The Localization of R-Spondin1 and R-Spondin 3 Peptides in Rat Hypothalamus: An Immunohistochemical Study / Localización de los Péptidos R-Spondin1 y R-Spondin3 en el Hipotálamo de Rata: Un Estudio Inmunohistoquímico

SUMMARY: The R-spondin protein family is a group of proteins that enhance Wnt/b-catenin signaling and have pleiotropic functions in stem cell growth and development. In the literature reviews, there is no histomorphological study showing the localization and distribution of R-spondins in different hypothalamic nuclei. For this reason, the purpose of this study was to determine the localization, distribution characteristics, and densities in the hypothalamic nuclei of neurons expressing Rspo1 and Rspo3 proteins. The free-floating brain sections of the male rats who were not exposed to any treatment were stained with the indirect immunoperoxidase method using the relevant antibodies. As a result of the immunohistochemical studies, it was determined that neurons expressing the Rspo1 protein were found in large numbers in the supraoptic nucleus (SON), the suprachiasmatic nucleus (SCh), anterior paraventricular nucleus, periventricular hypothalamic nucleus (PeV), anterior hypothalamic area, magnocellular preoptic nucleus (MCPO) and the lateral hypothalamic area (LH) from the hypothalamic nuclei, while they were localized in fewer numbers in the arcuate nucleus (ARC). Rspo3 protein expression was found in neurons localized in the hypothalamic nuclei SON, paraventricular nucleus (PVN), PeV, ARC, ventromedial nucleus (VMH), LH, anterior parvicellular nucleus, and zona inserta (ZI). In addition, neurons synthesizing both peptides were found in the cortex and hippocampus regions (H). Rspo1 and 3 proteins are expressed in hypothalamic energy homeostatic areas, thus these proteins may be involved in the regulation of food intake.

La familia de proteínas R-espondina es un grupo de proteínas que mejoran la señalización de Wnt/b-catenina y tienen funciones pleiotrópicas en el crecimiento y desarrollo de las células madre. En las revisiones de la literatura no existen estudios histomorfológicos que muestren la localización y distribución de las R-espondinas en diferentes núcleos hipotalámicos. Por esta razón, el propósito de este estudio fue determinar la localización, características de distribución y densidades en los núcleos hipotalámicos de neuronas que expresan las proteínas Rspo1 y Rspo3. Secciones de cerebro flotantes de ratas macho que no fueron expuestas a ningún tratamiento se tiñeron con el método de inmunoperoxidasa indirecta utilizando los anticuerpos pertinentes. Como resultado de los estudios inmunohistoquímicos, se determinó que las neuronas que expresan la proteína Rspo1 se encontraron en gran número en el núcleo supraóptico (SON), el núcleo supraquiasmático (SCh), el núcleo paraventricular anterior, el núcleo hipotalámico periventricular (PeV), el núcleo hipotalámico anterior área, núcleo preóptico magnocelular (MCPO) y el área hipotalámica lateral (LH) de los núcleos hipotalámicos, mientras que se localizaron en menor número en el núcleo arqueado (ARC). La expresión de la proteína Rspo3 se encontró en neuronas localizadas en los núcleos hipotalámicos SON, núcleo paraventricular (PVN), PeV, ARC, núcleo ventromedial (VMH), LH, núcleo parvicelular anterior y zona inserta (ZI). Además, se encontraron neuronas que sintetizan ambos péptidos en las regiones de la corteza y el hipocampo (H). Las proteínas Rspo1 y 3 se expresan en áreas homeostáticas de energía hipotalámicas, por lo que estas proteínas pueden estar involucradas en la regulación de la ingesta de alimentos.

Clinical Significance of Thrombospondin Type 1 Domain-Containing 7A and Neural Epidermal Growth Factor-Like 1 Protein in M-Type Phospholipase A2 Receptor-Negative Membranous Nephropathy / 中国医学科学院学报

Objective To investigate the clinical significance of thrombospondin type 1 domain-containing 7A (THSD7A) and neural epidermal growth factor-like 1 protein (NELL1) in phospholipase A2 receptor (PLA2R)-negative membranous nephropathy (MN). Methods A total of 116 PLA2R-negative MN patients treated in Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University from 2014 to 2021 were enrolled in this study.Immunohistochemistry was employed to detect THSD7A and NELL1 in the renal tissue.The pathological characteristics,treatment,and prognosis were compared between positive and negative groups. Results The 116 PLA2R-negative MN patients included 23 THSD7A-positive patients and 9 NELL1-positive patients.One patient was tested positive for both proteins.The THSD7A-positive group showed higher positive rate of IgG4 (P=0.010),more obvious glomerular basement membrane (GBM) thickening (P=0.034),and higher proportion of stage Ⅱ MN and lower proportion of stage I MN (P=0.002) than the THSD7A-negative group.The NELL1-positive group had lower positive rates of C1q and IgG2 (P=0.029,P=0.001),less obvious GBM thickening (P<0.001),more extensive inflammatory cell infiltration (P=0.033),lower proportion of deposits on multi-locations (P=0.001),and lower proportion of atypical MN (P=0.010) than the NELL1-negative group.One patient with THSD7A-positive MN was diagnosed with colon cancer,while none of the NELL1-positive patients had malignancy.Survival analysis suggested that THSD7A-positive MN had worse composite remission (either complete remission or partial remission) of nephrotic syndrome than the negative group (P=0.016),whereas NELL1-positive MN exhibited better composite remission of nephrotic syndrome than the negative group (P=0.015).The MN patients only positive for NELL1 showed better composite remission of nephrotic syndrome than the MN patients only positive for THSD7A (P<0.001). Conclusions THSD7A- and NELL1-positive MN is more likely to be primary MN,and there is no significant malignancy indication.However,it might have a predictive value for the prognosis of MN.

Rspo1 promotes osteoblast differentiation via Wnt signaling pathway.

R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo1 at transcript level during differentiating phase of human primary osteoblasts and human FOB1.19 cells. We also found that Rspo1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB1.19 cells. Similar additive role of Rspo1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo1 by transfected shRNA in hFOB1.19 cells. These results suggested the possibility of autocrine regulation by Rspo1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.