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Podocytes are highly specialized cells that play a pivotal role in the blood filtration process in the glomeruli of the kidney, and their dysfunction leads to renal diseases. For this reason, the study and application of this cell type is of great importance in the field of regenerative medicine. Hypertension is mainly regulated by the renin–angiotensin–aldosterone system (RAAS), with its main mediator being angiotensin II (ANG II). Elevated ANG II levels lead to a pro-fibrotic, inflammatory, and hypertrophic milieu that induces apoptosis in podocytes. The activation of RAAS is critical for the pathogenesis of podocyte injury; as such, to prevent podocyte damage, patients with hypertension are administered drugs that modulate RAAS signaling. A prime example is the orally active, non-peptide, selective angiotensin-II-type I receptor (AGTR1) blocker losartan. Here, we demonstrate that SIX2-positive urine-derived renal progenitor cells (UdRPCs) and their immortalized counterpart (UM51-hTERT) can be directly differentiated into mature podocytes. These podocytes show activation of RAAS after stimulation with ANG II, resulting in ANG II-dependent upregulation of the expression of the angiotensin-II-type I receptor, AGTR1, and the downregulated expression of the angiotensin-II-type II receptor 2 (AGTR2). The stimulation of podocytes with losartan counteracts ANG II-dependent changes, resulting in a dependent favoring of the specific receptor from AGTR1 to AGTR2. Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced the exclusive expression of 106 genes associated with DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, and calcium ion transmembrane transport. These findings highlight the usefulness of UdRPC-derived podocytes in studying the RAAS pathway and nephrotoxicity in various kidney diseases.

Acute kidney injury (AKI) is a major kidney disease with a poor clinical outcome. It is a common complication with an incidence of 10-15% of patients admitted to hospital. This rate even increases for patients who are admitted to the intensive care unit with an incidence of >50%. AKI is characterized by a rapid increase in serum creatinine, decrease in urine output, or both. Associated Symptoms include feeling sick or being sick, diarrhoea, dehydration, decreased urine output-although occasionally urine output remains normal, fluid retention-causing swelling in the legs or ankles, shortness of breath, fatigue and nausea. However, sometimes acute kidney injury causes no signs or symptoms and is detected by lab tests. Therefore, an urgent demand for non-invasive biomarkers for early detection of AKI are highly desirable. This might enable the prevention of the progression from AKI to CKD.In this study, we analysed the secretome of urine of an AKI patient cohort employing a kidney-biomarker cytokine assay. Based on these results we suggest, ADIPOQ, EGF and SERPIN3A as potential biomarkers, which might be able to detect AKI as soon as 24 h post-surgery. For the later stages, common biomarkers for the detection of AKI in both male and female patients we suggest, VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU. These markers in combination might present a robust strategy to identify the development of AKI as early as 24h or 72h post-surgery. Furthermore, we evaluated the effect of patient and healthy urine on human podocyte cells. We conclude that cytokines in the urine of AKI patients trigger processes which are needed to repair the damaged nephron and activate TP53 and SIRT1 to maintain the balance between proliferation, angiogenesis, and cell cycle arrest. In conclusion, the Renin-Angiotensin pathway seems to have major implications.

Chronic kidney disease (CKD) is a global health burden with a continuously increasing prevalence associated with an increasing incidence of diabetes and hypertension in aging populations. The CKD definition of a more than three months lasting low glomerular filtration rate (GFR) or other renal impairments including proteinuria implies that multiple factors may contribute to the disease. While there are indications of ethnic differences it is hard to disentangle these from confounding social factors. Usually, CKD is detected in later stages of the disease when irreversible renal damage has already occurred, thus suggesting a need for early non-invasive diagnostic markers.In this study, we explored the urine secretome of a CKD patient cohort from Ghana employing a kidney-injury and a more general cytokine assay.We identified panels of kidney-specific cytokine markers which were also gender-specific and a panel of gender-independent cytokine markers. The gender-specific markers are IL10 and MME for male and CLU, RETN, AGER, EGFR and VEGFA for female. The gender-independent cytokine markers were APOA1, ANGPT2, C5, CFD, GH1, ICAM1, IGFBP2, IL8, KLK4, MMP9 and SPP1 (up-regulated) and FLT3LG, CSF1, PDGFA, RETN and VEGFA (down-regulated).APOA1 – the major component of HDL particles – was up-regulated in Ghanaian CKD patients and its co-occurrence with APOL1 in a subpopulation of HDL particles may point to specific CKD-predisposing APOL1 haplotypes in patients of African descent – this however needs further investigation. The identified panels may lay down the foundation for CKD-biomarker assays to be confirmed in further studies with a larger cohort of patients.

