Drug inducted nephrotoxicity is a major cause of withdrawal of Food and Drug Administration (FDA) approved drugs. Animal models and 2D cell culture approaches do not faithfully replicate the structure and drug response associated with human renal tissues. Therefore, the goal of this study is to develop 3D organoid-based models of the kidney to provide a physiologically relevant platform to screen drugs for potential nephrotoxicity. In this study, kidney organoids were created using a process that involves mesodermal induction, organoid , and kidney differentiation. Immunofluorescence imaging was used to profile and quantitatively assess the presence of cells associated with the kidney including endothelial cells and renal tubular cells. Multicellular kidney organoids were then exposed to increasing concentrations (25–800 µM) of four nephrotoxic agents of varying levels of toxicity: cisplatin, gentamicin, dexamethasone, and probenecid. Cisplatin and gentamicin represent high toxicity drugs whereas, dexamethasone and probenecid represent low toxicity drugs. Cellular injury was assessed via evaluation of cell death. Our results show that drug-treated organoids exhibit dose-dependent increases in cell death with significant cell death associated with high toxicity drugs. These findings provide preliminary support for kidney organoids as valuable platforms to study nephrotoxic injury. Further optimization of both organoid maturation and toxicity assays can potentially enhance the reliability and physiological relevance of kidney organoid-based in-vitro platforms for nephrotoxicity testing.
Introduction: The cross-talk between transforming growth factor-beta (TGF-β)2/3 and fibroblast growth factor (FGF)2 during secondary palatal fusion is not fully understood. This study aimed to elucidate the details of cross-talk signaling of TGF-β2/3 and FGF2 during secondary palatal fusion using the small interfering RNA (siRNA) technique. Methods: Secondary palates of embryonic day 13 ICR mice were harvested, and the palates were cultured in the medium containing control siRNA, siTGF-β2, siTGF-β3, and siFGF2 (single or double knockdown). The expression levels of TGF-β2, TGF-β3, FGF2, and downstream genes were observed by western blotting and real-time RT-PCR. Results: Transfection of siRNA targeting the target gene resulted in a statistically significant decrease in expression levels. When the expression of either TGF-β3 or FGF2 was suppressed, a tendency for the expression of the other to increase was observed. Although Smad2 phosphorylation was not affected by siFGF2 transfection, siFGF2 treatment reduced MAPK phosphorylation levels of ERK, p38, and JNK. Following siFGF2 treatment, TGF-β3 expression increased, and suppression of its expression by siTGF-β3 also significantly increased FGF2 expression levels. At E13 + 72 h of culture, the siFGF2 treatment resulted in incomplete palatal fusion. In the TGF-β2/FGF2 and TGF-β3/FGF2 double knockdown groups at the stage, fusion defects were observed, particularly in the anterior region of the secondary palate. In the double knockdown of TGF-β/FGF2, the downstream gene expressions were also reduced. Conclusion: The results suggests that the TGF-β3 and FGF2 genes interact as a result of crosstalk, complement each other, and influence signal transduction to downstream genes.
Background: Microparticle hydrogels, or microgels, have emerged as promising therapeutics for nervous system repair because of their many beneficial properties. These properties include porosity, which leads to nutrient and waste exchange, large surface area to volume ratio compared to bulk biomaterials for cellular attachment and growth, and tailorable drug release to act as drug delivery depots. The potential of microgels as therapeutics in the nervous system is demonstrated by the key research being conducted in the field of nervous system repair. Summary: In this review, we discuss nervous system injuries and diseases that could benefit clinically from microgel therapeutics. Models of the in vitro nervous system that incorporate microgels are described, and in vivo models of nervous system injury or disease that use microgels as therapeutics are presented. Key Messages: The studies reported here show the promise of microgels in developing in vitro models to study the nervous system and their use as therapeutics in nervous system repair. Microgels have been shown to improve outcomes in many different animal models of nervous system injury or disease. These findings demonstrate the potential of microgels in nervous system repair and provide the rationale to continue to study and develop microgels to improve outcomes in the nervous system.
