As spermidine levels decline with age, spermidine supplementation is suggested to prevent or delay age-related diseases. However, valid pharmacokinetic data regarding spermidine remains lacking. Therefore, for the first time, the present study investigated the pharmacokinetics of oral spermidine supplementation. (2) Methods: This study was designed as a randomized, placebo-controlled, triple-blinded, two-armed crossover trial with two 5-day intervention phases separated by a washout phase of 9 days. In 12 healthy volunteers, 15 mg/d of spermidine was administered orally, and blood and saliva samples were taken. Spermidine, spermine, and putrescine were quantified by liquid chromatography–mass spectrometry (LC–MS/MS). The plasma metabolome was investigated using nuclear magnetic resonance (NMR) metabolomics. (3) Results: Compared with a placebo, spermidine supplementation significantly increased spermine levels in the plasma, but it did not affect spermidine or putrescine levels. No effect on salivary polyamine concentrations was observed. (4) Conclusions: This study’s results suggest that dietary spermidine is presystemically converted into spermine, which then enters systemic circulation. Presumably, the in vitro and clinical effects of spermidine are at least in part attributable to its metabolite, spermine. It is rather unlikely that spermidine supplements with doses <15 mg/d exert any short-term effects. Spermidine is a biogenic polyamine that plays a crucial role in cellular homeostasis, cell growth, proliferation, and autophagy ,2,3. ResPolyamines (including spermidine) in the gastrointestinal lumen originate from food, intestinal microbiota, pancreatic-biliary secretions, and degraded intestinal cells ,10, and The average daily nutritional intake of spermidine in European countries varies from approximately 10 to 15 mg . ExampleTo date, little is known about the mechanisms of intestinal absorption, presystemic metabolism, and the kinetics of polyamine transport systems in humans . The intLuminal polyamines are (at least in part) actively imported into intestinal epithelial cells (IECs) through the polyamine transport system (PTS) . In IECsSpermidine has an autophagy-inducing effect that makes it highly interesting for preclinical research ,22. As mMost prominent translational research with spermidine has focused on its possible efficacy in the context of brain health/aging, oncology, and SARS-CoV-2 infections. Regarding brain health and aging, spermidine-induced autophagy has been postulated to exert protective effects on neurodegenerative disorders and subjective cognitive decline and also to slow aging processes even in humans ,24,25. HIn fact, the administration of exogenous spermidine extends the lifespan in various models of aging through epigenetic modifications, autophagy induction, and necrosis suppression . In animIn oncology, due to its direct link with oncogenes, polyamine metabolism has long been a target for potential cancer therapeutic agents . For exaRecently, in vitro data revealed that an infection with SARS-CoV-2 leads to the viral inhibition of spermidine synthase; as a result, the intracellular spermidine concentration decreases, and autophagy as a cellular defense mechanism is reduced . IncubatTo probe these preclinical data in future clinical trials, detailed knowledge of spermidine pharmacokinetics is essential. Since SARS-CoV-2 replicates predominantly in the pharynx during the initial phase of infection, saliva concentrations of spermidine (as well as spermine and putrescine) after oral intake of spermidine are of major interest. Therefore, in addition to the blood plasma concentrations, we also analyzed the respective saliva concentrations. To the best of our knowledge, the concentrations of spermidine, spermine, and putrescine in saliva after oral intake of spermidine have never been investigated before.Thus, to foster translational research with spermidine, we determined the pharmacokinetics of orally administered spermidine, its precursor putrescine, and its metabolite spermine, as well as the effect of its multiple-day administration on the steady-state concentrations in blood and saliva in healthy volunteers.Additionally, also for the first time, we investigated the effect of orally administered spermidine on the human plasma metabolome using nuclear magnetic resonance (NMR) spectroscopy because spermidine has been postulated to act as a calorie restriction mimetic (CRM) in humans, which would imply effects on the plasma concentrations of various metabolites ,42.The study was designed as a randomized, placebo-controlled, triple-blinded, two-armed crossover trial with a washout phase of 9 days between the two intervention phases . The firThe subjects were recruited through the University of Luebeck’s email distribution list. Both supplementation and blood/saliva sampling were performed at the Institute of Nutritional Medicine. Randomization, blinding, and the production of the placebo and verum preparations were provided by the Center of Drug Absorption and Transport (C_DAT) of the University of Greifswald, Germany. Randomization was performed using random numbers generated in Microsoft Excel. With the command = random number, a random number between 0 and 1 was generated for each subject. Subsequently, a comparison was made in blocks of two to determine which subject had the higher random number. Those with the higher random number started with the verum arm, while subjects with the lower random number started with the placebo arm. This procedure ensured an equal distribution  of the two possible orders (V-P or P-V) among the 12 subjects. Thus, 6 subjects each start with verum or placebo. The capsules taken (verum or placebo) were provided by staff in Greifswald on the basis of the generated sequence for weeks 1 and 2, specifically for the subject-specific random number. Externally, verum and placebo are indistinguishable due to color-matched filling powder and capsules of the same size and color. For more information about products and capsules, see To detect possible dietary changes in the course of the study that might have influenced the results, the participants documented their meals throughout the study period in a validated nutritional log. Throughout the study course, subjects were advised to refrain from extraordinary physical work, dietary changes, and alcohol consumption of more than two standard glasses per day. In addition, the participants were advised to follow their usual activity and dietary habits.As this study was designed as an exploratory pharmacokinetic trial, case number calculation was not possible due to a lack of data. A sample size of 12 was considered appropriate as it is established in phase 1 clinical trials. Moreover, the robustness of the data was improved by the cross-over design with placebo control.The study was registered in the German Clinical Trials Register (DRKS) (DRKS00029397).2, acute diseases, a history of food allergy, intake of any medication, and dietary supplements during the 48-h period before the start of the study until the end of the study , use of illegal substances, pregnancy and breast-feeding, shift work, and adherence to a vegan diet.Study participants were recruited between December 2021 and April 2022. The inclusion criteria were age 18–45 and sufficient knowledge of the German language to understand the study instructions. The exclusion criteria were BMI < 18 or >29.9 kg/mIn total, 85 subjects were assessed for eligibility . After aChlorella algae powder and soybean extract were purchased, each containing 1.5 mg of spermidine per capsule. The capsules were vegan and consisted of hydroxypropyl methylcellulose (HPMC). The content indicated on the label was verified by an independent laboratory. Five administered hard capsules corresponded to a single dose of 7.5 mg of spermidine. Similar HPMC capsules  were used for the placebo and filled with inert and similar colored capsule filler consisting of microcrystalline cellulose  and iron oxide .Commercially available dietary supplements of the brand MoleQlar™  based on The intervention started with a standard dinner the evening before the first measurement day , placebo or verum capsules were administered to the subjects. The target dose in the verum arm of the study was 15 mg of spermidine per day (corresponding to five capsules in the morning and five in the evening). The same number of capsules were administered in the placebo arm. Subsequently, during the following 8 h, blood samples (9 mL of whole blood per sample) and saliva samples (after each blood sample) were taken under standardized dietary conditions. For the next 3 days, the respective capsules (verum or placebo) were self-administered by the participants every morning and evening, with one preprandial sampling in the morning. The second measurement day was on day 5 (similar to day 1). After a wash-out phase of 9 days, the second intervention phase started similarly to the first intervention phase.The water intake during the measurement days was standardized until noon. The participants drank 100 mL of non-carbonated water every full hour, directly after blood and saliva samples were taken. After lunch, they had water ad libitum.The calculation of pharmacokinetics was based on all blood and saliva samples. By contrast, only six samples of blood per subject were analyzed for the NMR metabolomics measurements .Standard dinner : a medium-sized margherita pizza from a local delivery service; non-carbonated water ad libitum.Standard breakfast : mixed bread, butter, jam, honey, and orange juice; non-carbonated water ad libitum.Standard lunch : a medium-sized salad  and one bun; non-carbonated water ad libitum.Standard snack : a piece of crumble cake; non-carbonated water ad libitum.The use of standard meals ensured that the study participants consumed a comparable amount of polyamines with their food before and during the sampling on the measurement days. The standard meals were taken at the Institute for Nutritional Medicine and were composed as follows:The meals between the measurement days were not standardized. However, study participants were instructed to refrain from relevant dietary changes during the study course. Compliance with this instruction was ensured by reviewing the nutritional logs provided by the participants.