Background: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5′ of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). Methods: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. Results: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. Conclusions: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided.

The aging process is manifested by a multitude of interlinked biological processes. These processes contribute to genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Together these are recognized as of the main risk factors of the world’s most prevalent diseases, such as neurodegenerative disorders, cancer, cardiovascular disease, and metabolic disease.The mammalian ortholog of the yeast silent information regulator (Sir2) SIRT1 is a NAD+-dependent class III histone deacetylase and has been recognized to be involved in many of the forementioned processes. Therefore, its activity is connected to aging via the regulation of apoptosis, cell differentiation, development, stress response, metabolism, and tumorigenesis. Furthermore, the physiological activity of several sirtuin family members has been connected to the regulation of life span of lower organisms (Caenorhabditis elegans and Drosophila melanogaster) as well as mammals. Aging in somatic cells of mammals is accompanied by mutations and other forms of DNA damage. These might manifest in transient cell cycle arrest associated with DNA repair, apoptosis, senescence, or cell differentiation. The activity of SIRT1 has previously been reported to be regulated by the DNA damage response pathway. On the one hand, SIRT1 is recruited from ATM to DBS and is required for DNA damage repair, but on the other hand, SIRT1 activity was also found to be negatively regulated by genotoxic stress via the interaction of ATM with Deleted in Breast Cancer 1 (DBC1). Increased levels of DBS are associated with downregulation of ATM and lower phosphorylation levels of AKT and GSK3ß, with significant implications for mesenchymal stem cell (MSC) maintenance and differentiation. In this proposed “stem cell checkpoint,” the ATM signalling pathway initiated by DBS maintains MSCs and blocks their differentiation. Based on this, it has already been established that in senescent mesenchymal stem cells, SIRT1 expression is decreased, while its overexpression delays the onset of senescence and loss of differentiation capacity/ability.In the present study, we provide evidence that SIX2-positive urine derived renal progenitor cells-UdRPCs isolated directly from human urine show typical hallmarks of aging when obtained from elderly donors. This includes the transcriptional downregulation of SIRT1 and its downstream targets AKT and GSK3ß. This transcriptional downregulation is accompanied by an increase in DNA damage and transcriptional levels several cell cycle inhibitors such as P16, reflecting possibly the ATM induced “stemness checkpoint” to maintain UdRPC stemness and differentiation capacity. We provide evidence that the renal progenitor transcription factor SIX2 binds to the coding sequence of SIRT1 and both factors mutually influence the transcription of each other. Furthermore, we show that the SIRT1 promoter region is methylation sensitive and becomes subsequently methylated in UdRPCs derived from aged donors, dividing them into SIRT1 high and low expressing UdRPCs. This downregulation might render the cells more vulnerable to endogenous noxae accelerating the accumulation of DNA damage and ultimately the accumulation of aging associated hallmarks.

Human urine is a non-invasive source of renal stem cells with regeneration potential. Urine-derived renal progenitor cells were isolated from 10 individuals of both genders and distinct ages. These renal progenitors express pluripotency-associated proteins- TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133, as well as the renal stem cell markers -SIX2, CITED1, WT1, CD24 and CD106. The transcriptomes of all SIX2+ renal progenitors clustered together, and distinct from the human kidney biopsy-derived epithelial proximal cells (hREPCs). Stimulation of the urine-derived renal progenitor cells (UdRPCs) with the GSK3β-inhibitor (CHIR99021) induced differentiation. Transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes- AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative negative regulator of self-renewal. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3 pathway. The urine-derived renal progenitor cells and the data presented should lay the foundation for studying nephrogenesis in human.

