful and cost-effective tool to achieve better BP control in many high-income countries but not much is known about its potential in low- and middle-income countries (LMICs). We used semi-structured questionnaires and focus groups in three LMICs; Peru, Cameroon and Malawi to examine perceptions and attitudes of patients diagnosed with essential hypertension towards living with hypertension, BP measurement and treatment, patient–physician relationship and opinions about self-management of high blood pressure. Results in all three countries were comparable. Patients showed varied levels of health literacy related to hypertension. BP measurement habits were mostly affected by resources available and caregiver support. Treatment and adherence to it were primarily affected by cost. Most patients were welcoming of the idea of self-management but skeptical about the ability to do self-monitoring accurately and the safety involving self-titration of medications. High blood pressure remains the leading preventable cause of premature death and disability worldwide, killing almost eight million people every year and is projected to increase by 60% to affect 1.6 billion adults worldwide by 2025, hyperteSelf-management of high blood pressure interventions include an array of activities including self-monitoring and lifestyle changes. Home blood pressure monitoring (HBPM) and self-titration of medications have proven to be cost-effective self-management strategies to control BP in higher-income countries–12. WhilIn this study, we aimed to understand current BP management practices in LMIC settings, as well as the feasibility of utilizing HBPM and self-titration of medications in three LMICs. We conducted a qualitative study examining hypertensive patients' experiences with hypertension and perspectives on self-management of BP. We reported and compared patients' attitudes and perspectives towards the diagnosis, measurement, and treatment of hypertension, and thoughts on HBPM and self-titration of medications.The BPMONITOR study was a multi-country pilot study developed to understand the feasibility of HBPM and development and execution of a stepped care self-titration medication plan with the treating doctor according to standard of care. The goals and protocol of BPMONITOR have been reported elsewhere. In thisData were collected between June 2016 and May 2017 in Lima, Peru, from March 2016 to May 2016 in Douala, Cameroon, and from October 2016 to February 2018 in Blantyre, Malawi using a standardized protocol and maximum variation sampling. In Peru, participants were recruited from cardiology and geriatric outpatient departments affiliated with Hospital Santa Rosa, a public tertiary hospital managed by the Ministry of Health located in the city center that serves a population of 67 000 people from five districts of Peru. In Cameroon, participants were recruited from the cardiology outpatient department from Deido District hospital, a public and primary care facility which serves the entire Deido district and four neighboring . In Malawi, participants were recruited from the hypertension outpatient department at Queen Elizabeth Central Hospital, a public hospital providing free services to more than 100 000 from surrounding areas. All three facilities are tertiary hospitals serving patients to better capture the diversity and heterogeneity between countries and not within them. These hospitals primarily serve low-income people in urban areas and tend to have high patient volumes, for which HBPM and self-management, including self-titration, might help improve BP control. Patients participated in this study while they were waiting for their appointments or after they have completed them.Adults diagnosed with essential or primary hypertension who had access to healthcare were recruited to participate in the study. To be eligible for study participation, a person had to be: (1) 18 years of age or older; (2) diagnosed with hypertension or a caregiver of a patient with hypertension; and, (3) able to voluntarily and independently consent to participate in the study. At each study site, an equal representation of men and women were sought, as well as time since diagnosis using a 1, 5 or 10 year threshold, given that there is evidence that health-seeking behaviors, self-maThe research team designed and piloted a semi-structured focus groups guide  for conds.d, 17 min) and FGDs was 30 min . In Peru, Interviews were conducted by a female research assistant with a degree in communications and previous experience in qualitative research. In Cameroon, FGDs were conducted by a male research assistant with previous training in qualitative data collection. In Malawi, focus group discussions were conducted in the native Chichewa language by two male research assistants (one primary and one as a backup) with clinical training and previous training in qualitative data collection; patients in the hypertension were approached as a group with a ‘waiting room talk,’ with individual follow-up with anyone expressing interest to join. All interviews were conducted in the local language at the site, performed at the health facilities and recorded, after obtaining written consent from all participants. In all three sites, data collection continued until saturation was reached where no new themes emerged[Participants first completed a short questionnaire designed to collect socio-demographic data including age, gender, marital status and years of education. Mean interview time mean was 45 min  with the majority only relying on getting their