® , corresponding to a total of 50 blood samples per subject by the end of the study.To ensure the validity of the time-dependent concentration determinations, blood and saliva samples were taken with a maximum time deviation of 5% of the planned time intervals. On days 1, 5, 15, and 19, blood samples were taken using a peripheral venous indwelling catheter placed in the crook of the subject’s elbow. At the end of each study day, the catheter was removed. On the shorter measurement days , blood samples were taken using a disposable cannula (butterfly). At any time point scheduled, 9 mL of peripheral blood was taken in an EDTA K3 Monovetteg for 10 min. It was ensured that the samples were never at room temperature for more than 10 min prior to refrigerated centrifugation. Immediately after centrifugation, the plasma supernatant was pipetted into ice-cooled microtubes. For each sample, 1000 μL of plasma was transferred into three different microtubes and frozen at −80 °C. Two samples were used for the time-dependent concentration determination and NMR metabolomics, respectively, and one sample was used as a retained sample.After collection, blood samples were centrifuged at 4 °C and 3000× The saliva samples were placed in appropriate microtubes by the study participants immediately after the respective blood sampling and frozen at −80 °C.N-(9-Fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu), boric acid, and formic acid were obtained from Sigma-Aldrich . Acetonitrile (super gradient grade) was obtained from VWR , while sodium hydroxide was provided by AppliChem . All solvents  and additives (formic acid) used for LC–MS/MS analysis were of LC–MS grade and obtained from VWR .Putrescine dihydrochloride and spermidine trihydrochloride were obtained from Acros Organics , and spermine tetrahydrochloride was purchased from Alfa Aesar . Putrescine-d4 dihydrochloride, spermidine-(butyl-d8) trihydrochloride, The determination of the concentrations of spermidine, spermine, and putrescine in plasma and saliva was performed by protein precipitation following derivatization with Fmoc-OSu based on the method of Xiong and Zhai . Isotopiv/v) and diluted with the same solvent unless stated otherwise. The quantitation range was 2–500 ng/mL for all polyamines in plasma. For saliva, a quantitation range of 10–2500 ng/mL was chosen for spermidine and spermine, while it was 0.4–100 µg/mL for putrescine. Each concentration of putrescine-d4 and spermidine-d8 in the internal standard solution was 2.5 µg/mL for plasma and 100 µg/mL and 2.5 µg/mL, respectively, for saliva analysis.Stock solutions, quality control spiking solutions, and internal standard solutions were prepared in methanol-water . The saliva samples were centrifuged for 10 min at 16,060× g , while the plasma samples were directly processed further. An aliquot of 100 µL of plasma or supernatant of centrifuged saliva, respectively, was transferred into a 1.5 mL microcentrifuge tube  and spiked with 25 µL of internal standard solution. Proteins were precipitated by adding acetonitrile containing 0.2% formic acid. After centrifugation for 10 min at 16,060× g and the transfer of an aliquot of the supernatant into a 1.5 mL amber glass vial , 200 mM borate buffer with a pH of 9.0 and 5 mM Fmoc-OSu solution in acetonitrile were added. After 30 min of incubation at room temperature, the derivatization reaction was stopped by adding formic acid (100%). This extract was analyzed using a LC–MS/MS system . The LC–MS/MS system consisted of an LC–40B X3 solvent delivery module, an SIL-40C X3 auto sampler, a CTO-40S column oven, an SPD-40 UV-Vis detector, an FCV-20AH2 valve unit, and an LC–MS-8060 mass spectrometer equipped with an ESI ionization unit. A detailed overview of the sample preparation can be found in ® 2.6 µm PS C18 100 Å 150 × 2.1 mm column  protected by a SecurityGuardTM ULTRA Cartridge , which was connected to a SecurityGuardTM ULTRA Holder . Acetonitrile + 0.1% HCOOH (A) and water + 0.1% HCOOH (B) were used as eluents for the mobile phase at a flow rate of 0.4 mL/min. A linear gradient was applied for the separation of the derivatized polyamines  mode . Further2 weighted least squares linear regression was used for all polyamines.Data acquisition and analysis were performed using LabSolutions (Version 5.97 SP1). The calibration curves consisted of eight calibration standards and were constructed by plotting peak area ratios of the analytes and internal standards against the concentration of the analytes. A 1/cThe method was validated with respect to intra- and inter-day accuracy and precision at three concentration levels  in six replicates on the same day and 18 replicates over 2 days, respectively. Stability  was evaluated at low (QC-L) and high (QC-H) quality control concentrations in four replicates. Furthermore, reinjection stability was investigated at three concentration levels  in six replicates after storage for 24 h in the autosampler. Plasma and saliva quality control standards were prepared by spiking QC working solutions  into the corresponding matrix. As no analyte-free plasma or saliva were available, the parameters of validation were evaluated by subtracting the concentration of nonspiked samples from spiked matrix samples. The matrix effect was evaluated by spiking the plasma and saliva of six individuals at low (QC-L) and high (QC-H) quality control concentrations and calculating the coefficient of variation (CV) of the measured concentration. As presented in Moreover, the stability investigation (data not presented) proved that the unprocessed plasma samples were stable for two freeze-thaw cycles, while unprocessed saliva samples were stable for three cycles. Unprocessed plasma and saliva samples were stable for a period of 6 weeks at −80 °C and for 3 h at room temperature. The reinjection of plasma and saliva samples after storage for 24 h in the autosampler revealed that the final extract was stable. The CV of the measured concentration of the six spiked individual plasma and saliva samples was below 15% at all investigated concentration levels (QC-L and QC-H).v/v, pH 7.4) by manual shaking and transferred into a 5 mm NMR tube. Prior to analysis, samples were stored at 6 °C in an automated SampleJet™. NMR analysis was performed on a Bruker 600 MHz Avance III HD nuclear magnetic resonance spectrometer with a TXI probe at 37 °C (310 K). Temperature calibration and quantification, as well as water suppression performance, were checked, and settings were optimized before starting the experiments if necessary. Based on the IVDr protocol, a one-dimensional (1D) NOESY experiment (pulse program: noesygppr1d) and a 1D Carr–Purcell–Meiboom–Gill spin-echo experiment  for the suppression of macromolecules and proteins were measured per sample. The evaluation and quantification of selected parameters were performed according to Bruker quantification in plasma/serum B.I.Quant-PS 2.0.0 and Bruker IVDr Lipoprotein Subclass Analysis (B.I.-LISA). In total, 39 metabolites  and 112 lipoprotein parameters were quantified automatically (Bruker BioSpin).Plasma samples were analyzed for their metabolites and lipoproteins by NMR spectroscopy according to Bruker’s standardized and certified in vitro diagnostic research protocol . Samples were processed and measured following a strict standard operating procedure, which was previously described by Dona et al. . In brie0-tlast), the maximal achieved concentration (cmax), and the time at which it appeared (tmax) were examined to compare the verum and placebo interventions. The data were plotted for each determined substance in plasma and saliva with their mean and SD for each sampling time point.The data analysis was conducted using GraphPad version 9.4.0. The normal distribution of the pharmacokinetic data was tested using the D’Agostino–Pearson (omnibus K2) and Anderson–Darling methods. The area under the curve (AUC) from the first to the last sample of the respective intervention phase .Sample 11 of the verum arm was compared with sample 11 of the placebo arm (8 h after first administration), and sample 25 of the verum arm was compared with sample 25 of the placebo arm (the last samples of each intervention phase). The respective samples from the two intervention phases (t = 0 h) were also compared to test the setup and baseline determinations. In addition, a Wilcoxon matched-pairs signed-rank test was performed to detect significant differences between the two intervention arms. All metabolites were plotted together in a forest plot to provide an overview of the changes in sample 11 of the verum arm versus sample 11 of the placebo arm, as well as in sample 25 of the verum arm versus sample 25 of the placebo arm . The relDue to unforeseen events , a total of four plasma and two saliva samples could not be taken. Only five (0.833%) of a total of 600 sampling times (50 per subject) were outside the targeted time interval. The largest time deviation was 7 min, while the shortest deviation was 2 min. Thus, more than 99% of the samples were taken within the scheduled time interval. No special relevance was attributed to these five samples, and they were analyzed like the other samples. One saliva sample and one plasma sample were not evaluable due to the analytical failure of the LC–MS/MS system.0-tlast, cmax, and tmax. Comparing placebo and verum, no significant differences between the plasma concentrations of spermidine and putrescine were found (0-tlast was significantly increased in the verum group (p = 0.0282).Initially, the mean concentrations of spermidine, spermine, and putrescine in the plasma and saliva in the placebo intervention of all 12 subjects combined over all time samples were determined as follows: spermidine (plasma) 12.41 ng/mL (SD 1.869), spermine (plasma) 5.102 ng/mL (SD 0.4646), and putrescine (plasma) 7.403 ng/mL (SD 2.130); spermidine  236.5 ng/mL (SD 181.5), spermine  201.0 ng/mL (SD 76.02), and putrescine  4.343 µg/mL. Subsequently, concentrations of spermidine, spermine, and putrescine in plasma and saliva for both the placebo and verum interventions were compared for AUCre found . For spe0-tlast plasma concentrations for spermine in the placebo intervention compared to the verum intervention (data not presented). Noteworthy, fasting plasma spermine concentrations were higher than the postprandial values in both the placebo and verum arms. The mean values of the fasting samples and the remaining postprandial samples of each subject were averaged to a mean value across the 12 subjects. The mean of the fasted matutinal sample’s placebo intervention for spermine in plasma was 5.720 ng/mL (SD 1.205); for the fasted matutinal samples of the verum intervention, it was 6.502 ng/mL (SD 1.492); for the postprandial samples of the placebo intervention, it was 4.940 ng/mL (SD 1.124); and for the postprandial samples of the verum intervention, it was 4.965 ng/mL (SD 1.324). Investigating whether oral spermidine supplementation increased fasting plasma spermine levels , no significant difference was found  was found, indicating no difference between matutinal concentrations on the days during the placebo treatment. A Dunn’s multiple comparison test did not find a significant change throughout the days when concentrations at 24, 48, 72, and 96 h were compared with starting spermine concentrations at 0 h during placebo treatment. Within the verum group, a p value of 0.0823 (ns) was found, indicating a strong trend of a change in fasted matutinal spermine concentrations during spermidine intake without reaching statistical significance. A Dunn’s multiple comparison test also found no significant change throughout the days when concentrations at 24, 48, 72, and 96 h were compared with starting spermine concentrations at 0 h, even though an increase in matutinal concentrations was observed during verum treatment.For both study arms, a Friedman test (nonparametric ANOVA) was performed to compare the fasted matutinal spermine plasma levels at 0, 24, 48, 72, and 96 h with each other within the specific study treatment (verum or placebo). Within the placebo treatment, a 0-tlast, cmax, and tmax . When comparing metabolite concentrations in the blood plasma, no significant differences were observed between the verum and placebo interventions . This al0-tlast plasma concentrations