Summary Background Natural amylin is a pancreatic hormone that induces satiety. Cagrilintide is a long-acting amylin analogue under investigation for weight management. We assessed the dose–response relationship of cagrilintide regarding the effects on bodyweight, safety, and tolerability. Methods We conducted a multicentre, randomised, double-blind, placebo-controlled and active-controlled, dose-finding phase 2 trial at 57 sites including hospitals, specialist clinics, and primary care centres in ten countries (Canada, Denmark, Finland, Ireland, Japan, Poland, Serbia, South Africa, the UK, and the USA). Eligible participants were adults aged at least 18 years without diabetes, with a body-mass index of at least 30 kg/m2 or at least 27 kg/m2 with hypertension or dyslipidaemia. Participants were randomly assigned (6:1) to subcutaneous self-injections of once-weekly cagrilintide (0·3, 0·6, 1·2, 2·4, or 4·5 mg), once-daily liraglutide 3·0 mg, or volume-matched placebo (for six placebo groups). The trial had a 26-week treatment period, including a dose-escalation period of up to 6 weeks, and a 6-week follow-up period without treatment. Participants and investigators were masked to the assigned study treatment with respect to active versus pooled placebo treatment, but not to different active treatments. The primary endpoint was the percentage change in bodyweight from baseline to week 26, assessed in all randomly assigned participants according to the trial product estimand (assuming all participants were adherent to treatment) and to the treatment policy estimand (regardless of adherence to treatment). Safety was assessed in all participants who received at least one dose of randomised treatment. This trial is registered with ClinicalTrials.gov , NCT03856047 , and is closed to new participants. Findings Between March 1 and Aug 19, 2019, we randomly assigned 706 participants to cagrilintide 0·3–4·5 mg (100–102 per dose group), 99 to liraglutide 3·0 mg, and 101 to placebo. Permanent treatment discontinuation (n=73 [10%]) occurred similarly across treatment groups, mostly due to adverse events (n=30 [4%]). In total, 29 participants (4%) withdrew from the trial. According to the trial product estimand, mean percentage weight reductions from baseline were greater with all doses of cagrilintide (0·3–4·5 mg, 6·0%–10·8% [6·4–11·5 kg]) versus placebo (3·0% [3·3 kg]; estimated treatment difference range 3·0%–7·8%; p Interpretation Treatment with cagrilintide in people with overweight and obesity led to significant reductions in bodyweight and was well tolerated. The findings support the development of molecules with novel mechanisms of action for weight management. Funding Novo Nordisk A/S.

High blood pressure is one of the major public health problems which causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin-angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory and -hypertrophic milieu that causes remodelling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CDK).In the past, disease modeling of kidney-associated malignancies was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of SIX2-positive urine derived renal progenitor cells (UdRPCs) into mature podocytes bearing typical foot processes. The UdRPCs-derived podocytes show the ability to execute Albumin endocytosis and the activation of the renin-angiotensin system by being responsive to ANGII stimulation. Our data reveals the ANGII dependent downregulation ofNPHS1andSYNPO, resulting in the disruption of the complex podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of α–ACTININ. In the present manuscript we confirm and propose UdRPCs as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within NBN, a DNA-damage repair protein. Hallmarks of NBS include several clinical manifestations such growth retardation, chromosomal instability, immunodeficiency and progressive microcephaly. However, the etiology of microcephaly in NBS patients remains elusive. Here, we employed induced pluripotent stem cell-derived brain organoids from two NBS patients to analyze the underlying mechanisms of microcephaly. We show that NBS-organoids carrying the homozygous 647del5 NBN mutation are significantly smaller in size with disrupted cyto-architecture Patient-derived organoids exhibit premature differentiation together with neuronatin (NNAT) overexpression and key pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. Moreover, we show that after exposure to bleomycin, NBS organoids undergo a delayed p53-mediated DNA damage response and aberrant trans-synaptic signalling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

Genes associated with immune response and inflammation have been identified as genetic risk factors for late-onset Alzheimer&acute, s disease (LOAD). The rare R47H variant within triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to increase the risk for developing Alzheimer&rsquo, s disease (AD) 2&ndash, 3-fold. Here, we report the generation and characterization of a model of late-onset Alzheimer's disease (LOAD) using lymphoblast-derived induced pluripotent stem cells (iPSCs) from patients carrying the TREM2 R47H mutation, as well as from control individuals without dementia. All iPSCs efficiently differentiated into mature neuronal cultures, however AD neuronal cultures showed a distinct gene expression profile. Furthermore, manipulation of the iPSC-derived neuronal cultures with an A&beta, S8C dimer highlighted metabolic pathways, phagosome and immune response as the most perturbed pathways in AD neuronal cultures. Through the construction of an A&beta, induced gene regulatory network, we were able to identify an A&beta, signature linked to protein processing in the endoplasmic reticulum (ER), which emphasized ER-stress, as a potential causal role in LOAD. Overall, this study has shown that our AD-iPSC based model can be used for in-depth studies to better understand the molecular mechanisms underlying the etiology of LOAD and provides new opportunities for screening of potential therapeutic targets.

