Journal: EBioMedicine

Article Title: Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

doi: 10.1016/j.ebiom.2019.01.006

Figure Lengend Snippet: ENaC protein and mRNA expression in PCK rats fed diets with varying sodium content. (a) Western blotting showing expression levels of α-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). Each lane on the blot is one animal. (b) mRNA expression for α-, β-, and γ-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets. (c) Western blotting showing expression levels of AQP2 in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to β-actin expression for the same samples). (d) miRNAs found to be differentially expressed ( p < .05) in the cortical tissues of the PCK rats fed a SD, NS and HS diets. *, p < .05, ** p < .01. (e) Western blots demonstrating total ENaC subunit expression in cultured mCCD cells transfected with a control scrambled miR sequence or miR9a-5p, with summary densitometry graphs (normalized to β-Actin and relative to control mCCD expression). Both β- and γ-ENaC are significantly decreased in mCCD cells over expressing miR9a-5p. * p < .05. Values in (b) and (c) were compared using a one-way ANOVA with a Tukey post-hoc test. For (d), differential expression was determined by DESeq2 method . False discovery rate within statistical analysis was controlled for using the Benjamini-Hochberg method, p value cutoff was 0.05. Graphs with whiskers show mean value in each group and SEM. In (e), following a Shapiro-Wilk normality test, control and experimental values were compared using an unpaired student t -test.

Article Snippet: Antibodies: α-ENaC (ext) – Alomone Cat #: ASC-030; AQP-2 – SCBT Cat # sc-9882; β-Actin – SCBT Cat # sc-1616 HRP, α-, β-, γ-ENaC –Stressmarq Cat#: SPC-403, SPC-404, and SPC-405 respectively.

Techniques: Expressing, Western Blot, Cell Culture, Transfection, Control, Sequencing, Quantitative Proteomics

Journal: EBioMedicine

Article Title: Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC)

doi: 10.1016/j.ebiom.2019.01.006

Figure Lengend Snippet: ENaC activity is altered in the miR-9a-5 overexpressed experimental group. (a) Representative gap-free current traces from cell-attached patches made on mCCD cells of naïve, untransfected cells (upper), control (cont) miR with a non-specific targeting sequence (middle) and miR-9a-5 (lower) group. The closed (c) and open (o) states are denoted with dashed lines. Currents were recorded at the membrane voltage of −60 mV. # denotes p < .05 between control group and miR9a-5 group. (b) Current-voltage relationship and representative examples of ENaC single-channel current at different membrane potentials made on mCCD cells of miR group. (c,d,e) Summary graphs of NP o (c), P o (d), and N (e) for ENaC activity in naïve (white), control miR (dark grey) and miR-9a-5 (black) groups. * P < .05, ** P < .01, ns – not statistically significant, miR9a-5 versus control-miR group by ANOVA . Values were compared using a one-way ANOVA with a Tukey post-hoc test. In box plots, the box is SEM, whiskers represent SD, and line within the box shows median value.

Article Snippet: Antibodies: α-ENaC (ext) – Alomone Cat #: ASC-030; AQP-2 – SCBT Cat # sc-9882; β-Actin – SCBT Cat # sc-1616 HRP, α-, β-, γ-ENaC –Stressmarq Cat#: SPC-403, SPC-404, and SPC-405 respectively.

Techniques: Activity Assay, Control, Sequencing, Membrane

Journal: Basic & Clinical Pharmacology & Toxicology

Article Title: Pleural Effusion Formation Linked to Altered Transporter Expression Involved in Alveolar Fluid Clearance: Insights From the Monocrotaline Model of Pulmonary Hypertension

doi: 10.1111/bcpt.70096

Figure Lengend Snippet: Relative protein expression of Scnn1a (A), Scnn1b (C) and Scnn1g (E) with representative immunoblot and total protein staining (B,D,F) in lungs. Data are presented as mean ± SEM; n = 6 per group; * p < 0.05.