of the mean values significantly differed when comparing the verum and placebo groups. Since four of the 12 subjects exhibited a spermine AUC0-tlast in the placebo intervention greater than in the verum intervention, a correlation analysis of the metabolome and spermine AUC0-tlast was performed. In the linear regression, the differences between the verum and placebo interventions in AUC0-tlast values of spermine plasma concentrations for each subject were compared with the corresponding differences in metabolite concentrations of the sample 25 . A significant difference was found for LDL-3/4/5, LDL FC, LDL PL, LDL-3/4/5 Chol, LDL-4 FC, LDL-3/4/5 PL, LDL-3/4/5 ApoB, and HDL-1 ApoA2.As demonstrated above, spermine AUCTo the best of our knowledge, this is the first randomized placebo-controlled pharmacokinetic study with spermidine in humans. Previous data on the pharmacokinetics of spermidine are either based on animal studies only or on clinical trials that do not meet the standards of pharmacokinetic studies. Most strikingly, we found that oral intake of spermidine at 15 mg/d for 5 days increased spermine levels in the plasma but not levels of spermidine or putrescine. Similarly, a nonsignificant trend indicated that spermidine supplementation increased fasted matutinal spermine plasma concentrations but not respective spermidine and putrescine concentrations.13C-labeled spermidine after jejunal instillation was 61–76% within 20 min [14C-labeled polyamines were found to be rapidly absorbed in the small intestine, passed into systemic circulation, and distributed to different tissues [Available pharmacokinetic data for spermidine are mainly limited to in vivo and animal models and suggest contradictory results. In a rodent ex vivo model, the portal vein recovery of n 20 min . In rats tissues . In a di tissues .In mouse models, prolonged administration of polyamine-rich chow was found to (partly sex-dependently) increase both spermidine and spermine blood levels ,47. In cInterestingly, early in vivo experiments in rats with dietary polyamines from the 1990s revealed differential effects of presystemic eliminations. After intragastrical administration, only 11–15% of putrescine was recovered in intestinal tissue, but 87–96% of spermidine and 79–82% of spermine were recovered . Based oPrevious analyses of spermidine pharmacokinetics in humans are scarce; published data are not based on randomized placebo-controlled interventions but rather on noninterventional data or food-based interventions. In 2018, the results of a randomized, placebo-controlled, double-blind phase II trial with supplementation of a polyamine-rich plant extract were reported . For 3 mMore recently, confirming data from rodent experiments , in a no0-tlast, cmax, nor tmax differed significantly between the verum and placebo treatments for the three polyamines investigated. Furthermore, no significant correlation between plasma and salivary concentrations of spermidine, spermine, or putrescine was detected. To our knowledge, no data on the salivary pharmacokinetics of spermidine, spermine, and putrescine after oral supplementation of spermidine have been published before. In our study, neither the salivary AUCPolyamines, including spermidine and putrescine, reportedly have diverse functions in the bacterial physiology of the oral microbiota . HoweverAs described earlier, a reduction of the viral replication of SARS-CoV-2 by 85% was demonstrated in vitro at a concentration of 333 µM spermidine, encouraging discussions about the preventive or even therapeutic potential of spermidine in the context of COVID-19 . Our resFurthermore, limited data are available on salivary concentrations of spermidine, spermine, and putrescine without oral spermidine supplementation. Cooke et al. investigated the time profiles of putrescine, cadaverine, indole, and skatole upon waking and during the day . In theiLi et al. measured the salivary concentration of various polyamines and amino acids in five healthy volunteers before and after they brushed their teeth . Before Although it has been speculated that intestinal polyamine uptake might be similar in rodents and humans, our data indicate that—different from rodent data—dietary spermidine is not quantitatively recovered in the systemic circulation but is subject to extensive presystemic metabolism. These findings are supported by previous studies. For instance, Soda et al. demonstrated millimolar concentrations of dietary polyamines in the duodenum and jejunum in humans after high-dose oral intake yielded maximal plasma concentrations of only 10–20 μM . Most liBoth the enterocytic and hepatic conversion of spermidine to spermine might explain the increased spermine concentration in blood plasma after oral spermidine intake, while spermidine concentrations remained unchanged. Alternative explanations could be a complete intraluminal degradation, a lack of intestinal absorption, or a rapid distribution to tissues and organs with peripheral uptake rates being similar to intestinal absorption, which would leave blood concentrations at a constant level. Since none of these mechanisms can explain the observed increase in spermine levels over time, our data strongly suggest that in humans, dietary spermidine is at least partly presystemically converted into spermine, which then occurs in the systemic circulation. Since no spermine pharmacokinetics were evaluable, these processes are probably concealed by other metabolic or distributing processes, as discussed above.Evidently, the pharmacokinetic relationship between oral spermidine intake and spermidine/spermine plasma concentrations is not simply additive, and the underlying mechanisms remain rather speculative. Obviously, dietary spermidine is subject to extensive presystemic metabolism. In this context, it has been suggested that the intestinal microbiota is involved in the presystemic metabolism of dietary polyamines. Matsumoto et al. reported that the administration of probiotic bifidobacteria increased spermine—but not spermidine—concentrations in the feces of animals and humans ,63. In fAs demonstrated above, a positive correlation of plasma spermine concentrations with several cholesterol fractions was found. A causal effect due to spermidine supplementation cannot simply be postulated, since no significant differences were observed between the verum and placebo interventions . While tSince spermidine supplementation has been postulated as a possible means of therapeutically targeting several diseases ,7, transFirst, our data demonstrate that a daily dose of 15 mg of spermidine significantly increases blood concentrations of spermine. In previous trials, daily spermidine doses of 0.9–1.2 mg have been used without any change in blood concentrations of polyamines or any clinical efficacy ,64. TakiSecond, it can be postulated that the in vitro and clinical effects of spermidine are not solely due to spermidine itself but may also be attributed to its metabolite, spermine, which is formed in the body prior to or in peripheral tissues . The conThird, the overall view of preclinical and clinical data, including the results presented here, suggests that the mechanisms of intestinal absorption as well as the mechanisms of presystemic spermidine metabolism are different in rodents and humans, thereby directly affecting the biological effects. Therefore, special caution is required when transferring experimental animal data with spermidine to humans. On the other hand, spermine might represent the predominant form of transport of spermidine in the blood, with dietary spermidine being absorbed into enterocytes and intracellularly converted to spermine, which is then extruded into the systemic circulation through the basolateral membrane, followed by reconversion to spermidine in tissues and organs. It is well known that the conversion of spermidine to spermine is not a one-way street but rather possible in both directions . NonetheFourth, oral spermidine intake of 15 mg/d does not affect salivary polyamine concentrations. Preclinical data implying an oropharyngeal effect of spermidine may not be translated into recommendations to supplement spermidine, as effective doses have yet to be determined. Systemic doses of 15 mg/d do not seem to be appropriate for yielding pharmacological concentrations in the saliva.Fifth, our findings challenge the concept of epidemiological spermidine studies based on statistical correlations between the spermidine content of food and biological effects without considering the variability of presystemic spermidine metabolism or dietary spermine intake.There are a number of limitations that should be considered in interpreting the current findings. Since this study was designed as an exploratory pharmacokinetic trial, case number calculation was not possible due to a lack of data. Accordingly, a sample size of 12 was considered appropriate as it was established in phase 1 clinical trials. Therefore, it cannot be excluded that the study was not sufficiently powered to detect minor concentration changes. However, the robustness of the data was improved by the cross-over design with placebo control, which can be considered the baseline. Validated analytic results of spermidine, spermine, and putrescine can be estimated as robust but were performed in plasma after protein precipitation. It is unclear whether the substances could be transported intracellularly in blood cells or bound to plasma proteins in relevant amounts, which would have led to an underestimation of the complete amount present in whole blood. It is also possible that there might be some significant effects of spermidine supplementation if taken by special subgroups, in higher doses, or simultaneously with certain foods. Moreover, the intervention phases were rather short (5-day interventions), and it cannot be excluded that a long-term intake of spermidine would show deviating results. The latter seems unlikely, as Soda et al. obtained very similar results on spermine concentrations with similar amounts of supplemented spermidine, even after long-term administration .The results presented here for the first time provide a pharmacokinetic basis for future translational research on spermidine. Oral spermidine intake of 15 mg/d for 5 days significantly increased spermine levels in the plasma but did not affect spermidine or putrescine levels. Our data strongly suggest that dietary spermidine is presystemically converted into spermine, which then occurs in the systemic circulation. Consequently, we postulate that the in vitro and clinical effects of spermidine are (at least in part) not attributable to spermidine itself but rather to its metabolite, spermine. It is rather unlikely that spermidine supplements with doses <15 mg/d exert any effect. Moreover, even spermidine doses of 15 mg/d do not affect salivary polyamine concentrations; in particular, pharmacological spermidine concentrations in the saliva that, due to preclinical data, might be effective at inhibiting oropharyngeal SARS-CoV-2 replication are not remotely achieved.Finally, epidemiological studies that have correlated dietary spermidine intake with biological effects are challenged by our results, as such studies have not considered the variability of presystemic spermidine metabolism and dietary spermine intake.