AIM To investigate the mechanisms behind the lower postprandial glucose (PPG) concentrations achieved with fast-acting insulin aspart (faster aspart) than with insulin aspart (IAsp). MATERIALS AND METHODS In a randomized, double-blind, crossover trial, 41 people with type 1 diabetes received identical subcutaneous single faster aspart and IAsp doses (individualized for each participant), together with a standardized mixed meal (including 75 g carbohydrate labelled with [1-13 C] glucose). PPG turnover was determined by the triple-tracer meal method using continuous, variable [6-3 H] glucose and [6,6-2 H2 ] glucose infusion. RESULTS Insulin exposure within the first hour was 32% greater with faster aspart than with IAsp (treatment ratio faster aspart/IAsp 1.32 [95% confidence interval {CI} 1.18;1.48]; P < .001), leading to a 0.59-mmol/L non-significantly smaller PPG increment at 1 hour (ΔPG1h ; treatment difference faster aspart-IAsp -0.59 mmol/L [95% CI -1.19; 0.01]; P = .055). The trend towards reduced ΔPG1h with faster aspart was attributable to 12% greater suppression of endogenous glucose production (EGP; treatment ratio 1.12 [95% CI 1.01; 1.25]; P = .040) and 23% higher glucose disappearance (1.23 [95% CI 1.05; 1.45]; P = .012) with faster aspart than with IAsp during the first hour. Suppression of free fatty acid levels during the first hour was 36% greater for faster aspart than for IAsp (1.36 [95% CI 1.01;1.88]; P = .042). CONCLUSIONS The trend towards improved PPG control with faster aspart vs IAsp in this study was attributable to both greater early suppression of EGP and stimulation of glucose disappearance.

Background and Objectives Degarelix is a gonadotropin-releasing hormone antagonist registered for the treatment of advanced hormone-dependent prostate cancer. Treatment causing androgen deprivation is associated with QT prolongation and this study investigated whether degarelix at supratherapeutic concentrations has an intrinsic effect per se on cardiac repolarisation and the QT interval. Methods This was a single-centre, randomised, crossover study comparing the effect of degarelix, placebo, and the positive control moxifloxacin on the QT interval. Degarelix and placebo treatments were double-blind, whereas moxifloxacin treatment was open-label. Eighty healthy men, aged 18–45 years, received single intravenous doses of degarelix 2.8 mg, and placebo, as well as a single oral dose of moxifloxacin 400 mg. Electrocardiograms were collected up to 24 h after the start of administration, with the QT interval assessed and plasma concentrations of degarelix concomitantly analysed. Results Time-matched, one-sided 95% upper confidence boundaries for baseline-corrected average changes from placebo for the QT interval, corrected using the Fridericia method (ΔΔQTcF), did not exceed 10 ms at any timepoint, with maximum degarelix concentrations reaching approximately threefold the concentrations seen in the treatment of prostate cancer. Furthermore, concentration-exposure analysis indicated absence of any QT prolongation effects of degarelix. No significant effect on any other cardiac parameter was observed. The lower bound of the 98.3% confidence interval for moxifloxacin ΔΔQTcF exceeded 5 ms, thus verifying assay sensitivity. Conclusion The results showed that the study was validated to detect a significant effect on the QT interval, and that degarelix by itself does not have any effect on the QT interval and cardiac repolarisation at supratherapeutic concentrations.

Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p

Background Fast-acting insulin aspart (faster aspart) is insulin aspart (IAsp) in a new formulation aiming to mimic the fast endogenous prandial insulin release more closely than currently available insulin products. In a post hoc analysis of pooled data from six clinical pharmacology trials, the pharmacological characteristics of faster aspart and IAsp were compared. Methods The analysis included 218 adult subjects with type 1 diabetes from six randomised, double-blind, crossover trials in the faster aspart clinical development programme. Subjects received subcutaneous dosing (0.2 U/kg) of faster aspart and IAsp. In three trials, a 12-h euglycaemic clamp was performed (target 5.5 mmol/L; 100 mg/dL) to assess pharmacodynamics. Results The pharmacokinetic and pharmacodynamic profiles were left-shifted for faster aspart versus IAsp. Onset of appearance occurred 4.9 min earlier (95% confidence interval [CI] faster aspart-IAsp: [−5.3 to −4.4], p

1 Background Faster-acting insulin aspart (faster aspart) is insulin aspart (IAsp) in a new formulation with additional excipients (L-arginine and niacinamide). In adults, faster aspart provides faster onset and greater early exposure and action vs IAsp. 2 Aim This randomized, double-blind, 2-period crossover trial investigated the pharmacological properties of faster aspart vs IAsp in 12 children (6-11 years), 13 adolescents (12-17 years), and 15 adults (18-64 years) with type 1 diabetes mellitus. 3 Methods Subjects received 0.2 U/kg subcutaneous dosing (mean of 8.3, 12.8, and 15.6 U, respectively) immediately prior to a standardized meal (17.3 g carbohydrate/100 mL; amount adjusted by body weight). 4 Results Consistently across age groups, onset of appearance occurred approximately twice-as-fast (5-7 minutes earlier) and early exposure (AUCIAsp ,0-30min; area under the IAsp curve from 0 to 30 minutes) was greater (by 78%-147%) for faster aspart vs IAsp, with no treatment differences in total exposure (AUCIAsp ,0-t) or maximum concentration (C max). Two-hour postmeal plasma glucose excursion was reduced for faster aspart vs IAsp (although only reaching statistical significance in children). In accordance with the absolute dose administered for each age group, AUCIAsp ,0-t for faster aspart was lower in children (estimated ratio children/adults [95% confidence interval]: 0.59 [0.50;0.69], P

