Journal: Cells

Article Title: Kidney-Specific Membrane-Bound Serine Proteases CAP1/Prss8 and CAP3/St14 Affect ENaC Subunit Abundances but Not Its Activity

doi: 10.3390/cells12192342

Figure Lengend Snippet: CAP3/St14 knockout mice showed altered protein abundancies for ENaC subunits, NCC, and NKCC2 and downregulation of CAP1/Prss8 upon Na + deprivation. ( A ) Representative Western blot analyses of CAP3, CAP1, αENaC, αENaC CL (cleaved), βENaC, γENaC, γENaC CL (cleaved), NCC, pNCC, NHE3, NKCC2, ROMK and BK on kidney lysates from controls (black circles, n = 6) and CAP3/St14 knockout (CAP3/St14 Pax8LC1 , white circles, n = 6). Kidney lysates of control and renal tubular-specific knockouts of αENaC, βENaC, and γENaC served as negative (-ctl) and positive (+ctl) controls, respectively. ( B ) Quantification of data. Results are presented as mean ± SEM. Data were analyzed using an unpaired two-tailed Welch’s t -test, and p values <0.05 were considered statistically significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used with dilution 1/1000: αENaC, βENaC, NCC, pNCC (T53), NHE3, NKCC2 [ , ], γENaC (StressMarq, Biosciences, Victoria, Canasa) [ , ], CAP3/St14 (R&D systems AF3946, Minneapolis, MN, USA), BK/Maxi-Ki, Kca1.1 (APC-151, Alomone labs, Jerusalem, Israel).

Techniques: Knock-Out, Western Blot, Control, Two Tailed Test

Journal: Cells

Article Title: Kidney-Specific Membrane-Bound Serine Proteases CAP1/Prss8 and CAP3/St14 Affect ENaC Subunit Abundances but Not Its Activity

doi: 10.3390/cells12192342

Figure Lengend Snippet: CAP1/Prss8; CAP3/St14 double knockout mice showed normalized ENaC subunit but still lowered pNCC and BK protein abundances under a low Na + diet. ( A ) Representative Western blot analysis of CAP3, CAP1, αENaC, αENaC CL (cleaved), βENaC, γENaC, γENaC CL (cleaved), NCC, pNCC, NHE3, NKCC2, ROMK, and BK on kidney lysates from controls (black squares, n = 5) and CAP1/Prss8; CAP3/St14 double knockout (CAP1/Prss8; CAP3/St14 Pax8LC1 , n = 7) mice. Kidney lysates of control and renal tubular-specific knockouts of αENaC, βENaC, and γENaC served as negative (−ctl) and positive (+ctl) controls, respectively. ( B ) Quantification of the data. Results are presented as mean ± SEM. Data were analyzed using an unpaired two-tailed Welch’s t -test. p values < 0.05 were considered statistically significant; * p < 0.05, *** p < 0.001.

Article Snippet: The following primary antibodies were used with dilution 1/1000: αENaC, βENaC, NCC, pNCC (T53), NHE3, NKCC2 [ , ], γENaC (StressMarq, Biosciences, Victoria, Canasa) [ , ], CAP3/St14 (R&D systems AF3946, Minneapolis, MN, USA), BK/Maxi-Ki, Kca1.1 (APC-151, Alomone labs, Jerusalem, Israel).

Techniques: Double Knockout, Western Blot, Control, Two Tailed Test

Journal: Kidney international

Article Title: Renal tubular dysfunction in patients with American cutaneous leishmaniasis.

doi: 10.1038/ki.2011.251

Figure Lengend Snippet: Figure 2 | Western blotting analysis in the urinary exosome fraction, normalized to urinary creatinine, in controls and in patients with ACL. Abundance of (a) AQP2 and (b) NKCC2. ACL, American cutaneous leishmaniasis; AQP2, aquaporin 2; NKCC2, Na-K-2Cl cotransporter.