BP measured only during pre-scheduled clinic appointments . There were concerns raised about the lack of resources to monitor their blood pressure either at home or at the clinics. Among those who monitored their BP at home, the frequency of home BP monitoring seemed to be low and tied to either: (1) their belief that they are adherent to their medications, thus there was no need to check their BP, (2) the fact that their doctor did not instruct them to do so, or (3) they feel unwell on a certain day which makes them check their BP. Of these, 4 out of 15 participants in Peru said that they measure their BP in other places such as pharmacies located near their home or healthcare facility which were private and managed by the district municipality. They mostly do so because they want to either confirm their BP readings which were measured at the clinic, or check their BP more frequently. Complaints about the costs associated with getting their BP checked outside the clinic or several times were brought up in the discussions. When asked ‘Does anyone in your family help you check your blood pressure?’ only those who were likely to monitor their BP at home expressed a positive role for peers or family members in encouraging more frequent home BP measurements. Examples of relevant quotes from participants are shown in medications should be available’ . One of the 31 participants in Malawi described that he is on one antihypertensive agent only because the others that were prescribed to him are out of stock. Additionally, there were challenges with resupply when traveling and not having their medical records on them (either lost or not properly documented), and when they are unsure of drug names. Example quotes from three countries are shown in When asked ‘are you taking your medications for blood pressure?’ participants revealed that they mostly do. In Peru, two of the 16 patients expressed that they do take more than one pill of one medicine when they are feeling specifically ill that day. In Malawi, patients described that the high pill burden ‘for those on treatment for other chronic diseases, such as HIV,’ are lead causes for their missed dosages. Participants expressed a linkage between access and adherence to medications. Reasons for compromised adherence fell into four categories; (1) unintentional missed doses, (2) access issues to medications and healthcare, (3) health beliefs and (4) medication side effects. In Peru, two out of the 16 participants stated that another perceived limitation is when the doctor is on vacation or occasionally when they are on strike. After a doctor's holiday or strike, it is harder to obtain an appointment because the quotas per doctor are filled faster. Patients described two sources of access issues. Medication stock-outs were a significant concern although there is an agreement that, ‘Many participants felt that their provider interactions are not thorough enough in terms of either physical assessments or level of discussion and counseling while a few explained that they had developed a close relationship with their doctor over the years and described that as ‘friendship.’ Almost quarter of the participants in Malawi specifically highlighted the lack of continuity in service provision by the same clinician as a challenge in understanding the status of their hypertension. In Peru, almost 50% of the study participants relied on the information provided by their doctor for managing their BP. They stated that they did not seek health advice from other people or looked for information from other sources while in Cameroon and Malawi, the Internet and consulting with peers was a go-to source for some participants. In Cameroon, some patients explained that they had developed a close relationship with their doctor over the years, which in some cases is described as ‘friendship.’ Participants stated that when they have doubts about anything such as having new symptoms, they ‘simply’ ask their doctors. In Peru, four patients said that they have never been told exactly what is hypertension or its associated risks if not managed well. Some patients reported that he had never been asked to monitor his BP on a daily basis.the same like those you get at the doctor's office.’ Participants voiced that a shift to home BP monitoring could reduce burden on both patients and the healthcare system. On a patient-level, patients thought that home BP monitoring reduces the need to go to clinic replacing that with calls to the doctor's office which thus saves them transportation money, time and effort. On a system-level, participants stated a possible reduction in the number of patients going to and waiting in clinics just to get their BP checked, which improves the ‘efficiency’ of the entire healthcare system.A broad range of key themes ranging from patient empowerment and engagement to seeing no added value emerged when an intervention that includes HBPM and self-management, including self-titration of medications was proposed. The majority of participants agreed that the intervention would allow people to take more responsibility for their disease progression, specifically ‘motivating’ patients to engage in care more consistently, and increase control of their BP. Some felt that HBPM could serve as an early warning system to visit a health facility sooner than later in case of abnormal readings, which could lead to quicker attention to disease progression and medical care. However, there was a minority in both Malawi and Cameroon that saw no value in home BP monitoring since the readings