Conclusions: Tai chi effectively enhanced dynamic postural control in people with PN. However, no better effects on postural control from Tai Chi than from other rehabilitation approaches were observed in this study. Further high-quality trials are needed to better understand Tai Chi’s effects on individuals with PN.Background: Effects of Tai Chi on people with peripheral neuropathy (PN) are not yet apparent. This systematic review was conducted to evaluate the effects of Tai Chi on postural control in people with PN. Methods: Literature was screened in seven databases for relevant randomized controlled trials. The reports and methodological quality were evaluated. A meta-analysis was performed using RevMan5.4 software. Results: Ten reports were included, involving a total of 344 subjects. The meta-analysis found that Tai Chi therapy for people with PN resulted in a smaller sway area, in the double-leg stance with eyes closed test (SMD = −2.43, I Peripheral neuropathy (PN) is one of the most common neurological diseases in the elderly population, affecting 20% of the elderly . PN damaImpaired postural control in PN leads to higher risks of falls, with the incidence of falls in PN patients being as much as 20 times higher than in healthy patients ,8. Over Tai Chi, with its low impact and slow motion, is one of the exercise therapies that effectively prevent falls in the elderly ,16,17. OTai Chi has been proposed as an effective therapy for partially restoring neuromuscular function in people with PN. Li and coworkers  observedTai Chi training can improve postural control in people with PN. It is part of the comprehensive rehabilitation of people with PN, closely related to functional rehabilitation. In recent years, more researchers have paid attention to the role of Tai Chi in improving the static postural control ,29,30,31It is unclear if Tai Chi is effective in the postural control of people with PN. Although there are many studies investigating the effects of Tai Chi on postural control in people with PN, there is a lack of relevant systematic reviews. Previous systematic reviews concentrated on the postural control benefits of Tai Chi, focusing on the non-PN population. Therefore, a new systematic review to determine the efficacy of Tai Chi, comprehensively and systematically, in the postural control of people with PN is necessary to assist healthcare providers, policymakers, and other decision-makers in making their decisions. This review’s specific objective was to evaluate Tai Chi’s effect on postural control in people with PN.This systematic review was registered with INPLASY  and follows applicable Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 (PRISMA2020) ,34. The Details of the search strategy used for each database are shown in the The reports retrieval process consisted of the following steps: (1) Relevant studies were retrieved from various databases, such as Web of Science, CNKI, and others, using keywords based on the obtained literature; (2) Databases were searched using all relevant keywords, and the titles and abstracts read. If the inclusion criteria were met, the entire paper was searched and read; (3) The references of all included reports were checked for further relevant publications.Participant, intervention, comparison, and outcome (PICO) criteria were set for literature retrieval, inclusion, screening, and exclusion.2.Participants with a clinical diagnosis of PN based on symptoms had not practiced Tai Chi in the past four months before the trials. They had an increased vibrating perception threshold (VPT), a decreased nerve conduction velocity (NCV), and engaged in moderate intensity exercise at least once a week. The body mass indices (BMIs) of the participants were between 10 and 35 kg/mThe experimental group received Tai Chi alone or with other resistance training. In contrast, the control group received other forms of exercise, such as postural control or routine rehabilitation training. We did not discriminate against different types of Tai Chi intervention since we could not find evidence differentiating the effects of different types of Tai Chi intervention.Comparisons between groups and between before and after intervention were conducted. To assess the effects of Tai Chi on static postural control, the One-leg standing test (OLST) and double-leg stance balance with both eyes closed were utilized .To assess improvement in dynamic postural control the Berg balance scale, the ABC balance confidence scale, the six-minute walking distance test (6MWD) (m), and the Timed Up-and-Go Test (TUG) were utilized ,35.Studies were excluded if the full-text article could not be retrieved in English or Chinese, if the articles involved animal experiments, or if the articles were meeting abstracts, case reports, methodological experimental designs, or reviews.Two trained reviewers used a multi-step screening approach to assess the studies independently and recorded the main reasons for exclusion. The author, publication year, and title were reviewed in the first screening to determine which studies to include. In the second screening, titles and abstracts were reviewed to exclude studies that did not meet the inclusion criteria. In the final screening, the full texts were reviewed to determine the outcome indicators and other relevant data, such as author, publication year, sample size, selection criteria, intervention measures, frequency and intensity, intervention duration, follow-up time, causes of loss to follow-up, results, and conclusions.Disagreements were resolved through discussion. The final decision was made by a third author if consensus could not be reached through discussion. This data was then extracted for use in the meta-analysis by one author independently and was confirmed by another author.In addition, the following literature characteristics of each included report were documented: reasons for duplicative systematic reviews, number of actual applications versus previous reviews, experimental methods of inclusion, demographic data and characteristics. Heterogeneity and subgroup analysis of the main studies were also documented, along with review conclusions as to whether Tai Chi was effective in improving the balance function of people with PN.Methodological quality evaluation for papers included in the meta-analysis was conducted by two researchers independently, using the Cochrane Collaboration’s tool to assess the risk of bias (updated 2022) . The evaPapers were classified into three levels based on the potential for bias. A paper was rated as grade A if it met all of the quality standards in the Cochrane Collaboration’s tool for assessing the risk of bias and the potential for bias was low. A paper was rated as grade B if it met some of the standards and the potential for bias was moderate. A paper was rated as grade C if it did not meet any of the standards, and the potential for bias was high. After independently evaluating the quality of each paper, the researchers discussed their assessments and decided whether to include or exclude the paper from the analysis. A third author made the final decision if this was required.2, with I2 values of ≤25% representing no to low heterogeneity, 25% < I2 ≤ 50% representing moderate heterogeneity, 50% < I2 ≤ 75% representing high heterogeneity, and I2 > 75% representing very high heterogeneity. If the I2 value was less than 50%, a fixed-effects model was used for combination, and if the I2 was greater than, or equal to, 50%, a random-effects model was used [The meta-analysis was performed using RevMan 5.3.0  for data processing, merging of data, performing a sensitivity analysis on the outcome indicators, determining risk of bias, and the drawing of forest plots. Continuous variables were analyzed in a meta-analysis using the standard mean difference (SMD) with a 95% confidence interval (CI), calculated as the effect size of the outcome. SMD was interpreted as follows: small effect, 0.2–0.6; medium effect; 0.6–1.2, large effect, 1.2–2.0; and very large effect, >2.0 . Heterogwas used ,38. SensFollowing title/abstract screening, the rest of the studies were carefully screened for inclusion, and there were still 359 articles. Only 9 studies ,39,40,41A total of 8 articles ,31,32,39The quality of 1 study, Patricia AQ, was evaluated as “A”, and the quality of the remaining 9 studies as “B”. Five papers were low risk, 5 papers could not be judged as low risk or high risk in random sequence generation, Five papers introduced the specific methods of allocation concealment and 7 papers had no the allocation concealment scheme. Nine studies had no relevant information about the blinding of participants and personnel and blinding of outcome assessment. Eight articles reported complete data and missing data, with reasons described. Seven articles were low risk in selective reporting and 7 papers could not be judged as low risk or high risk .p for heterogeneity = 0.10, I2 = 52%). There were no significant differences in the times for the OLST between the Tai Chi and the control groups . Due to the moderate heterogeneity between studies, we performed a sensitivity analysis. Firstly, a lower-quality study was excluded [2 = 54%, p = 0.84). Secondly, a study with a small sample size was excluded [2 = 55%, p = 0.20). Finally, excluding one study with results significantly different from several others [2 = 53%, p = 0.13). A total of three sensitivity analyses were performed and similar results were obtained, indicating that the results presented in this paper are stable.Four meta-analysis studies ,29,30,31excluded . This exexcluded , but thip for heterogeneity = 0.97, I2 = 0%) and the fixed-effect model was used. Compared with the control group, the swaying area of the Tai Chi group after training was significantly reduced .p for heterogeneity = 0.89, I2 = 0%). The balance scale scores were not different between the Tai Chi group and the control .Three studies ,31,39 rep for heterogeneity = 0.95, I2 = 0%). The distances of the 6MWD did not differ between the Tai Chi and control groups   B.p for heterogeneity = 0.69; I2 = 0%). There were no differences in TUG time between the Tai Chi group and the control group   C.p = 0.06). The heterogeneity between studies was low and a fixed-effect model was used   A.p for heterogeneity = 0.11, I2 = 50%). The TUG time of the Tai Chi group was significantly shorter after training   B.This systematic review with meta-analysis aimed to assess the effect of Tai Chi intervention on postural control among people with PN. The main findings from this review suggest that Tai Chi had a high clinical effect (SMD = −2.43) on the improvement of static postural control, as measured by changes in the sway area test of double-leg stance with eyes closed, compared with the control group, and a small clinical effect  in dynamic postural control, as measured by changes in 6MWD and TUG, compared with baseline. However, Tai Chi had no better effect than other rehabilitation approaches of the control group on OLSD and dynamic postural control.The sample size included in this meta-analysis included 199 males and 145 females, mainly aged over 60 years. The duration of intervention was mainly 60 min and ranged from 8 weeks to 24 weeks. The types of Tai Chi were 16, 24, and 27-style. Types of intervention in the control group included aerobics, stretching, routine rehabilitation, balance training, medication, and 24-style Tai Chi with resistance training.This study reviewed 10 ,39,40,41The impact of research conclusions on the quality of the literature is as follows. Firstly, due to the low quality of the included randomized controlled trials, most researchers came to relatively definite conclusions. Higher-quality RCTs help provide scientific evidence. This review observed that the overall methodological quality, reporting quality, and evidence quality of the included RCTs varied, indicating limitations in the reliability of the conclusions of these RCTs. Secondly, scholars mostly used the term “Tai Chi” to represent all types of Tai Chi. There are various types of Tai Chi, including “Chen, Yang, Wu, Sun,” and others. Different types of Tai Chi have different effects on rehabilitation. This study did not further analyze and compare different types of Tai Chi. A subgroup analysis for different types of Tai Chi is necessary. Furthermore, the diversity of Tai Chi, including differences in training style, exercise form, frequency, and duration, may be a source of heterogeneity in RCTs. This also requires further analysis.Both Double-leg stances with eyes closed and OLST were used to evaluate the improvement of static postural stability of Tai Chi in people with PN ,30,40,41There may be several reasons. First, compared to the double-leg stance with eyes closed test, OLST is a more challenging stability test, due to the small support surface in postural control research . The resThe result of the double-leg stance with eyes closed in this study indicated that Tai Chi may enhance postural control and reduce the risk of falls. A recent study showed that Tai Chi could improve lower extremity strength . For peoA previous study reported that people with PN exhibit abnormal walking patterns, including increased loading times on the heel and metatarsal and decreased loading on the hallux . The BalThe other assessment of the Tai Chi effect on dynamic control in this study showed that Tai Chi training improved indicators of dynamic postural control such as walking faster and farther after training than baseline ,24,31,32The effect of Tai Chi on postural control has been confirmed in older adults  and peopThe mechanisms of the results are not very clear. Some studies showed that Tai Chi effectively improves postural control by focusing on transferring the center of mass (COM) and stabilizing the ankle swing, enhancing the function of the sensorimotor system and knee joint extension strength ,56. OtheTai Chi is well-tolerated, safe, economical, and enjoyable for older adults . The resThis study has the following limitations that must be considered: (1) The scales used for outcome indicators in the 10 studies were subjective, leading to differences in the operation of the same scale evaluation, thereby reducing the reliability of the meta-analysis; (2) The treatment durations in the included articles ranged from 8 to 24 weeks, and factors, such as training time, training period, method of determining PN, and outcome indicators, may have impacted the results. With a small number of RCTs included, the study was unable to conduct further stratified analysis based on indicators such as age, gender, and intervention methods; (3) The term “Tai Chi” was used in this study to represent all types of Tai Chi without specifying the different types, leading to a lack of information on specific Tai Chi styles. As a result, this review cannot recommend using a particular type of Tai Chi.Tai chi effectively enhanced dynamic postural control in people with PN. However, no better effects on postural control from Tai Chi than those derived from the other rehabilitation treatments were observed in this meta-analysis. Despite this, the limited quality of the studies suggests there is a need for further high-quality trials to confirm the long-term benefits of Tai chi on individuals with PN.