Article Snippet: We obtained the peptide-derived polyclonal antibodies specific to AQP2, NKCC2, NHE3, and pendrin from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot

Journal: Cell reports

Article Title: Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

doi: 10.1016/j.celrep.2020.108616

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-NKCC2 , Cell Signaling Technologies , Cat# 38436, RRID:AB_2799134.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Chem

Article Title: Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

doi: 10.1016/j.chempr.2020.06.017

Figure Lengend Snippet: In Vitro Selection of the Selective NKCC1 Inhibitor ARN23746 as a Lead Compound (A) Schematic representation of the intervention point in bumetanide’s structure for synthesizing novel bumetanide analogs. (B) Quantification of the inhibitory activity of bumetanide (Bume) and bumetanide analogs (10, 100 μM) in NKCC1-(left) or NKCC2-(right) transfected HEK293 cells in the Cl − influx assay. Data are presented as a percentage of the respective control DMSO. Data represent mean ± standard error of the mean (SEM) from 3–4 independent experiments (Kruskal-Wallis one way ANOVA on Ranks, NKCC1 10 μM: H = 84.898, DF = 6, p < 0.001; NKCC1 100 μM: H = 86.799, DF=6, p < 0.001; NKCC2 10 μM: H = 40.700, DF = 6, p < 0.001; NKCC2 100 μM: H = 70.569, DF = 6, p < 0.001, Dunn’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (C) Representation of the ligand-based computational strategy to discover novel molecular scaffolds that inhibit NKCC1. The obtained bumetanide pharmacophore ( 1 ) consists of three H-bond acceptor (HBA) features (red spheres), three H-bond donor (HBD) interactions (blue spheres), one lipophilic feature (green sphere), and one stacking feature (brown sphere) anchored around the central aromatic core. Ligand disposition was then implemented by superimposing other known unspecific NKCC1 inhibitors ( 2 ), revealing shared features and different dihedral dispositions of substituent around the central aromatic core. This model was used as a search filter for the virtual screening ( 3 ) of our internal chemical collection (∼15,000 compounds). Results generated from in vitro testing of the 165 initial hits ( 4 ) were then used to retrain the model ( 5 ) and perform a second more specific screening of our chemical library and commercial chemical libraries (∼135,000 compounds). This iterative computational cycle led to hit compounds ( 6 ) ARN22393 and ARN22394. (D) Quantification of the inhibitory activity of the indicated compounds (10, 100 μM) in NKCC1-transfected HEK293 cells (Cl − influx assay). Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 3–4 independent experiments (Kruskal-Wallis one way ANOVA on Ranks, 10 μM: H = 37.119, DF = 3, p < 0.001; 100 μM: H = 33.724, DF = 3, p < 0.001, Dunn’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (E) Chemical structures of NKCC1 inhibitors with novel scaffold. (F) Left, example traces obtained in the Cl − influx assay on NKCC1-transfected HEK293 cells for each compound (100 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC1-mediated Cl − influx. Right, quantification of the NKCC1 inhibitory activity of the indicated compounds (10, 100 μM) in experiments such as those on the right. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 3–4 independent experiments (10 μM: one way ANOVA, F (4,84) = 33.048, p < 0.001, Dunnett's post hoc test, ∗p < 0.05, ∗∗∗p < 0.001; 100 μM: Kruskal-Wallis one way ANOVA on Ranks, H = 50.796, DF = 4, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001. (G) Left, example traces obtained in the Ca 2+ influx assay on 3DIV hippocampal mouse neurons for each tested compound (100 μM). The arrows indicates the addition of GABA (100 μM) and KCl (90 mM). Right, quantification of the effect of the indicated compounds (10, 100 μM) in the Ca 2+ influx assay on 3DIV neurons. Data are presented as a percentage of the control DMSO. Data represent mean ± SEM from three independent experiments. 10 μM: one-way ANOVA, F (5,161) = 77.184, p < 0.001, Dunnett's post hoc test ∗∗p < 0.01, ∗∗∗p < 0.001; 100 μM: Kruskal-Wallis One ANOVA on Ranks, H = 134.681, DF = 5, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001). (H) Amplitude change average and single cell data points of GABA-evoked currents obtained by voltage-clamp recordings of 12–20 DIV hippocampal mouse neurons before (gray example recordings in the inset above) and after (black example traces) bath application of the indicated drugs (10 μM). Data are presented as mean ± SEM (Paired t test, ∗p < 0.05, ∗∗p < 0.01). Scale bars: 250 pA, 250 ms. See also A–S1D; and .