will likely be ‘Concerns about an intervention that includes HBPM revolved around three main issues. First, for a few, the intervention sounded burdensome, of no or a small value or time-consuming. This may be indirectly linked to another theme that emerged around a perceived increase in stress and anxiety in the face of self-management BP. Second, two practical challenges provided by the participants included the lack of adequate numbers of clinicians to receive calls from every patient; and a concern that the intervention would only last for a period of time and then the current state of BP care and control would resume. Third, some expressed that it would be a form of injustice to the other patients who are not to be included in the study since they will not be able to self-monitor their BP. When asked ‘what worries you about the intervention,’ participants expressed associated costs, risks of self-titration by doing ‘mistakes’ as the main issues that worry them .Table 7In three LMIC settings, Peru, Malawi and Cameroon, we found unique distinctions in the context in which HBPM and self-management, including self-titration may be used to improve BP control. These distinctions were clear in the experiences and attitudes related to hypertension, measurement of BP, patient–physician relationship and feasibility of future HBPM interventions.With regard to experiences and attitudes related to the diagnosis of hypertension, we found that in all three countries, there is a clear role for health literacy and the source of information that the patients primarily rely on in shaping their perceptions of illness, treatment and future HBPM interventions. A potential HBPM strategy in this case may include focusing on increasing awareness by engaging providers and caregivers as well and paying attention to certain dietary and cultural attributes to BP control that differ at each setting. Together lifestyle modifications and self-management through HBPM and self-titration of medications achieve better BP control.Measurement of BP was closely concomitant with what is available and affordable. Some participants expressed that they have not adhered to measuring their BP regularly because their physicians have not asked them to. These patients were not likely to self-monitor their BP or see the value in doing so. This is in contrast to a meta-analysis paper that identified disagreement with clinical recommendations as the most common barrier among health care providers. It is iThe role patient–physician relationship plays in hypertension care and medication adherence is well established in the literature, 29. Patm Hg reduction in BP in groups that use a home BP monitor combined with automated interactive voice response messages, in comparison to controls[Although self-management is a relatively ‘new’ concept for many LMICs health systems, this study has found that patients are mostly welcoming of the idea had they been educated on the details of the intervention and on hypertension. Engagin controls. There i controls.et al., suggest that more than 80% of themes in qualitative studies are discoverable within two to three focus groups[Doing a multi-site study across three very different countries using three different languages to collect data introduces certain limitations to this study. Some nuances might have been lost in the translation. However, the opportunity to discuss our findings have reduced the possibilities of misinterpretation of the data. First, the demographic data was self-reported. Since the study was designed to establish a baseline understanding about perceptions, a limited number of questions were included in the study protocol. The choice of conducting semi-structured interviews or focus group discussions at each study site was dictated by the time allowed without affecting the clinic's flow at each site and bearing in mind the time conflicts for participants. The number of focus groups was small, yet Guest us groups. Also, aus groups. FGs risThis study presents foundational research that explores patient attitudes and perceptions of hypertension diagnosis, measurement, treatment, patient–physician relationship and self-management of blood pressure in three different LMICs. We also examined the readability of introducing HBPM in these different parts of the world drawing on patients favoring HBPM and a medication self-titration plan to address cost as long as safety is taken into consideration. Our study revealed important knowledge gaps in the causes and consequences of hypertension, BP measurement and adherence to treatment that need to be addressed before introducing a HBPM intervention. There is a critical need to understand the local context at each setting and identify the best ways to promote HBPM practices to ultimately achieve better BP control.