Human urine is a non-invasive source of renal stem cells with regeneration potential. Urine-derived renal progenitor cells were isolated from 10 individuals of both genders and distinct ages. These renal progenitors express pluripotency-associated proteins- TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133, as well as the renal stem cell markers -SIX2, CITED1, WT1, CD24 and CD106. The transcriptomes of all SIX2+ renal progenitors clustered together, and distinct from the human kidney biopsy-derived epithelial proximal cells (hREPCs). Stimulation of the urine-derived renal progenitor cells (UdRPCs) with the GSK3β-inhibitor (CHIR99021) induced differentiation. Transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes- AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative negative regulator of self-renewal. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3 pathway. The urine-derived renal progenitor cells and the data presented should lay the foundation for studying nephrogenesis in human.

Background Fast-acting insulin aspart (faster aspart) is an ultra-fast-acting formulation of insulin aspart (IAsp). This post hoc analysis investigated the pharmacokinetics of faster aspart versus IAsp, measured as free or total IAsp, and the relationship between anti-IAsp antibodies and the pharmacokinetics/pharmacodynamics of faster aspart and IAsp. Methods Free and total IAsp concentrations and anti-IAsp antibodies were determined in adults with type 1 diabetes mellitus receiving subcutaneous faster aspart and/or IAsp in four single-dose clinical pharmacology trials (n = 175) and a 26-week phase IIIa trial (n = 1040). Pharmacodynamics were assessed by euglycaemic clamp or meal test, respectively. Results The pharmacokinetic profile was left-shifted and early exposure was greater with faster aspart versus IAsp independent of free or total IAsp assay. The faster aspart-IAsp difference in the time to 50% of maximum IAsp concentration in the early part of the pharmacokinetic profile (tEarly 50 % Cmax) [95% confidence interval (CI)] was − 8.8 [− 10.0 to − 7.5] and − 7.6 [− 8.8 to − 6.4] min for free and total IAsp, respectively. The faster aspart/IAsp ratio for the area under the concentration–time curve (AUC) for IAsp from time zero to 30 min (AUCIAsp,0–30 min) [95% CI] was 1.88 [1.74–2.04] and 1.77 [1.64–1.90] for free and total IAsp. Higher anti-IAsp antibody levels were associated with a lower ratio of free/total IAsp for the total AUC for IAsp (AUCIAsp,0–t). Early glucose-lowering effect (AUC for the glucose infusion rate [GIR] from time zero to 60 min [AUCGIR,0–60 min]) was greater by 25–44% for faster aspart versus IAsp independent of anti-IAsp antibody levels. Total glucose-lowering effect (total AUC for GIR [AUCGIR,0–t]) in a clamp and 1-h postprandial glucose increment in a meal test appeared essentially unaffected by anti-IAsp antibodies. Conclusions Faster aspart provides accelerated pharmacokinetics versus IAsp regardless if based on free or total IAsp assay. Higher anti-IAsp antibodies increase total IAsp concentrations but do not influence faster aspart nor IAsp pharmacodynamics. ClinicalTrials.gov identifiers NCT01618188, NCT02003677, NCT01934712, NCT02568280, NCT01831765. Electronic supplementary material The online version of this article (10.1007/s40262-018-0718-6) contains supplementary material, which is available to authorized users.