Article Snippet: To assess NKCC1 and NKCC2 activity (Cl − influx assay), 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL Opti-MEM, 8 μg of DNA plasmid coding for NKCC1 (PRK-NKCC1 obtained from Medical Research Council and the University of Dundee), NKCC2 (OriGene plasmid #RC216145) subcloned in PRK5 plasmid, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl − -sensitive variant of the mbYFPQS (Addgene plasmid #80742), and 32 μL of Lipofectamin 2000.

Techniques: In Vitro, Selection, Activity Assay, Transfection, Generated

Journal: Chem

Article Title: Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

doi: 10.1016/j.chempr.2020.06.017

Figure Lengend Snippet: ARN23746 Restores [Cl − ] i in DS Neurons, Does Not Inhibit NKCC2 in HEK Cells and Shows Improved Brain Penetration In Vivo in Comparison to Bumetanide (A) Left, representative pseudo-color images (colored scale below) of the [Cl − ] i measured with the MQAE Cl − -sensitive dye, in WT and Ts65Dn hippocampal neurons at 15 DIV, after treatment with control DMSO (0.1%) and the indicated compounds (10 μM). Scale bar: 70 μm. Right, quantification of the indicated compounds (10 μM) in modulating [Cl − ] i in experiments such as those on the left. Data represent mean ± SEM from three independent experiments (two-way ANOVA, F interaction (2,30) = 3.815, p = 0.033, Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (B) Left, example traces obtained in the Cl − influx assay on NKCC2-transfected HEK293 cells for each compound (10 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC2-mediated Cl − influx. Right, quantification of the NKCC2 inhibitory activity in experiments such as those on the right. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from four independent experiments (Kruskal-Wallis one way ANOVA on Ranks, H = 16,962, DF = 2, p < 0.001, Dunn’s post hoc test, ∗∗∗p < 0.001). (C) Left, example traces obtained in the Tl influx assay on KCC2-transfected HEK293 cells for each compound (10 μM). The arrows indicate the additions of TlSO 4 and NaCl. Right, quantification of the KCC2 inhibitory activity in experiments such as those on the left. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from 4 independent experiments (one way ANOVA, F (2,51) = 10.676, p < 0.001, Dunnett's post hoc test ∗∗∗p < 0.001). (D) Quantification of the level of bumetanide and ARN23746 in the brain at diverse time points (5, 15, 30, 60, 120 min) after injection in C57BL/6N mice (BioLASCO Taiwan). (Two-way ANOVA, F treatment (1,50) = 6.510, p = 0.014, Tukey’s post hoc test, ∗p < 0.05.). (E) Quantification of the ratio between brain and plasma concentration of bumetanide and ARN2346 5 min after the injection (two-tailed t test, t = 7.915, p < 0.001). See also E–S1G.

Article Snippet: To assess NKCC1 and NKCC2 activity (Cl − influx assay), 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL Opti-MEM, 8 μg of DNA plasmid coding for NKCC1 (PRK-NKCC1 obtained from Medical Research Council and the University of Dundee), NKCC2 (OriGene plasmid #RC216145) subcloned in PRK5 plasmid, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl − -sensitive variant of the mbYFPQS (Addgene plasmid #80742), and 32 μL of Lipofectamin 2000.

Techniques: In Vivo, Transfection, Activity Assay, Injection, Concentration Assay, Two Tailed Test