INTRODUCTION:Human eyelid development begins during the embryonic period. Although the general outline of early eyelid development has been described, many structures and developmental phenomena remain insufficiently investigated or controversial mainly due to the scarcity of human fetal specimens. The purpose of this study is to clarify these unresolved issues and to determine the precise developmental timing of each eyelid structure. METHODS:Fifty-six embryos (Carnegie stage (CS) 18-23) and six fetuses (early 9th week of development) from the Kyoto Collection were included in this study. The anatomical structures of the eyelid were observed using serial histological sections and external photographs, and the findings were described according to each developmental stage. RESULTS:The medial and lateral canthi formed between CS19 and 21. Mesenchymal condensations corresponding to the prospective upper and lower eyelid retractor regions were first observed at CS18 and acquired a sheet-like configuration by the 9th week of development. Mesenchymal condensation at the future location of the medial palpebral ligament was first observed at CS20. The orbicularis oculi muscle primordium emerged at the 9th week. Eyelid fusion initiated on the nasal side at CS22. It then progressed from both the nasal and temporal sides towards the center, until completion at the 9th week. The lacrimal canaliculi first appeared at CS19. CONCLUSIONS:Using the rich specimens of the Kyoto collection, we newly identified the developmental timing of the prospective lower eyelid retractor complex and medial palpebral ligament regions, as well as the initiation of eyelid fusion on the nasal side. Furthermore, we established a detailed timeline of early human eyelid morphogenesis. Our findings may guide future research on human eyelid development, anatomy, and the mechanisms underlying congenital eyelid disorders.
PURPOSE:To propose that progressive neuronal and satellite glial cell death within the pterygopalatine ganglion (PPG) is an underrecognized upstream mechanism in chronic aqueous-deficient dry eye, with particular relevance to diabetes mellitus, and to examine whether this ganglionic lesion also unifies broader secretory and cerebrovascular abnormalities into a single "diabetic secretory denervation syndrome". METHODS:A narrative, hypothesis-driven synthesis was performed across ocular surface disease, autonomic neuroscience, diabetic neuropathy, regulated cell death, surgical denervation models, and cerebrovascular physiology. PubMed was searched using both "pterygopalatine ganglion" and "sphenopalatine ganglion" as synonyms, combined with terms for cell death, diabetes, vidian neurectomy, and cerebrovascular function. Reference lists were hand-searched. RESULTS:The PPG is the sole parasympathetic relay for lacrimal, nasal, palatal, and conjunctival secretion, and a major source of vasodilator drive to the anterior cerebral circulation. Surgical interruption of this pathway reliably causes aqueous-deficient dry eye. In streptozotocin-induced diabetic rats, nitrergic PPG neurons undergo TUNEL-confirmed apoptosis during an irreversible second phase of diabetic nitrergic neuropathy, driven by the synergistic action of advanced glycation end products and endogenous nitric oxide through a caspase-3-dependent mechanism. Additional cell-death modalities (necroptosis, pyroptosis, ferroptosis, and satellite glial cell apoptosis) are predicted by analogy with other autonomic ganglia under equivalent metabolic stress. Its degeneration is therefore predicted to cause coordinated multi-territory secretory failure rather than isolated dry eye. CONCLUSION:PPG neurodegeneration has been directly demonstrated in the diabetic rodent ganglion and mechanistically anchored in AGE-RAGE (advanced glycation end products and their receptor, RAGE)-mediated apoptosis. Extending this experimentally grounded finding, we hypothesise that these deficits (diabetic dry eye, impaired nasal mucociliary clearance, palatine xerostomia, goblet cell depletion, and reduced cerebral perfusion) may share a common upstream ganglionic lesion, a unifying proposal that remains to be tested directly. The hypothesis generates testable predictions and reframes therapy toward neuroprotection of the PPG.