BackgroundHuman urine is now recognised as a non-invasive source of stem cells with regeneration potential. These cells are mesenchymal stem cells but their detailed molecular and cellular identities are poorly defined. Furthermore, unlike the mouse, the gene regulatory network driving self-renewal and differentiation into functional renal cellsin vitroremain unresolved.MethodsWe isolated urine stem cells from 10 individuals from both genders and distinct ages, characterized them as renal progenitor cells and explored the gene regulatory network sustaining self-renewal.ResultsThese cells express pluripotency-associated proteins-TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133. Expression of pluripotency-associated proteins enabled rapid reprogramming into iPSCs using episomal-based plasmids without pathway perturbations. Transcriptome analysis revealed expression of a plethora of nephrogenesis-related genes such asSIX2, OSR1, CITED1, NPHS2, NPHS1, PAX2, SALL1, AQP2, EYA1, SLC12A1andUMOD. As expected, the cells transport Albumin by endocytosis. Based on this, we refer to these cells as urine derived renal progenitor cells-UdRPCs. Associated GO-term analysis of UdRPCs and UdRPC-iPSCs underlined their renal identity and functionality. Upon differentiation by WNT activation using the GSK3β-inhibitor (CHIR99021), transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes-AXIN2, JUN and NKD1.Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative regulator of self-renewal and differentiation in UdRPCs. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3 pathway.ConclusionThisin vitromodel and the data presented should lay the foundation for studying nephrogenesis in man.Significance StatementHuman urine is a non-invasive source of stem cells with regeneration potential. Here, we investigated the cellular and molecular identities, and the gene regulation driving self-renewal and differentiation of these cellsin vitro. These cells express pluripotency-associated markers enabling easy reprogramming. Based on the expression of renal associated genes, proteins and functionality, we refer to these cells as urine derived renal progenitor cells-UdRPCs. CHIR99021-induced differentiation of UdRPCs activated WNT-related genes-AXIN2, JUN and NKD1.Protein interaction network identified JUN as a putative regulator of differentiation whereas self-renewal is maintained by FGF2-driven TGFβ-SMAD2/3. Our data will enhance understanding of the molecular identities of UdRPCs, and enable the generation of renal disease modelsin vitroand eventually kidney-associated regenerative therapies.

Objective The aim of this study was to characterise the population pharmacokinetics of FE 999049, a novel recombinant human follicle-stimulating hormone (FSH), after multiple dosing in healthy women, taking into account endogenous FSH levels and the reproductive hormone dynamics. Methods Longitudinal measurements of FSH, luteinising hormone, progesterone, estradiol, and inhibin B levels were collected after repeated subcutaneous dosing with 225 IU of FE 999049 in 24 gonadotropin downregulated healthy women. The FSH data were described using nonlinear mixed-effects modelling. Results The measured FSH levels were modelled as a sum of endogenous FSH and FE 999049. The FE 999049 population pharmacokinetics were best described using a one-compartment model with first-order absorption and elimination, and a transit model for delayed absorption. The apparent clearance and volume of distribution increased with body weight in accordance with an allometrically scaled power exponent of 0.75 and 1, respectively. Endogenous FSH levels were lower in individuals with higher progesterone levels at baseline and were further suppressed over time with increasing inhibin B levels. Conclusions This characterisation of FE 999049 population pharmacokinetics after repeated dosing is in line with previous findings after single-dose administration. The results provide a basis for study design and data evaluation in the future development of recombinant FSH products, and show it can be of importance to account for endogenous FSH levels and its variation over time for accurate estimation of exogenously administered FSH pharmacokinetic parameters. Thus, correcting FSH concentrations by the observed endogenous FSH baseline value at all time points may be incorrect.

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Aging associated DNA hypomethylation of LINE-1 and Alu retroelements may be a crucial determinant of loss of genomic integrity, deterioration and cancer. In peripheral blood LINE-1 hypomethylation has been reported to increase during aging, but other studies did not observe significant changes. We hypothesized that these apparently inconsistent reports might relate to differences between cellular and cell-free DNA. Using the technique of idiolocal normalization of real-time methylation-specific PCR (IDLN-MSP) for genetic imbalanced DNA specimens we obtained evidence that LINE-1 hypomethylation in cell-free DNA, but not cellular DNA from peripheral blood is an epigenetic biomarker for human aging. Furthermore, hypomethylation of cell-free DNA is more extensive in smokers, suggesting that it might be used as a surrogate marker for monitoring the improvement of smoking-induced adverse effects after cancelling smoking. Keywords: Epigenetics, DNA methylation, Cell-free DNA, Retroelements, LINE-1

This study aimed to estimate the relationship between pharmacokinetics and the antidiuretic effect of desmopressin. In the investigator-blind, randomized, parallel group study, 5 dose groups and 1 placebo group, each consisting of 12 healthy, overhydrated, nonsmoking male subjects 18–55 yr of age were infused intravenously over 2 h with placebo or 30, 60, 125, 250, and 500 ng desmopressin in 50 ml of normal saline. Plasma desmopressin and urine osmolality rose by variable amounts during the infusions of 60, 125, 250, and 500 ng desmopressin. Plotting mean urine osmolality against the concurrent mean plasma desmopressin yielded a temporal delay between pharmacokinetic (PK) and -dynamic (PD) responses in all dose groups. Using simulation from the indirect-response model, assuming a constant (4 ng/ml) desmopressin concentration, this delay between PK and PD was estimated at 4 h (10th-90th percentile: 1.8–8.1). Within each group, however, there were large individual variations (2- to 10-fold) in the magnitude and duration of the antidiuretic effect. The antidiuretic effect of intravenous desmopressin in water-loaded healthy adults varies considerably due largely to factors other than individual differences in pharmacokinetics. The antidiuretic effect is time as well as dose dependent and may be self-amplifying. The most likely explanation for these findings is that the time required for a given level of plasma desmopressin to exert its maximum antidiuretic effect varies markedly from person to person due to individual differences in the kinetics of one or more of the intracellular mechanisms that promote the reabsorption of solute-free water by principal cells in renal collecting tubules.