Membranous nephropathy is an autoimmune disease affecting the glomeruli and is one of the most common causes of nephrotic syndrome. In the absence of any therapy, 35% of patients develop end-stage renal disease. The discovery of autoantibodies such as phospholipase A2 receptor 1, antithrombospondin and neural epidermal growth factor-like 1 protein has greatly helped us to understand the pathogenesis and enable the diagnosis of this disease and to guide its treatment. Depending on the complications of nephrotic syndrome, patients with this disease receive supportive treatment with diuretics, ACE inhibitors or angiotensin-receptor blockers, lipid-lowering agents and anticoagulants. After assessing the risk of progression of end-stage renal disease, patients receive immunosuppressive therapy with various drugs such as cyclophosphamide, steroids, calcineurin inhibitors or rituximab. Since immunosuppressive drugs can cause life-threatening side effects and up to 30% of patients do not respond to therapy, new therapeutic approaches with drugs such as adrenocorticotropic hormone, belimumab, anti-plasma cell antibodies or complement-guided drugs are currently being tested. However, special attention needs to be paid to the choice of therapy in secondary forms or in specific clinical contexts such as membranous disease in children, pregnant women and patients undergoing kidney transplantation. Membranous nephropathy (MN) is an autoimmune disorder affecting the kidney glomerulus and is one of the most prevalent diseases causing nephrotic syndrome (NS) in adults . Its preThe 2021 Kidney Disease Improving Global Outcomes (KDIGO) guidelines recommend the use of rituximab (RTX) or a calcineurin inhibitor (CNI) as the initial treatment for patients at moderate risk of disease progression, while the use of first-line cyclophosphamide (CYC) is recommended for high-risk patients. Conversely, patients at low risk of progression do not require immunosuppressive therapy and should be treated only with support care . HoweverMN is an immunocomplex disease. Pathogenesis begins with the binding of circulating antibodies to antigens present on podocytes. In the secondary forms, however, the initiating trigger could be the deposition of immune complexes in the subepithelial space .The first research on the pathogenesis of the disease dates back to 1980, from studies conducted on mouse models of Heymann’s nephritis; however, the antigen responsible for this disease, megalin, was not found to be present in humans. The first data in humans were found in 2002 following a case of neonatal MN, in which the mother, who had been immunized during a previous pregnancy, had transferred the antibodies directed against neutral endopeptidase (NEP) to the newborn. In 2009, the discovery of antigens directed against phospholipase A2 receptor 1 (PLA2R1) represented a milestone in understanding the pathogenesis responsible for about 70% of adult MN cases. In 2014, a second antigen, thrombospondin, was identified  and NELLPLA2R is a transmembrane receptor of the mannose receptor family whose role in human cells is still unknown. Anti-PLA2R antibodies, which have a prevalence of 70–80% in primary MN, have diagnostic, prognostic and predictive value. Positivity to these antibodies demonstrated a sensitivity of 78% and a specificity of 99% in the diagnosis of NM; moreover, the specificity reaches 100% when the search for PLA2R in serum is complemented by staining of the kidney biopsy. These discoveries made it possible to diagnose MN even in those situations where a biopsy is complicated, such as in patients with a single kidney or with an increased risk of bleeding. Anti-PLA2R antibodies are able to anticipate the disappearance of proteinuria in response to therapy by several months and, conversely, in disease relapses. Finally, higher antibody titers correlate with a worse prognosis and a slower response to therapy . As provThrombospondin-1-domain-containing 7 A (THSD7A) is a transmembrane protein localized in podocytes. The prevalence of antithrombospondin antibodies is up to 5% of all NM cases and up to 10% of cases without PLA2R antibodies. Several studies have reported an association with malignancy; therefore, more intensive malignancy screening is required in these patients. In addition, the presence of these antibodies in tumor cells has been described as well as, more importantly, a reduction in antibody titer and proteinuria following chemotherapy . Like PLNELL -1, a glycoprotein poorly expressed in adult tissues but essential for ossification during child growth , is the However, how these autoantibodies are formed is still largely unknown. Genetics plays a major role, and an association with a single nucleotide polymorphism at the HLA-DQA1 locus has been described. Chinese studies that have found a different prevalence between rural and industrialized areas also highlight that environmental pollution may play a role. The development of such autoantigens can likely be attributed to the loss of peripheral or central tolerance, altered expression of antigens or molecular mimicry mechanisms . NonetheAccording to the latest KDIGO guidelines, patients with NS who test positive for anti-PLA2R antibodies do not require a biopsy to diagnose the disease; however, in some specific cases, it can be considered. Data on anti-THS7DA are insufficient to extend the same recommendation to this antibody [However, a kidney biopsy may be performed for purposes other than diagnosis alone. A Mayo Clinic retrospective study covering the period from 2015 to 2018 analyzed 97 patients with primary MN who were positive for PLA2R antibodies but still underwent biopsy. Of these, 60 had an eGFR greater than 60 mL/min, and renal biopsy did not alter the therapy or patient management in any way, even in the two cases diagnosed with superimposed diabetic nephropathy or focal and segmental glomerulosclerosis (FSGS). On the other hand, the discovery of additional findings, such as acute interstitial nephritis and crescents in the group of patients with an eGFR below 60 mL/min, actually had an impact on therapy, suggesting more aggressive therapy and additional vasculitis antibody testing . Also, tn = 5), interstitial nephritis (n = 5) and IgA nephropathy (IgAN) (n = 2). Of note, patients with an additional diagnosis were those with the highest creatinine and lowest eGFR values. Interestingly, the proportion of patients with an additional diagnosis other than MN was slightly higher in patients who were negative for anti-PLA2R antibodies [Similarly, Wiech et al. analyzed 263 patients with a histological diagnosis of MN, including 194 who were positive for anti-PLA2R antibodies. Of the latter, twelve (6%) received an additional diagnosis such as diabetic nephropathy , serological abnormalities or when patients do not respond to immunosuppressive therapy and show a progressive decrease in eGFR or persistent NS despite the disappearance of PLA2R antibodies. In any case, the possibility of a renal biopsy must always be assessed on a case-by-case basis, taking into account both the costs and the risks to the patient .Regardless of the degree of proteinuria, renal function and extent of NS, all patients with MN should receive the best possible supportive care and be treated to prevent possible complications from the disease . Such trThe treatment of edema includes both the use of diuretics and dietary salt restriction. Loop diuretics administered twice daily are the first-line therapy . As the Angiotensin-converting enzyme inhibitors (ACEi) or angiotensin-II-receptor blockers (ARBs) are the first-line therapy for blood-pressure control thanks to their additional effect in reducing proteinuria. Reaching the target blood pressure  can both protect against cardiovascular risks and slow down the loss of the GFR. On the other hand, loss of renal function can be prevented if proteinuria is reduced to less than 0.5 g per day or slowed to less than 1.5 g per day . In addiHyperlipidemia must be treated if the patient has other risk factors for cardiovascular disease, including diabetes, smoking, hypertension or being overweight. The first step in treating lipid abnormalities is diet and lifestyle modification. Statins are effective in lowering lipid levels, and there is some evidence that atorvastatin may also reduce proteinuria  comparedAmong the different forms of glomerulonephritis, MN is the one that carries the greatest risk of thromboembolic events, especially deep-vein thrombosis and renal-vein thrombosis , and theThe first drugs used to treat MN were exclusively glucocorticoids, although trials conducted with prednisone showed no or only transient benefit ,39. In aIn 1984, a multicenter Italian RCT assigned 67 patients with MN to symptomatic treatment or to an alternating combination of chlorambucil and glucocorticoids for six months. At months 1, 3 and 5, 1 g of intravenous methylprednisolone was administered for 3 days and 0.5 mg/kg oral prednisone for a subsequent 27 days, while chlorambucil at 0.2 mg/kg/day was given at months 2, 4 and 6. The treatment group showed a stabilization of renal function and an improvement in proteinuria, and these results were confirmed 10 years later . This apEarly indications of a potential role for cyclosporine in the treatment of MN came from several observational studies in which patients achieved partial or complete remission ,46,47. HSince patients suffering from MN can spontaneously experience remission, it is crucial to determine which patient would benefit more from immunosuppressive therapy and who can be treated only with supportive therapy. Hence, the latest KDIGO guidelines  suggest Consequently, it is suggested to wait 6 months while using maximal anti-proteinuria therapy since spontaneous remission can occur. On the other hand, if high-level proteinuria, high-tier PLA2R auto-antibodies or low-molecular-weight proteinuria are present, an earlier re-evaluation is mandatory, while if kidney function is rapidly decreasing and if nephrotic syndrome is unresponsive to symptomatic therapy, an early course of immunosuppressive therapy should be started at soon as possible. The chance of spontaneous remission is higher in patients with proteinuria below 4 g/day compared with those below 8 g/day and 12 g/day  and in patients with lower tiers of anti-PLA2R antibodies compared with higher ones. Moreover, in addition to single values, it is fundamental to evaluate the trajectory of such parameters.Consequently, immunosuppressive therapy should be practiced only in patients at risk of progressive kidney injury. It is not required if proteinuria is not in nephrotic range , if serum albumin is greater than 30 g/L and if the eGFR is above 60 mL/min. Such patients usually have low risk of thromboembolic complications and have few or any symptoms and can therefore be managed only with conservative therapy. Immunosuppressive therapy in such patients would add risks without any potential benefits.On the other hand, if there is at least one risk factor for disease progression, immunosuppressive