Activation of dopamine D2 receptor promotes pepsinogen secretion by suppressing somatostatin release from the mouse gastric mucosa

In vivo administration dopamine (DA) receptor (DR)-related drugs modulates gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D2 receptor (D2R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D2Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D2R-/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D1R, D2R, and D5R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D2R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D2R-/- mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D2R-/- mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D2R-mediated suppression of somatostatin release.

Physiological activation of the nephron central command drives endogenous kidney tissue regeneration

Tissue regeneration is limited in several organs including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest existing remodeling capacity. This study uncovered a novel endogenous tissue remodeling mechanism in the kidney that is activated by the loss of body fluid and salt and involves a unique niche of chief cells called macula densa (MD) that control resident progenitor cells via secreted angiogenic, growth and extracellular matrix remodeling factors, cytokines and chemokines. Serial intravital imaging, MD Wnt mouse models and transcriptome analysis provide functional and molecular characterization of this newly identified MD program for kidney regeneration complemented with human and therapeutic translation. The concept that chief cells responding to organ-specific physiological inputs control local progenitors and direct them to remodel or repair tissues may be applicable to other organs and diverse tissue regenerative therapeutic strategies.

Chief cell plasticity is the origin of metaplasia following acute injury in the stomach mucosa

ObjectiveMetaplasia arises from differentiated cell types in response to injury and is considered a precursor in many cancers. Heterogeneous cell lineages are present in the reparative metaplastic mucosa with response to injury, including foveolar cells, proliferating cells and spasmolytic polypeptide-expressing metaplasia (SPEM) cells, a key metaplastic cell population. Zymogen-secreting chief cells are long-lived cells in the stomach mucosa and have been considered the origin of SPEM cells; however, a conflicting paradigm has proposed isthmal progenitor cells as an origin for SPEM.DesignGastric intrinsic factor (GIF) is a stomach tissue-specific gene and exhibits protein expression unique to mature mouse chief cells. We generated a novel chief cell-specific driver mouse allele, GIF-rtTA. GIF-GFP reporter mice were used to validate specificity of GIF-rtTA driver in chief cells. GIF-Cre-RnTnG mice were used to perform lineage tracing during homoeostasis and acute metaplasia development. L635 treatment was used to induce acute mucosal injury and coimmunofluorescence staining was performed for various gastric lineage markers.ResultsWe demonstrated that mature chief cells, rather than isthmal progenitor cells, serve as the predominant origin of SPEM cells during the metaplastic process after acute mucosal injury. Furthermore, we observed long-term label-retaining chief cells at 1 year after the GFP labelling in chief cells. However, only a very small subset of the long-term label-retaining chief cells displayed the reprogramming ability in homoeostasis. In contrast, we identified chief cell-originating SPEM cells as contributing to lineages within foveolar cell hyperplasia in response to the acute mucosal injury.ConclusionOur study provides pivotal evidence for cell plasticity and lineage contributions from differentiated gastric chief cells during acute metaplasia development.

Mist1+ gastric isthmus stem cells are regulated by Wnt5a and expand in response to injury and inflammation in mice

Background and aimsThe gastric epithelium undergoes continuous turnover. Corpus epithelial stem cells located in the gastric isthmus serve as a source of tissue self-renewal. We recently identified the transcription factor Mist1 as a marker for this corpus stem cell population that can give rise to cancer. The aim here was to investigate the regulation of the Mist1+ stem cells in the response to gastric injury and inflammation.MethodsWe used Mist1CreERT;R26-Tdtomato mice in two models of injury and inflammation: the acetic acid-induced ulcer and infection with Helicobacter felis. We analysed lineage tracing at both early (7 to 30 days) and late (30 to 90 days) time points. Mist1CreERT;R26-Tdtomato;Lgr5DTR-eGFP mice were used to ablate the corpus basal Lgr5+ cell population. Constitutional and conditional Wnt5a knockout mice were used to investigate the role of Wnt5a in wound repair and lineage tracing from the Mist1+ stem cells.ResultsIn both models of gastric injury, Mist1+ isthmus stem cells more rapidly proliferate and trace entire gastric glands compared with the normal state. In regenerating tissue, the number of traced gastric chief cells was significantly reduced, and ablation of Lgr5+ chief cells did not affect Mist1-derived lineage tracing and tissue regeneration. Genetic deletion of Wnt5a impaired proliferation in the gastric isthmus and lineage tracing from Mist1+ stem cells. Similarly, depletion of innate lymphoid cells, the main source of Wnt5a, also resulted in reduced proliferation and Mist1+ isthmus cell tracing.ConclusionGastric Mist1+ isthmus cells are the main supplier of regenerated glands and are activated in part through Wnt5a pathway.