In order to investigate whether high-protein medium-fat diets (HP) with larger relative content of high-status foods such as meat are more socially and culturally acceptable than high-carbohydrate medium-fat diets (HC) a survey was conducted among participants (overweight men and women 18–56 y) in a strictly controlled dietary intervention in Denmark. Questionnaires were distributed at baseline, and at six and 12 months after start of intervention trial. Health and well-being improved during trial independent of diet group. However, in both groups, participants missed foods which were restricted in their own diet (carbohydrate-rich foods and protein-rich foods respectively) whereas difference in satisfaction with amount of fatty foods was not significant. The study suggests that in spite of higher amount of high-status foods, high-protein medium-fat diets are no more acceptable than high-carbohydrate medium-fat diets. Both deviate from cultural norms for meal formats and dietary composition and may therefore be difficult to comply with in a long-term perspective. Health programmes promoting dietary change must address cultural norms about meal structures.

The pharmacokinetics of a new selective oestrogen receptor modulator levormeloxifene was investigated in mice, rats, cynomolgus monkeys and humans by compartmental pharmacokinetics. Levormeloxifene was administered as an oral solution in all studies. Allometric scaling was used to predict human pharmacokinetic parameters and the performance of the approach was evaluated. Mean values of clearance confounded by F(CL/F) were 0.073, 0.29, 3.18 and 2.4 l/h in mice, rats, monkeys and humans, respectively. Values of distribution volume at steady state confounded by F(Vss/F) were 0.073 and 7.5 l in mice and rats. In monkeys, values of the central volume F(Vc/F) and volume at steady state F(Vss/F) were 28.9 and 57.9 l, respectively. In humans, values of Vc/F and Vss/F were 106 and 587 l, respectively. Predicted CL/F and Vss/F showed a linear relationship when plotted vs BW on a log–log scale; for CL/F, r was 0.95–0.98 and for Vss/F, r was 0.99. Using allometric scaling the predicted human Vss/F deviated 3-fold from the experimentally determined values. Observed values of CL/F deviated 21–25 fold from the predicted, the latter depending on the scaling method. Confidence intervals for the predicted parameters showed major lack of precision for all the allometric scaling methods. Copyright © 2003 John Wiley & Sons, Ltd.

The paper will provide an overview of the problem of heterogeneity in consumer data and various ways of coping with it analytically. It will present a new model that combines latent class regression analysis with random coefficient regression models together with principal components regression. Finally, the model approach is illustrated, using a data set on consumer evaluations of frozen peas.

Increased age and female gender are well-known risk factors for the development of desmopressin-induced hyponatremia. However, little focus has been on exploring gender differences in the antidiuretic response to desmopressin. Based on an exploratory analysis from three clinical trials, we report a significant gender difference in the effects of desmopressin on nocturnal urine volume that could not be explained by pharmacokinetic differences. Mean desmopressin concentration profiles were tested for covariates, and age and gender were not statistically significant and only weight was significant for log(Cmax) ( P = 0.0183) and borderline significant for log(AUC) ( P = 0.0571). The decrease in nocturnal urine volume in nocturia patients treated with desmopressin over 28 days was significantly larger for women at the lower desmopressin melt doses of 10 and 25 μg than for men. The ED50 for men was modeled to be 43.2 μg and 16.1 μg for women, with the ED50 men/women estimated to be 2.7 (1.3–8.1 95% CI), corresponding to significantly higher sensitivity to desmopressin in women. An increasing incidence of hyponatremia with increasing dose was found, and at the highest dose level of 100 μg decreases in serum sodium were approximately twofold greater in women over 50 yr of age than in men. A new dose recommendation stratified by gender is suggested in the treatment of nocturia: for men, 50- to 100-μg melt is an efficacious and safe dose, while for women a dose of 25 μg melt is recommended as efficacious with no observed incidences of hyponatremia. Areas for further research are proposed to uncover pathophysiological mechanism(s) behind these gender differences.