therapy is recommended. The choice of the specific therapy should account for the patients’ characteristics, drug availability, preference from patients and physicians and the specific side effect of each drug. The use of MMF is not included since KDIGO 2012 argued against its utility. Many randomized control trials (RCTs) and cohort studies proved that rituximab (RTX) and tacrolimus/cyclosporine can increase the rate of both complete and partial remissions, with a better safety profile than cyclophosphamide. However, the use of calcineurin inhibitors (CNIs) is hampered by their high relapse rate and, consequently, they can be used in monotherapy only in the case of a moderate risk of progression. Cyclophosphamide (CYC), on the other hand, reduces the risk of kidney failure to a greater extent, but it should be used only in high-risk patients due to its toxicity burden. The cumulative dose of the latter should not exceed 36 g in order to avoid malignancy or 10 g to preserve fertility.The definition of relapsing MN varies among different authors. While some authors define relapse as an increase in proteinuria above 3.5 g/d, the working group of KDIGO 2021 defined a relapse as an increase in proteinuria with a coexistent decrease in serum albumin. If a patient experiences a relapse after a first course of rituximab, a second course of the drug can be administered. On the other hand, if a relapse occurs after a course of CNI + steroid, rituximab can be added alone or in combination with CNI. Finally, if the first-line therapy consisted of cyclophosphamide + glucocorticoids, a second course of the same schedule can be repeated, accounting for the maximum dose tolerated, or, alternatively, CNI + rituximab or rituximab alone may represent a second-line therapy.There is no consensus, on the other hand, about the proper definition of resistant disease. If anti-PLA2R antibodies are detectable before first-line therapy, the disease can be defined as resistant if the antibody titer is still high after one course of therapy. Conversely, proteinuria evaluation cannot be used since it can persist for up to 24 months after therapy. If patients are not PLA2R-positive, persistence of the nephrotic syndrome can define a resistant disease. In evaluating resistant disease, the first step consists in checking the adherence to therapy by measuring the levels of B cells, anti-rituximab antibodies, CNI and IgG or the development of leukocytopenia in patients using cyclophosphamide.If the first line-line therapy consists of RTX, CNI can be added to the therapy if the eGFR is stable. If the eGFR is decreasing or if even CNI + RTX fails to achieve a response, a trial of cyclophosphamide + glucocorticoids can be made. On the other hand, if CNI represents the first attempt, RTX can be administered if the kidney function is stable; however, if it is unstable or if RTX did not achieve a response, an alkylating agent must be tried. If the latter represents the first-line therapy, RTX can be added before trying another course of alkylating agents. If patients fail to respond to either RTX or CYC, their enrolment in trials with experimental drugs is suggested (see “Novel therapeutic approaches for Membranous Nephropathy” below).In our center, intravenous methylprednisolone is usually administered in an inpatient setting, while RTX can be administered to both outpatients and inpatients.2 RTX administered intravenously on days 1 and 8 (n = 37) or non-immunosuppressive therapy alone (n = 38). After 6 months, 13 patients in the NIAT-RTX group and 8 patients in the NIAT group achieved remission. At the prolonged follow-up, with a median follow-up of 17 months, the difference was significant, with remission occurring in 64.9% of patients in the NIAT-RTX group and only 34.2% in the NIAT group (p < 0.01). In addition, PLA2R Ab-depletion rates in the NIAT-RTX and NIAT groups at 3 months were 56.0% and 4.3%, respectively (p < 0.001) [n = 36) achieved complete or partial remission in the corticosteroid–cyclophosphamide subgroup and 58.1% (n = 25 patients) in the tacrolimus-RTX subgroup, with complete remission after two years in 60% of patients (n = 26 patients) in the corticosteroid–cyclophosphamide subgroup and in 26% (n = 11 patients) of the tacrolimus-RTX subgroup. The rate of patients achieving an immunological response, i.e., a disappearance of anti-PLA2R antibodies, was significantly higher in the corticosteroid–cyclophosphamide group than in patients receiving tacrolimus and rituximab, both at three months (77% vs. 45%) and six months (92% vs. 70%). Relapsing disease was observed in three patients in the tacrolimus-RTX subgroup and only in one patient in the corticosteroid–cyclophosphamide subgroup. No difference was found in the rate of serious adverse events between the two subgroups [2 administered one to four times weekly as an alternative first-line option for the initial treatment of patients carrying a moderate or high risk of progressive disease. A study including 12 patients with idiopathic MN and NS receiving titrated B-cell treatment was followed with the aim of determining the ideal dosage compared to a group of 24 patients receiving the standard protocol of four weekly doses of 375 mg/m2. The results showed that only one patient required a second dose to achieve complete depletion of CD20 cells. After 12 months, the progression of NS manifestations and the proportion of patients achieving disease remission (25%) were identical in both groups. All patients achieved sustained CD20 cell depletion [2 while the second group of 16 patients received two doses. RTX represented the first-line therapy for 56% (n = 19) and the second-line therapy for 44%(n = 15) patients. Follow up was conducted for 12 months; at the end, 14.7% (n = 5) achieved a complete response, 29.4% (n = 10) a partial response and 55.8% (n = 19) no response. The response occurred approximately 6 months after dosing. The outcome was similar for one and two doses of RTX, with 55.5 and 56% of non-responders respectively [n = 112) developed low IgG levels for ≥4 months after more than one cycle [n = 20) of the CNI group (p = 0.06) [p < 0.001), both serious  and non-serious . The cumulative incidence of a first event  and serious  or non-serious  event was significantly lower with RTX [The last drug included in KDIGO for the treatment of MN was RTX, a chimeric IgG1 monoclonal antibody that exerts its B-cell depleting effect through its binding to CD20. To date, numerous studies have been conducted to evaluate the efficacy of this treatment in monotherapy and in combination with the drugs already in use. The GEMRITUX randomized trial, published in 2017, evaluated 6 months of non-immunosuppressive drug therapy (NIAT) in combination with 375 mg/m< 0.001) . The difubgroups . The Itaepletion . Anotherepletion . To confectively . In termne cycle . More re = 0.06) ). Althouwith RTX . Neverthwith RTX .Immunosuppressive therapy can be associated with serious and potentially fatal side effects. In addition, 20–30% of patients do not achieve a complete or even a partial response to therapy. If response has not been achieved after two or more lines of therapy, alternative therapies may be considered . FindingMycobacterium tuberculosis and the much rarer Mycobacterium shimoidei, have been described without overt renal tuberculosis. Also, in this case, the initiation of pathogen-specific antibiotic therapy against these mycobacteria, namely clarithromycin, rifampicin and ethambutol, can significantly improve proteinuria and the renal status [Treponema pallidum, may allow for the rapid improvement of the NS and avoid the use of steroid therapy. Intramuscular benzathine penicillin once a week for three weeks has been shown to be effective [Secondary MN accounts for almost 25% of MN. It includes forms of disease caused by infections, malignancies, autoimmune diseases and drugs . Recognil status . In hepal status . Still, ffective ,102. In ffective . If paroffective . In addiffective ,106. Anoffective . Membranffective . Howeverffective . Finallyffective .2) [2 once a week for 4 weeks have been reported. In addition, in the case of rituximab-resistant disease or in patients suffering from rituximab serum sickness [Clostridium butyricum preparations reduced the recurrence of nephrotic syndrome compared with patients treated with placebo and that, in the same group, fewer patients received rituximab for recurrences [MN is a rare disease in children. It accounts for less than 2% of biopsies in American children , althoug2) . A repor2) . In thissickness , new antsickness . Finallyurrences .During pregnancy, idiopathic MN is a rare cause of NS; there are few case reports in the literature . In womeIn kidney transplant recipients MN can occur either as a recurrent disease, when the diagnosis of MN has already been made in native kidneys, or as de novo MN (DNMN), when the cause of kidney failure is a different disease. It is not always possible to distinguish between these two identities because the cause of the primary failure may be unknown, in which case other authors use the term transplant MN . Beyond Determining the exact incidence of MN is difficult because indications for performing biopsies differ between centers; in addition, not all centers perform electron microscopy and immunofluorescence routinely. However, recurrent membranous nephropathy (RMN) can occur in up to 40% of cases, while DNMN is reported in up to 2% in the adult population and up to 9% in the pediatric population . Of noteAnother important issue concerns the costs of such therapies; in fact, it is essential to evaluate the impact that these treatments have on the healthcare budget . Also, cMoreover, the modified Ponticelli regimen requires various hospital admissions to perform steroid and cyclophosphamide infusions, while RTX can be administered in an outpatient setting.To the best of our knowledge, currently no studies have systematically evaluated patients’ choice over the various therapeutical opportunities proposed for MN. In our experience, CYC raises concern due to the risk of malignancy and the effect on fertility; similarly, other patients wish to avoid steroids or CNI administration for their side effects. Rituximab, conversely, appears to be well tolerated, especially when administered in an outpatient setting.In this review, we have summarized the most important points on the treatment of MN. Although the discovery of new autoantibodies may allow for diagnosis without a renal biopsy and provide important prognostic and therapeutic clues, currently, up to 30% of patients do not respond to therapy despite supportive care and appropriate immunosuppressive therapy, leading to ESRD. The use of new drugs targeting plasma cells and the complement system, which are currently under investigation, could help to significantly reduce this percentage. On the other hand, further studies on specific contexts such as childhood, pregnancy and kidney transplantation are needed to improve diagnosis and to choose the most appropriate treatment.


