In this extensive study, real-time reverse transcriptase-polymerase chain reaction was used to analyze the expression levels of all 19 Wnt genes and their 11 potential antagonists in mouse blastocysts, pregastrula, gastrula, and neurula stages. By complementing these results with in situ hybridization, we revealed new expression domains for Wnt2b and Sfrp1, respectively, in the future primitive streak at the posterior side and in the anterior visceral endoderm before the initiation of gastrulation. Moreover, the anterior visceral endoderm expresses three secreted Wnt antagonists (Sfrp1, Sfrp5, and Dkk1) in partially overlapping domains. We also identified expression patterns for the Wnt1, Wnt3a, Wnt6, Wnt7b, Wnt9a, Wnt10b, and Sfrp1 genes at the blastocyst stage. In particular, the expression of Wnt1 and Sfrp1 predominantly in the inner cell mass and of Wnt9a in the mural trophoblast and inner cell mass cells surrounding the blastocoele suggests new roles for the Wnt pathway in preimplantation development. This article is the first report on the regional expression of Wnt genes in the mouse blastocyst.
Aims/hypothesisIslet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.MethodsHuman islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1−/− mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.ResultsCFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).Conclusions/interpretationThe ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.Electronic supplementary materialThe online version of this article (10.1007/s00125-018-4672-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Aims/hypothesis We investigated whether heterogeneous nuclear ribonucleoproteins F and K (hnRNP F, hnRNP K) mediate insulin inhibition of renal Agt expression and prevention of hypertension and kidney injury in an Akita mouse model of type 1 diabetes. Methods Adult male Akita mice (12 weeks old) were treated with insulin implants and killed at week 16. Untreated nonAkita littermates served as controls. The effects of insulin on blood glucose, systolic BP (SBP), renal proximal tubular cell (RPTC) gene expression and interstitial fibrosis were studied. We also examined immortalised rat RPTCs stably transfected with control plasmid or with plasmid containing rat Agt promoter in vitro.Results Insulin treatment normalised blood glucose levels and SBP, inhibited renal AGT expression but enhanced hnRNP F, hnRNP K and angiotensin-converting enzyme-2 expression, attenuated renal hypertrophy and glomerular hyperfiltration and decreased urinary albumin/creatinine ratio, as well as AGT and angiotensin II levels, in Akita mice. In vitro, insulin inhibited Agt but stimulated Hnrnpf and Hnrnpk expression in high-glucose media via p44/42 mitogen-activated protein kinase signalling in RPTCs. Transfection with Hnrnpf or Hnrnpk small interfering RNAs prevented insulin inhibition of Agt expression in RPTCs. Conclusions/interpretation These data indicate that insulin prevents hypertension and attenuates kidney injury, at least in part, through suppressing renal Agt transcription via upregulation of hnRNP F and hnRNP K expression in diabetic Akita mice. HnRNP F and hnRNP K may be potential targets in the treatment of hypertension and kidney injury in diabetes.
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