Population approaches are more robust estimators of insulin sensitivity (SI) and glucose effectiveness (SG) with the minimal model of glucose kinetics during an intravenous glucose tolerance test (IVGTT). We assessed the performance of 3 population methods, iterative two-stage (ITS), Bayesian hierarchical Markov chain Monte Carlo (MCMC), and NONMEM first-order conditional estimation (FOCE) with interaction (NM), and made a comparison with the standard two-stage method (STS) employing the weighted nonlinear regression analysis. To evaluate accuracy of individual and population estimates, 40 simulated insulin-modified frequently sampled IVGTTs (IM-FSIVGTT) were derived from real IM-FSIVGTTs (0.3 g glucose per kg body weight with 0.02 U/kg insulin at 20 minutes; 30 samples over 180 minutes) performed in 40 healthy Caucasian subjects (male/female, 22/18; age, 46 +/- 9 years; body mass index [BMI], 26.7 +/- 5.7 kg. m(-2); mean +/- SD). The population methods assumed a log-normal population distribution of parameters. All methods gave a similar but overestimated population SG by 9% to 13%. Population SI was underestimated to a different degree by the methods (STS 6%, ITS 10%, MCMC 13%, and NM 7%). The between-subject variability of SG was overestimated by STS and underestimated by the population methods (true 33%, STS 40%, ITS 19%, MCMC 24%, NM 24%; coefficient of variation). For SI, this quantity was well estimated by all methods (true 79%, STS 80%, ITS 82%, MCMC 83%, NM 82%). The results for individual estimates indicate that STS performs better than the population methods when estimating SI (STS 12%, ITS 16%, MCMC 16%, NM 16%; 1 outlying subject excluded; root mean squared error expressed as percent of mean) but worse for SG (STS 28%, ITS 21%, MCMC 20%, NM 19%). We conclude that the robust performance of population approaches, preventing parameter estimation failures associated with the nonlinear regression analysis, is not required with IM-FSIVGTT in subjects with normal glucose tolerance. The standard two-stage technique is the preferred method under such circumstances.

The preferred approach to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of insulin analogues is the euglycemic glucose clamp. Currently non-compartmental data analytical approaches are used to analyze data. The purpose of the present study is to propose a novel compartmental-model for analysis of data from glucose clamp studies. Data used in this trial only involved 18 of the 20 originally treated subjects. Data was obtained from a crossover trial where 18 healthy subjects each received a single subcutaneous (s.c.) dose of 1.2 nmol/kg (body weight) insulin aspart (IAsp) or 1.2 nmol/kg human insulin (HI) during a euglycemic glucose clamp after overnight fast. Serum insulin and glucose concentrations were measured and the glucose infusion rate (GIR) was adjusted after dosing, to maintain blood glucose near basal levels. Individual model parameters were estimated for IAsp, HI, and the corresponding glucose and GIR data. We found statistically significant differences between most of the HI and IAsp pharmacokinetic parameters, including the sigmoidicity of the time course of absorption (1.5 for HI vs. 2.1 for IAsp (unit less), P = 0.0005, Wilcoxon Signed-rank test), elimination rate constant (0.010 min-1 for HI vs. 0.016 min-1 for IAsp (P = 0.002)). The PD model parameters were mostly not different, except for the rate of insulin action (0.012 min-1 for HI vs. 0.017 min-1 for IAsp (P = 0.03)). The model may provide a framework to account for different PK properties when estimating the PD properties of insulin and insulin analogues in glucose clamp experiments.

Objective The purpose of this analysis was to develop a population pharmacokinetic model for a novel recombinant human follicle-stimulating hormone (FSH) (FE 999049) expressed from a human cell line of foetal retinal origin (PER.C6®) developed for controlled ovarian stimulation prior to assisted reproductive technologies. Methods Serum FSH levels were measured following a single subcutaneous FE 999049 injection of 37.5, 75, 150, 225 or 450 IU in 27 pituitary-suppressed healthy female subjects participating in this first-in-human single ascending dose trial. Data was analysed by nonlinear mixed effects population pharmacokinetic modelling in NONMEM 7.2.0. Results A one-compartment model with first-order absorption and elimination rates was found to best describe the data. A transit model was introduced to describe a delay in the absorption process. The apparent clearance (CL/F) and apparent volume of distribution (V/F) estimates were found to increase with body weight. Body weight was included as an allometrically scaled covariate with a power exponent of 0.75 for CL/F and 1 for V/F. Conclusions The single-dose pharmacokinetics of FE 999049 were adequately described by a population pharmacokinetic model. The average drug concentration at steady state is expected to be reduced with increasing body weight.