Overnutrition among adolescents is becoming a major public health concern, with all the adverse consequences associated with unhealthy eating behaviors. Hence, clear evidence linking dietary consumption with the risk of overnutrition is crucial for targeted dietary recommendations using a robust statistical approach. This study assessed the link between dietary consumption patterns and the risks of overnutrition among adolescents in Ethiopia.via proportional allocation. Dietary consumption was captured using a validated and contextualized 80-item food frequency questionnaire over the past month through a face-to-face interview. Weight and height were measured under a standard procedure. Body Mass Index for Age Z-score (BAZ) was calculated, and BAZ above +1 was considered overnutrition. The frequency measures were standardized into daily equivalents, and dietary patterns were derived using exploratory factor analysis after checking for assumptions. A bivariable and multivariable binary logistic regression model was fitted with an odds ratio and 95% confidence intervals.A community-based survey was conducted on a random sample of 510 adolescents selected using a stratified random sampling A total of 510 participants were enrolled. Four major dietary patterns , explaining 66.6% of the total variation, were identified. The overall prevalence of overnutrition was 29.0% (27–31%), where 22.5 and 6.5% were overweight and obese, respectively. Physical inactivity , maternal literacy , habit of snacking , skipping meals , cereals and discretionary food dietary pattern , and protein–rich and vegetable dietary pattern  were important factors associated with odds of overnutrition.Overnutrition is a public health concern affecting one-third of adolescents, and it is closely linked with dietary consumption patterns, eating behaviors, wealth status, literacy, and level of physical activity. Therefore, public health interventions targeting unhealthy eating and lifestyles are urgently needed to curb the increasing burden of overnutrition among adolescents and its future complications. These hOvernutrition is characterized by excessive accumulation of fat as a result of excessive energy intake or decreased daily energy expenditure. The World Health Organization (WHO) defines adolescent overnutrition as body mass index (BMI) for age Z-score (BAZ) above +1, including overweight (BAZ between +1 and + 2) and obesity (BAZ above +2) . Due to The global prevalence of overweight and obesity is rising among adolescents by 14% over the past decades, affecting over 340 million adolescents, and the rate of increase is alarming in developing countries (30%) . SimilarStudies conducted so far have mainly focused on the effects of individual food consumption and some aspects of diet on overnutrition. However, diet could be better characterized using a robust statistical approach called “dietary pattern analysis.” It is a better approach to characterize habitual consumption and overall dietary consumption –23. Diet2.2.1.This study was conducted in Dire Dawa, which is located at a latitude of 9° 35′ 35″ N, a longitude of 41° 51′ 57″ E, and 1,204 m above sea level in eastern Ethiopia. According to the Dire Dawa health bureau, the region has a total population of 535,685, and the majority of the population lives in urban areas (72%). An estimated 59,871 adolescents reside in the region, both at home in their communities and at primary and secondary schools. In urban and rural classifications, the region has nine and more than 30 kebeles (third level administrative units in Ethiopia) in urban and rural settings. The study was conducted from February to March 2022 using a primary data collection method.2.2.A community-based cross-sectional study was conducted on a randomly selected adolescent group in Dire Dawa town to assess the relationship between various dietary patterns and the risks of overnutrition. The study findings targeted all adolescent girls in the study area, and these could indicate the burden of overnutrition and its predictors. Hence, we excluded those with vertebral deformities, spinal curvatures, and diagnosed chronic illnesses, which might affect the proper height measurement and may restrict an individual’s dietary intake.2.3.p = 26.1)  , at a 95A stratified sampling technique with a proportional allocation method was employed for this study. First, four kebeles were randomly selected using simple random sampling, and then, four blocks or villages were selected from each kebele, where the sample size is proportionally allocated relative to the number of adolescents in each village. Finally, we employed a systematic random sampling technique with a sampling fraction calculated from the total number of adolescents and the allocated sample size. With the assistance of community-based health extension workers, adolescents were tracked in the selected households.n” is the total sample size calculated, “N” is the total adolescent population (target population), “Ni” is the total number of adolescents in a particular stratum, and “in” the sample size for each stratum.The proportional allocation formula 2.4.The dependent variable was overnutrition, which is a composite indicator calculated from BAZ scores from weight and height mesurements at appropriate cutoff points. While the major dietary patterns identified from the dietary consumption captured through food frequency (FFQ) were the major independent variables of this research, socio demographic variables , eating behaviors , sedentary lifestyles , and level of physical activity  were also assessed as confounding variables in assessing the association between major dietary consumption patterns with the risks of overnutrition among adolescents.2.5.In this study, “skipping meals” was defined as the omission or lack of consumption of one or more of the traditional main meals  throughout the day . In addiBased on the standardized BAZ score, a BAZ score above +2 was classified as having obesity, while a BAZ score between + 1 SD and − 2 SD was classified as overweight adolescents and otherwise normal. Hence, the burden of overnutrition was quantified as the sum of overweight and obesity prevalence among adolescents .On the other hand, the level of physical activity was assessed using the standard and highly valid General Physical Activity Questionnaire (GPAQ) and scored as a metabolic equivalent (MET) depending on the intensity and duration of each activity. Hence, we defined a physically active adolescent when the total physical activity MET minute per week is at least 600 and physically inactive otherwise . Thus, m2.6.Data were collected using a combined approach through face-to-face interviews using a structured and pretested questionnaire, anthropometric measurements of height and weight using a standard procedure. The questionnaire was prepared in English and the respective local languages. The tool is composed of socio-demographic characteristics, eating behavior, dietary pattern, and lifestyle. The data was collected by a trained pair of recently graduated health professionals.More importantly, we employed a validated 80-item FFQ used in the Ethiopian context. The tool is modified and contextualized for the study setting, with the inclusion of important food items and the exclusion of unnecessary items. The semiqualitative FFQ includes relevant and specific food items from cereal products, legumes, fruit, vegetables, meat, milk and milk products, sweet foods, snacks, and other food consumption over the past 1 month, where the frequency is ranked from “never” to “everyday” from one to seven , 28. We The 16-item GPAQ validated tool developed by WHO for physical activity surveillance was employed to assess the level of physical activity of adolescents in three domains, including activity at work, travel to and from places, recreational activities, and sedentary behavior, through face-to-face interviews. The activity level of the study participants was evaluated according to the standard WHO total physical activity calculation guide, and the level of total physical activity was categorized as physically active or inactive as detailed in the previous section , 30.The height was measured using a Seca standing height measuring device by a trained and reliable anthropometric measurer to the nearest 0.1 cm. The weight was measured using a calibrated adult electronic scale, taking two consecutive measurements, and the average weight was recorded to the nearest 0.1 kg. Hence, the BMI was calculated by dividing the weight in kilogram by the height in meters squared. Based on this, the BMI is transformed into a standardized BAZ to rank the nutritional status of adolescents.2.7.Data were entered into controlled data entry software (Epi Info) with validity and consistency checks. Close supervision was employed during data collection, in addition to a two-days training given to data collectors and supervisors to maintain the quality of the data. Further training of data collectors was done on proper anthropometric measurements using a standard method. Moreover, pretesting was conducted on 10 samples to evaluate the tool and assess the reliability and validity of the data collector. The technical error measurement (TEM) was calculated for each data collector, and those with a TEM above 1.5 and 2% for intra- and inter-observer measurements were excluded from collecting the data .2.8.Data were entered and coded using EPI–info version 7.2.1.0 for data exploration and cleaning. The cleaned data was exported to SPSS version 21 for data transformation and statistical analysis. The data were presented using frequency, percent, mean, standard deviations, statistical tables, and graphs. The data were checked for outliers, distribution skewness, and collinearity.The age, sex, height, edema, and weight of the adolescents were further exported to WHO Anthroplus version 1.0.4 to calculate the BAZ score, where they were further exported and merged with the primary data. The BAZ score was recoded as overnourished (overweight and obesity), otherwise normal as per the standard cutoff point .p-value was below 0.05. A minimum load value above 0.3 was considered adequate for the set of variables , transport, and leisure time-related physical activity (moderate and vigorous). Based on the GPAQ tool, each