P0882STUDY ON MECHANISM OF PTH SECRETION IN SECONDARY HYPERPARATHYROIDISM BY OPTOGENECTICS

Background and Aims Hyperparathyroidism (SHPT) which caused by disorders of PTH metabolism is one of the most common complications in CKD patients. In the progression of SHPT, expression of CaSR in the parathyroid gland decreases, which impairs the regulation of PTH secretion. It is technically challenging to precisely manipulate PTH secretion in hyperplastic human parathyroid tissue. As an emerging technology, optogenetics allows the reversible control of neuronal activity and cellular precesses using lights. In this study, we establish an optogenetic approach that bypasses CaSR to regulate the level of PTH secretion in human parathyroid cells. It is hoped to provide a new idea for the prevention and treatment of SHPT. Method First, we isolated and identified human parathyroid gland by SHPT patients. The primary cell culture of chief cells were performed and identification. After that, the cells were transfected with the lentivirus carrying ChETA-eYFP or eYFP gene. Lights stimulation of the cultured human parathyroid cells was performed after ChETA expression. The PTH level was measured by ELISA before and after stimulation. Meanwhile, Fura-2/AM was loaded to normal or ChETA expressing chief cells for calcium imaging by microscope. Results The parathyroid glands of three SHPT patients were obtained after total parathyroidectomy (Fig.1A). Hematoxylin & Eosin (HE) staining was performed. The tissue showed typical histological characteristics of SHPT: asymmetric enlargement of the chief cells and the nodular distribution (Fig.1B). Parathyroid cells were cultured by primary culture method. By the 7th day of cultivation, the chief cells were grown in uniform size and aggregation state (Fig. 2A). The cells were stained with immunofluorescence, results showed that most of the isolated cells expressed parathyroid hormone (PTH), calcium-sensing receptors (CaSRs) and Vitamin D receptors (VDR) (Fig. 2B). We used lentivirus carrying the CMV-ChETA-eYFP construct to transfect cultured human parathyroid cells. Comparing with eYFP, most of the chief cells were labeled and expressed green fluorescence successfully after 48 hours (Fig.3). We constructed a blue light-emitting diode to stimulated the transfected human parathyroid chief cells for 0.5h (20 Hz with stimulus intervals of 50 ms). Culture medium was collected before and after stimulation for 1h and 6h, and PTH was determined by ELISA. The results showed that there was no significant difference in PTH level between ChETA group and eYFP group before stimulation, however, PTH decreased significantly after 1 hour of stimulation in ChETA group, and gradually returned to the control group after 6 hours (Fig.4). After that, we used a calcium fluorescence assay to reveal light-induced changes of intracellular Ca2+. In ChETA group, blue light stimulation led to a significant increase in fluorescent signals, but a significant decrease when turned off, and the signal strength increased again when it was turned on in a rhythmic manner. But the fluorescent signal of eYFP group remained stable throughout the whole process (Fig.5). Conclusion We used optogenetics to bypass CaSR rhythmic suppression of PTH secretion in human parathyroid cells. It is hoped that this study will provide new strategies for the treatment and prevention of SHPT

Evaluation of the metabolism properties of choline kinase alpha in neoplasms of the parathyroid glands. A pilot study

BACKGROUND: Primary hyperparathyroidism (PHPT) is a widespread endocrine disease characterized by excessive production of parathyroid hormone (PTH) due to parathyroid gland hyperplasia (PGH) or tumor lesions (adenoma or cancer of the parathyroid gland (PG) in 80% and 15% of cases respectively). Choline kinase alpha (XK) overexpression is described in tumors of different localization, but there is no data on its expression in PG tumors. AIMS: To study the character of XK expression in PG neoplasms and its relationship with clinical, laboratory, and visualization characteristics (positron emission tomography combined with computed tomography (PET/CT) with 18Ffluorocholine (18FFC)). MATERIALS AND METHODS: The material for the study was based on tissue samples from 10 patients of 3470 years old (Me = 61.5; [48; 66]), with a laboratoryconfirmed diagnosis of PHT. An immunohistochemical study (IHC) was carried out on materials from 2 patients with hyperplasia of the main cells, from 5 patients with adenoma of PG, from 1 patient with atypical adenoma and 1 with carcinoma of PG; in 1 case the metastasis of cancer of the neck with lymph node was examined. RESULTS: The expression of XK is spotted in all types of PG cells (chief cells: active and inactive forms), transitional forms between the chief cells and oxyphil; oxyphil cells, but it was most intense in active chief cells. The expression of XK was observed in neoplasms of PG of various degrees of malignancy. In the most numerous group of PG formations with a favorable prognosis (11 samples from 7 patients), no statistically significant correlation (p 0.05) was obtained between the intensity expression of the XK, of the PTH and the proliferative activity index Ki67, the level of radiopharmaceutical accumulation in PET/CT with 18FFC (SUVmax) and laboratory data (PTH, Ca, Ca++). CONCLUSIONS: In the majority of investigated cases, moderate and intensive expression of the XK was detected in PG cells. A small amount of studied cases does not allow us to identify the connection between the intensity of XK expression and the malignant potential for the formation of PG.