The escalating prevalence of adrenal incidentaloma (AI) has been associated with the improvement of radiologic techniques and widespread imaging in aging population. It is currently unclear whether patients with obesity more likely develop AI and the current rise in the prevalence of AI could be at least partly associated with the respective rise in obesity. We compared the prevalence and characteristics of non-functional (NF) and autonomous cortisol secreting (ACS) adrenal incidentalomas (AIs) after the study population was stratified by different body mass indexes (BMI) and age groups.N = 290) and ACS (N = 142), of median age 63.4 (54.0–71.6) years and median BMI 28.6 (25.5–31.7) kg/m2. The data collection contained 11.132 points including demographic, anthropometric, radiologic, hormonal and metabolic parameters.Retrospective cross-sectional study comprising of 432 patients  with NFAI (P = 0.008), with higher basal cortisol (P < 0.001), lower basal DHEAS (P = 0.001), lower suppression DHEAS (P = 0.027) and higher aldosterone (P = 0.039). AIs with ACS were larger than NFAI (P < 0.001). Interestingly, ACS group had lower body mass (P = 0.023) and did not differ in BMI, blood pressure, heart rate, lipid profile, fasting glucose and presence of diabetes mellitus type 2 when compared to NFAI., By contrast to the similarity of metabolic profiles in ACS and NFAI, some components of adverse metabolic traits were rather associated with higher BMI and older age, in particular in NFAI.We observed 68–87% higher prevalence of AI across different age groups in NFAI and ACS in obese/overweight compared to normal weight subjects. Patients with ACS were older (The prevalence of NFAI and ACS were significantly higher in overweight/obese subgroup across the age distribution. Stratification by age and BMI displayed significant differences in some metabolic traits, in particular in NFAI. Adrenal incidentaloma (AI) is an asymptomatic adrenal mass detected on imaging not performed for suspected adrenal disease . It has The majority of AIs are characterized as nonfunctional (NFAI) , 9, 10. It is currently unclear whether patients with obesity and IR more likely develop AI and the current rise in the prevalence of AI could be at least partly associated with the respective rise in obesity.We aimed to evaluate the prevalence and characteristics of NFAI and ACS after the study population stratification by different BMI and age groups.We conducted a retrospective cross-sectional study including all patients with AI referred to University Medical Centre from January 2005 to January 2012. After that period, the majority of patients with AI were managed in our outpatients’ clinics. The study was conducted in accordance with the Declaration of Helsinki, approved by the National Ethical Committee.We identified 769 adult patients with documented diagnosis of AI. The medical records of all patients were reviewed by the authors to confirm the diagnosis of AI based on European Society of Endocrinology Clinical Practice Guideline for management of AIs  and diviThe following data were collected from the hospital documentation system: patient gender, age at diagnosis, weight, height, method of radiological assessment of AI , size of AI, site of AI , Hounsfield units, blood pressure, heart rate, baseline cortisol and cortisol after ODST, baseline ACTH, baseline DHEAS, DHEAS after ODST, aldosterone, PRA, TSH, fasting glucose, lipids . Normal levels of baseline cortisol were defined as 138–690 nmol/l, cortisol after ODST < 50 nmol/l, ACTH < 10.2 pmol/l, DHEAS 3.6–12.9 μmol/L, aldosterone 0.134–0.751 nmol/l, PRA 0.6–4.84 μg/l/h, TSH 0.59–4.29 mE/l, fasting glucose 3.6–6.1 mmol/liter, cholesterol < 5.2 mmol/liter, HDL > 1 mmol/liter, LDL < 3.4 mmol/liter, triglycerides < 1.7 mmol/liter. Among additional biochemical parameters we included creatinine, natrium and leucocytes count since they have been included into the risk estimator for adrenal tumor functionality . The datCortisol was determined using IMMULITE® 2000 Cortisol assay. Intra-assay variations were ≤ 15% and inter-assay variations were ≤ 20% for the applied methods. DHEAS was measured by specific double antibody RIA using 125 I-labeled hormones . Intra-assay coefficient variation (CV) for DHEAS ranged from 4.9 to 9.8% and inter-assay CV from 7.9 to 13.0%. Aldosterone was determined using ACTIVE® Aldosterone RIA for serum samples. Intra-assay variations were ≤ 4.5% and inter-assay variations were ≤ 9.8% for the applied methods. PRA was determined using Angiotensin I RIA KIT for serum samples. Intra-assay variations were ≤ 8% and inter-assay variations were ≤ 10% for the applied methods. ACTH was determined using IMMULITE® 2000 ACTH system. Intra-assay variations were ≤ 9.5% and inter-assay variations were ≤ 10% for the applied methods. TSH was determined using ADVIA Centaur CP TSH3-Ultra assay. Intra-assay variations were ≤ 16.7% and inter-assay variations were ≤ 22.3% for the applied methods. Fasting glucose levels were determined using the standard glucose oxidase method . Lipids were determined using Adiva 1800, Siemens analyzer. Intra-assay variations ranged from 1.6 to 6.3%, and inter-assay variations ranged from 5.8 to 9.6% for the applied methods. Creatinine, natrium, leucocytes count were assessed with routine biochemical evaluation after 12-h overnight fast. CT evaluation included maximum diameter of incidentaloma, laterality, presence of necrosis, calcifications, atypical characteristics. MR report included evaluation of maximum diameter of the tumor and laterality. BMI was calculated as the weight in kilograms divided by square of height in meters. Blood pressure was measured with sphygmomanometer using appropriate cuff size.https://pxweb.stat.si/) database was used. P values below 0.05 were considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics, version 21.0 .The results for continuous variables are presented as means and standard deviation (SD) or median and interquartile range. The results for categorical variables are presented as frequencies. Spearman’s rho was used to assess correlation between continuous variables, while Fisher’s exact test was used to assess the association between categorical variables. To compare continuous variables between different groups, nonparametric Mann-Whitney test or Kruskal-Wallis and pairwise comparisons with post hoc Bonferroni corrections were used. To calculate prevalences, population data from SI-STAT  suppression test between 50 nmol/l and 138 nmol/l and 14 had cortisol levels post dexamethasone > 138 nmol/L.The study population consisted of 432 patients (179 (41.4%) male, 253 (58.6%) female) of median age 63.4 (54.0–71.6) years, median body mass 77.6 (67.4–88.8) kg and median BMI 28.6 (25.5–31.7) kg/mP-values > 0.05).In majority of subjects, AI was diagnosed by CT (388 (92.2%)), in the rest by MRI [11 missing data]. 