Journal: International Journal of Molecular Sciences

Article Title: Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics

doi: 10.3390/ijms23031428

Figure Lengend Snippet: NKCC-1 transporter expression in renal lymphatic vessels and vessels with PAN proteinuric injury. ( A ) Immunostaining of afferent renal lymphatic vessels demonstrated NKCC1 transporter expression, particularly prominent in lymphatic endothelial cells. ( B ) NKCC1 mRNA levels in extra-renal lymphatic vessels were significantly greater in PAN-injured rats vs uninjured controls. Results are mean ± SEM for 7 rats per group analyzed by unpaired t test * p < 0.05.

Article Snippet: After blocking with 2.5% normal horse serum, the tissue was incubated with anti-NKCC1 antibody (Boster Bio, 1:1000, Pleasanton, CA, USA) overnight and then incubated with anti-rabbit secondary antibody.

Techniques: Expressing, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics

doi: 10.3390/ijms23031428

Figure Lengend Snippet: High Na + environment regulated NKCC-1 signaling pathway in lymphatic endothelial cells (LECs). ( A ) Cultured LECs exposed to a high-sodium environment showed greater expression of NKCC1 mRNA, ( B ) while expression of phosphorated NKCC-1 protein decreased. ( C ) High-sodium environment decreased protein expression of SPAK, an upstream activating kinase of NKCC1. Experiments were performed independently 3 times using 3 wells per treatment and analyzed by ANOVA followed by Dunnett multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with 2.5% normal horse serum, the tissue was incubated with anti-NKCC1 antibody (Boster Bio, 1:1000, Pleasanton, CA, USA) overnight and then incubated with anti-rabbit secondary antibody.

Techniques: Cell Culture, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Claudin-18 Loss Alters Transcellular Chloride Flux but not Tight Junction Ion Selectivity in Gastric Epithelial Cells

doi: 10.1016/j.jcmgh.2020.10.005

Figure Lengend Snippet: Loss of Cldn18 results in dysregulation of gastric mucosal chloride transporter expression . ( A ) mRNA expression for gastric mucosal chloride transporters that ( A ) normally display a specific basolateral or apical distribution pattern and ( B ) significantly differed (Log2, P < .0001) between tissues from wild-type and Cldn18- KO mice in the RNASeq analysis. Normalized counts data were determined by using Kruskal-Wallis test with post hoc comparisons performed using Dunn’s method and are plotted as means ± SE from n = 3 (3 M) wild-type and n = 3 (3 M) Cldn18 -KO mice. ( C ) Confocal microscopy analysis of NKCC1 expression and localization in tissues from Cldn18 -KO mice (n = 4; 2 M/2 F) shows strong membrane localization in intestinalized SPEM cells (iSPEM) at base of gastric glands, a position that was shown to express Cftr. Inset ( i ) is higher magnification image of the gland base to better show NKCC1 localization. HOE, Hoechst 33342 to identify nuclei; PC, parietal cells. Scale bars : 50 μm (low mag); 20 μm ( inset i ). ( D ) Confocal microscopy analysis of NKCC1 expression and localization in tissues from wild-type mice (n = 4; 2 M/2 F) shows strong membrane localization along the basolateral membrane of PC with no membrane localization in chief cells (CC) at the base of gastric glands. Scale bar : 50 μm. ( E ) mRNA expression for effectors that regulate NKCC1 in tissues from wild-type and Cldn18- KO mice. P values represent the Log2-fold change in expression between wild-type and Cldn18- KO mice. ( F ) Confocal microscopy analysis of NaK-ATPase expression and localization in tissues from Cldn18 -KO mice (n = 4; 2 M/2 F) shows strong membrane localization in iSPEM at the base of gastric glands. Inset ( i ) is higher magnification image of PC, demonstrating thickened signal along the basolateral membrane ( arrows ). At the gland base, NaK-ATPase localizes in a homogeneous manner to the basolateral membrane of iSPEM cells ( arrows ). Scale bars : 50 μm (low mag); 10 μm ( insets i and ii ). ( G ) Confocal microscopy analysis of NaK-ATPase expression and localization in tissues from wild-type mice (n = 4; 2 M/2 F) shows strong membrane localization in all cells in the gastric corpus mucosa. Inset ( i ) is higher magnification image of PC and neck mucous cells (MNC), demonstrating a beaded localization of signal along the basolateral membrane ( arrows ). In contrast, NaK-ATPase localizes in a homogeneous manner to the basolateral membrane of CCs ( arrows ). Note that in CCs, NaK-ATPase does not localize to the basal membrane ( asterisks ). Scale bars : 50 μm (low mag); 10 μm ( insets i and ii ).

Article Snippet: The same strategy was used for paraffin sections from male (n = 2) or female (n = 2) wild-type or Cldn18 -KO mice stained with anti-NKCC1 (LSBio, Seattle, WA; LS-C313276-100) or anti-NaK-ATPase (Abcam, Cambridge, UK; ab76020 [EP1845Y]).

Techniques: Expressing, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

doi: 10.3389/fimmu.2018.02049

Figure Lengend Snippet: Expressions of p-NKCC1 in rat lung tissues and MLE-12 cells. (A) Representative images of p-NKCC1 immunofluorescence staining (FITC-labeled green; original magnification ×400) of rat lung. Nuclei were counterstained with DAPI (blue). (B) T-NKCC1 and p-NKCC1 expressions in MLE-12 cells determined by western blot analysis ( n = 5 per group). (C) Histogram of p-NKCC1 determined by flow cytometry in MLE-12 cells ( n = 3 per group). BMT attenuated the activation of epithelial p-NKCC1 in IR-ALI. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD. * P < 0.05 compared with the control group; # P < 0.05 compared with the HR group.

Article Snippet: The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.

Techniques: Immunofluorescence, Staining, Labeling, Western Blot, Flow Cytometry, Activation Assay, Control

Journal: Frontiers in Immunology

Article Title: Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

doi: 10.3389/fimmu.2018.02049

Figure Lengend Snippet: The mechanisms of NKCC1 modulating alveolar fluid clearance and inflammation in IR-ALI. IR stress causes phosphorylation of NKCC1 and activation of TRAF6, which result in cell swelling and inflammation of alveolar epithelium. Inhibition of NKCC1 by bumetanide reciprocally modulates epithelial TRAF6 expression. This interaction suppresses downstream p38 MAPK and NF-κB pathways, which attenuates the reduction of AFC via upregulating α-ENaC expression and reduces the alveolar inflammation.

Article Snippet: The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Expressing