level of physical activity was transformed into MET in minutes per week, and then the sum of sub-activities’ MET minutes per week was calculated. Then, we multiplied the number of days involved in a certain level of physical activity per week by the daily duration (in minutes) and the metabolic equivalents considering the physical activities in minutes done in a week  , 38. Henp-value below 0.05)  and an inflated standard error (SE > 2) were considered to assess the possibility of multicollinearity between independent variables . Importaow 0.05) , 41. We . A letter of cooperation was taken to the respective offices before data collection. We gave a detailed and translated information sheet, and assent was obtained from the parents or guardians of adolescents aged below 18 years, while informed written consent was obtained from them directly for those aged at least 18 years. Participation in the study was on a voluntary basis, and no enforcement was implied. We conducted the interview in a secure place, and the collected data were not shared with a third party to assure confidentiality.3.3.1.Of the total sample of 510 respondents, 48% were male. In addition, the majority, 208 (40.8%) and 326 (63.9%), were aged 10 to 13 years and were Muslims. Regarding maternal education, about 12.0, 16.5, 42.0, and 29.6% of them had no formal education, primary education, secondary education, or had at least a college education, respectively. Concerning occupational status, about 30.2% were housewives. About 200 (39.2%) and 161 (31.6%) of adolescents were from low and medium socioeconomic class families, respectively. Moreover, more than half (53.9%) of adolescents live in families where there are 3–5 family members .3.2.2 = 101, df = 21, value of p below 0.0001). Four major dietary patterns were identified, and these explained 67% of the total variation in the dietary consumption of adolescents. The identified dietary patterns had a factor loading above 0.5 in each food group, which indicates a significant correlation  of 0.5, and there was a significant correlation among items (Xrelation .Food group items with a complex structure (a higher loading for more than one factor) were excluded, and we derived four major dietary patterns with dominant food item loadings for each component. These are cereal, energy, and discretionary calorie foods; fatty and milk products; protein-rich foods and vegetables; and fruits. A factor score was generated using the Bartlett procedure, which is a robust and unbiased estimate of the true factor score. Then, the factor score was categorized into three terciles . Four dietary patterns, including “cereal, energy, and discretionary calorie foods,” “oil, fatty foods, and milk products,” “protein-rich foods, and vegetables,” and “fruits,” were identified as indicated in 3.3.In this study, a total of 115 (22.5%) and 33 (6.5%) were found to be affected by overweight and obesity, respectively. The combined prevalence of overnutrition (both overweight and obesity) was 29% (95% CI: 27–31%). Related to these, 2% of adolescents were found to be thin. When disaggregated by certain relevant factors, the burden of overnutrition was higher among females (15.6%) and among mid-adolescents aged 14–16 years (10.3%) as compared to males (13.1%) and older adolescents (7.1%), respectively. More importantly, the burden was significantly higher among private schools (22.5%) as compared to those who attend public schools (3.6%), excluding those who did not attend school during the study.3.4.value of p of less than 0.05.A bivariable binary logistic regression was conducted, and the association between socio-demographic, dietary/eating habits, physical activity, sedentary lifestyle, and dietary consumption pattern related factors with overnutrition among adolescents was evaluated. Hence, the state of overnutrition was significantly associated with maternal education, higher wealth status, family size, habits of snacking, skipping breakfast, being physically inactive, sedentary life, and a dietary pattern characterized by cereal and energy-rich foods at a Older adolescents , females , and those from the wealthiest families  had a higher risk of overnutrition as compared to their counterparts. In addition, adolescents from literate families  and smaller family sizes  were significantly associated with a higher burden of overnutrition compared to adolescents from illiterate families and larger family sizes (> 5). Furthermore, snacking  and skipping breakfast  significantly increased the risk of overnutrition by 2.7- and 2.6-folds, respectively. Physical inactivity (MET below 600 MET minutes per week)  and sedentary lifestyle  were significant factors positively associated with the risks of overnutrition among adolescents .Regarding the association between major dietary patterns and the risks of overnutrition, low and medium percentiles of cereal and energy food consumption patterns  and higher percentiles of fatty food consumption patterns  were associated with a higher risk of overnutrition among adolescents as compared to those with low terciles of energy and fatty food consumption. While those with low  and medium protein-rich foods and vegetable consumptions  were positively associated with a higher odd of overnutrition among adolescents .value of p < 0.0001), indicating an improved model. Variables with a significant decline in model fitness on removal were retained in the final model despite a higher value of p as shown in Variables associated with overnutrition in bivariable analysis and important factors explored in previous literature were included and tested in multivariable binary logistic regression analysis. A Hosmer and Lemeshow’s goodness of fit value of 0.98 was identified, which indicates a fitted regression model for a given dataset. In addition, the effects of further addition or removal of factors on the model fitness were evaluated using an omnibus test  and less extended families  had an almost twofold increased risk of overnutrition compared to their counterparts. Physical inactivity (600 MET minutes per week) was associated with a more than sixfold increase in the risk of overnutrition  when compared to those who were physically active. Adolescents from educated families (literate mothers) were statistically significantly associated with a higher occurrence of overnutrition , indicating the majority of the overnutrition cases were concentrated among adolescents from educated families. Moreover, those with a habit of snacking  and skipping breakfast  were significantly associated with a twofold increased risk of overnutrition compared to those without habits of snacking and those with a regular meal. As compared to those with higher percentiles of cereals and discretionary food consumption patterns, adolescents with low  and medium  cereal consumption had 69 and 128% higher risks of being overnourished, respectively. Adolescents with a relatively medium tercile of protein-rich and vegetable consumption (medium) have a 2.3-times higher risk of overnutrition  as compared to those with a higher percentile of protein-rich and vegetable consumption. Hence, a higher tercile of protein-rich and vegetable consumption lowered the risks of overnutrition among adolescents .4.Overnutrition is considered a global epidemic, and the burden is increasing , 2. It ip <0.0001). It is also indicated that the impact of wealth on the risk of obesity is higher for those with lower educational status (AOR = 1.78) than for those who are well-educated   , 20. A se <0.05) . Adolesce <0.05) . Howevere <0.05) , 23. On e <0.05) , 25. Hene <0.05) .In this study, we found that lower consumption of cereals and higher consumption of discretionary food dietary patterns determine the risk of overnutrition. In addition, lower tercile of protein rich and vegetable dietary patterns were important factors associated with overnutrition among adolescents. A study from China also indicated that subjects in the highest quartiles had a higher risk of obesity . It is cPhysical activity (PA) and a sedentary lifestyle, are among causes of overweight or obesity in teenagers. A sedentary lifestyle is quite relaxed, including sitting, lying down, etc., every day at work  and at home  , 36. TheGenerally, dietary consumption patterns, physical activity level, snacking, skipping meals, and maternal literacy were associated with the risks of overnutrition among adolescents. Proper characterization of the diet is critical in linking dietary risk factors with overnutrition among adolescents , 43. DieAlthough this study generates valid information quantifying the relationship between eating behavior and overnutrition among adolescents using a reliable method, the use of cross-sectional data might make it difficult to establish causality , 46. The5.Overnutrition is a public health concern affecting one-third of adolescents, and it is closely linked with dietary consumption patterns, skipping breakfast, the habit of snacking, wealth status, literacy, and level of physical activity. Therefore, public health interventions to curb overnutrition among adolescents targeting unhealthy eating patterns and lifestyles are urgently needed to halt its emergence. Furthermore, parents of adolescents should promote an enabling environment for healthy dietary consumption and a better nutritional status for their children. Schools and the health bureau should strengthen access to physical activity facilities and regular physical activities through school competitions and healthy clubs.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Dire Dawa Univesity Institutionl Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin.FG participated in conceptualization, design, data curation, project administration, data analysis, writing a report, and reviewing and approving the manuscript. AO participated in conceptualization, design, validation, supervision, methodology, data acquisition, data preparation, data visualization, and formal data analysis. In addition, AO substantially contributed to writing the draft manuscript, manuscript preparation, reviewing, and submitting the manuscript. ID participated in drafting the manuscript, critically reviewing it, and editing the drafted manuscript. All authors contributed to the article and approved the submitted version.