183 (43.9%) of patients presented with right-sided AI, 147 (35.3%) with left sided AI. In 87 (20.9%) AI was observed bilaterally [15 missing data]. Median size of right sided AI was 25 –30 mm anP-values > 0.05). In ACS group 102 subject had one-side AI, 40 had bilateral adrenal mass. One-sided ACS had lower cholesterol (P = 0.021) and left-sided AI in patients with bilateral ACS were smaller than left-sided AI in patients with unilateral ACS (P = 0.025). One-sided ACS did not differ from bilateral ACS in any other anthropometric, clinical, hormonal, biochemical or radiologic characteristics .In NFAI group 243 subjects had one-side AI, 47 had bilateral adrenal mass. One-sided NFAI did not differ from bilateral NFAI in any of the anthropometric, clinical, hormonal, biochemical or radiologic characteristics , with higher basal cortisol level (P < 0.001), lower basal DHEAS (P = 0.001), lower DHEAS after ODST (P = 0.027) and higher aldosterone (P = 0.039). They had slightly lower weight (P = 0.023). AIs with ACS were significantly larger than NFAI (P < 0.001). There was no significant difference in gender distribution, ACTH, PRA, TSH, creatinine, sodium and leukocytes between patients with ACS and NFAI . We observed no significant difference between NFAI and ACS groups regarding BMI, blood pressure, heart rate, lipid profile, fasting glucose and presence of diabetes mellitus type 2 .The comparison of selected parameters between NFAI and ACS is outlined in Table P = 0.032). The data are provided in Table P < 0.001), with highest values in patients aged 60–69 years and lowest values in patients over 79 years. There were no differences in leukocyte concentration (P = 0.958).In NFAI group, younger patients have lower blood pressure, lower body mass, LDL, fasting glucose, lower creatinine, basal DHEAS and DHEAS after ODST in comparison with older patients. There were significantly more younger women than man P = 0.03. The datP < 0.001), with highest values in patients aged 60–69 years and lowest values in patients aged 70–79 years. There were no differences in leukocyte concentration (P = 0.425).In ACS group, younger subjects significantly differ from older in blood pressure, cholesterol, LDL, fasting glucose, creatinine. The data are provided in Table P = 0.287). BMI was not correlated with serum cortisol after ODST  in the whole study population.There was no significant difference between NFAI and ACS groups regarding BMI , patients with NFAI and higher BMI, had higher fasting glucose , lower HDL , higher triglycerides , higher creatinine  (P = 0.023) and higher leukocytes . There were significantly more patients with diabetes mellitus in higher BMI groups (P = 0.002).When stratified by BMI (≤ 25 kg/m2 vs. 25–30 kg/m2 vs. > 30 kg/m2), differed in TSH , HDL  and creatinine . Patients with ACS across the three BMI groups  did not differ in age, basal cortisol, basal DHEAS and DHEAS after ODST, aldosterone, PRA, TSH, blood pressure, heart rate, fasting glucose, cholesterol, triglycerides, LDL, sodium and leukocytes. There was no significant difference in gender distribution between groups (P > 0.05). Regression analysis did not show any significant correlation between BMI and presence of diabetes mellitus in this group .When stratified by BMI patients with ACS and different BMI . Further subdivision including second cut off for cortisol levels post dexamethasone > 138 nmol/L was not performed since only 14 out of 142 patients in ACS would have been characterized for this subgroup. Moreover, there might be a selection bias since the obesity and its complications, in particular among younger patients could be the reason for performing a CT scan and the detection of AIs. However, we excluded all symptomatic patients including some of those imaging indications that are closely related to obesity such as symptomatic cholecystolithiasis and pancreatitis.By our knowledge, comparisons of the patients’ and tumor’s characteristics according to the triple stratification based on incomplete post-dexamethasone cortisol suppression, age and BMI have not yet been performed. Although our study does not prove causality of relationship between obesity and development of AIs, it eloquently portrays the importance of considering the primary role of BMI when addressing the prevalence as well as patients’ and tumor’s characteristics in both, ACS and NFAI groups. We encourage longitudinal studies aiming to investigate the potential development of AIs and potential growth of pre-existent AIs in subjects with obesity. Furthermore, interventional studies in overweight/obese patients with AI should assess the potential impact of insulin sensitizers and weight reduction on the size, growth, functionality and metabolic consequences of AIs. Translational studies evaluating the molecular phenotype of AI related to relevant signaling cascades in IGF-1 and MAPK pathways might also provide further insights into this potential bidirectional relationship between obesity and AIs.