GRP78 contributes to the beneficial effects of SGLT2 inhibitor on proximal tubular cells in DKD

Beneficial effects of SGLT2 inhibitors on kidney function are well-known; however, their molecular mechanisms are not fully understood. We focused on 78 kDa glucose-regulated protein (GRP78) and its interaction with SGLT2 and Integrin β1 beyond the chaperone property of GRP78. In STZ-induced diabetic mouse kidneys, GRP78, SGLT2, and Integrin β1 increased in the plasma membrane fraction, while they were suppressed by canagliflozin. The altered subcellular localization of GRP78/Integrin β1 in STZ mice promoted epithelial mesenchymal transition (EMT) and fibrosis, which were mitigated by canagliflozin. High glucose conditions reduced intracellular GRP78, increased its secretion, and caused EMT-like changes in cultured HK2 cells, which were again inhibited by canagliflozin. Urinary GRP78 increased in STZ mice, and in vitro experiments with recombinant GRP78 suggested that inflammation spread to surrounding tubular cells and canagliflozin reversed this effect. Under normal glucose culture, canagliflozin maintained SERCA activity, promoted endoplasmic reticulum (ER) robustness, reduced ER stress response impairment, and protected proximal tubular cells. In conclusion, canagliflozin restored subcellular localization of GRP78, SGLT2 and Integrin β1 and inhibited EMT and fibrosis in DKD. In non-diabetic CKD, canagliflozin promoted ER robustness by maintaining SERCA activity and preventing ER stress response failure, and contribute to tubular protection.</p

Chemically Activatable Alkyne Tag for Low pH-Enhanced Molecular Labeling on Living Cells

Stimuli-responsive “activatable” reactive tags are applicable to selective labeling of biomolecules in a defined area or environment in living systems, yielding new insights into cellular processes through molecular imaging and fishing. Here, we developed a chemically activatable alkyne tag that can be incorporated into biological molecules and labeled with azide-tagged functional molecules through the alkyne–azide cycloaddition “click” reaction after chemical activation. Formation of the alkyne tag from the precursor moiety was confirmed to proceed in physiological aqueous media and was particularly enhanced under mildly acidic pH. The tag was successfully applied to low-pH sensitive labeling of a cholesterol analogue with azide-tagged biotin on living mammalian cells. Our results provided proof of principle that the present activatable alkyne tag can be used as a tool to selectively analyze molecules of interest in low-pH regions in living systems

Domain scores of the website quality measurements (n = 83).

Domain scores of the website quality measurements (n = 83).</p

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

<div><p>The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues <i>in vitro</i>. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated <i>in vitro</i> by 3D MSC constructs incorporated with HUVECs.</p></div

genotype

This file contains the genotype data of 788 tea accessions used in this study

<i>In silico</i> analyses of the reticulate process of HUVECs.

<p>The HUVEC model (red polygons) was set in those of the hMSCs models (blue polygons) at 0 MCS. In absence of VEGF, HUVECs proliferated and colonized near their initial position (A). By contrast, in the presence of VEGF, HUVECs responded and spread across a wide area with the reticulate structure (B).</p

Illustration of reference frames and ground reaction forces in the axial plane.

<p>White arrows indicate ground reaction force in the flat insole condition, and black arrows indicate ground reaction force in the laterally wedged insole condition. TF, tibial frame; FF, femoral frame. When the reference frames are aligned parallel in the axial plane (a), the lever arm of the ground reaction force is smaller in the laterally wedged insole condition than in the flat insole condition for both TF and FF. When the reference frames are rotated with each other (e.g. external rotation of the knee)(b), the lever arm in the laterally wedged insole condition is the smaller than in the flat insole condition for TF, while the lever arm in the laterally wedged insole condition is larger than in the flat insole condition for FF.</p

Flow chart of website selection.

The top square indicates the initial website selection. The lower squares show the excluded websites and the numbers for analysis.</p

Immunofluorescence stained images of hMSC/HUVEC 3D constructs for angiogenic factors.

<p>(A–D) VEGF and (E–H) HGF were diffused within each cell construct. White dotted lines indicate outline of cell construct.</p

Self-organization of mineralized matrices in the 3D cell constructs after 50 days of culture.

<p>(A) Mineral deposition (M) was generated in the cytoplasm in the 100:0 cell constructs. (B) Mineral deposition resulted in a cancellous bone-like porous structure in the 95:5 cell constructs.</p