Article Snippet: Membranes were blocked with 5% non‐fat dried milk in TBST for 1 h at room temperature and subsequently incubated with primary antibodies against the subunits of ENaC ( Scnn1a [#bs‐2957R, Bioss], Scnn1b [#ASC‐019, Alomone Labs], Scnn1g [#PA5‐110347, Invitrogen]), the subunits of Na + /K + ‐ATPase ( Atp1a1 [#MA3928, Invitrogen], Atp1a2 [#ARC2458, Invitrogen], Atp1b1 [#PA5‐102831, Invitrogen]) and Aqp3 [#PA5‐78811, Invitrogen].

Techniques: Expressing, Western Blot, Staining

Journal: Frontiers in Physiology

Article Title: Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

doi: 10.3389/fphys.2021.656460

Figure Lengend Snippet: Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 μg for α-ENaC, β-ENaC, and γ-ENaC and 20 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) α-ENaC, (B) cleaved α-ENaC, (C) β-ENaC, (D) cleaved γ-ENaC. The values represent individual measurements and the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. 2K; ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. 2K-1C; &&& P < 0.001 and &&&& P < 0.0001 vs. 2K-1C + E; $$ P < 0.01 and $$$$ P < 0.0001 vs. 2K-1C + L.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% non-fat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and the following specific primary antibodies overnight (4°C): polyclonal antibody against podocin (1:1,000; Santa Cruz Biotech, sc-21009), polyclonal antibody against nephrin (1:1,000; Abcam, Cambridge, MA, Ab 58968), polyclonal antibody against megalin (1:50,000; a gift from Dr. Daniel Biemesderfer) , polyclonal antibody against cubilin (1:1,000; Santa Cruz Biotech, sc-20609), polyclonal antibody against the ClC-5, H + /Cl – exchange transporter 5 (1:1,000; Alpha Diagnostic International, CLC51-A, San Antonio, TX, United States) , polyclonal antibody against α-ENaC (1:1,000; Alomone Labs, ASC-030, Jerusalem, Israel), polyclonal antibody against β-ENaC (1:1,000; StressMarq Biosciences, SPC-404, Victoria, British Columbia, Canada), polyclonal antibody against cleaved γ-ENaC (1:1,000; Alomone Labs, ASC-011), or actin (1:5,000; Abcam ab179467).

Techniques: Expressing, Isolation, Control, Membrane, SDS Page, Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Antibodies used (alphabetical order)

Article Snippet: ENaCβ , StressMarq Biosciences (SPC-404, Victoria, BC, Canada) , WB, 1:5,000.

Techniques: Generated

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Iron does not transcriptionally regulate ENaC alpha. The kidneys of 12-week-old WT or iron overloaded Hamp -/- mice were analyzed by real time PCR for gene expression of ENaC alpha. There was no significant difference in ENaC alpha gene expression of WT or spontaneously iron overloaded Hamp -/- mice under basal conditions ( A ). We confirmed that iron can regulate ENaC alpha gene transcription in mpkCCD cells, a cortical collecting duct cell line that is known to express ENaC alpha. 200 × 10 5 cells were serum-starved for 12 h and then cultured for 12 more hours in vehicle or FAC (100 uM). RNA was purified and then cDNA was synthesized. ENaC alpha gene expression was measured. Iron loading did not regulate ENaC alpha gene expression in mpkCCD cells ( B ), though it increased oxidative stress ( C ). One hundred uM FAC did not activate cell death program under the experimental conditions ( D ). Data from 2 independent experiments were pooled and analyzed. The statistical significance was determined by a 2-tailed Mann–Whitney test and is plotted as mean ± SEM. ** p < 0.001.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Synthesized, MANN-WHITNEY

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC alpha and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in WT control mice. ( B ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Estrogen Impairs Adipose Tissue Expansion and Cardiometabolic Profile in Obese-Diabetic Female Rats

doi: 10.3390/ijms222413573

Figure Lengend Snippet: Ovariectomy and E2 treatment effects on adiponectin and mediators of adiponectin signaling pathway in female ZDF rats. ( A ) Serum adiponectin levels. WAT protein levels of ( B ) adiponectin, ( C ) ADIPOR1 and ADIPOR2, ( D ) APPL1, ( E ) PPARG. ADIPOR, adiponectin receptor protein; APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1; PPARG, peroxisome proliferator activated receptor gamma. Protein bands were normalized to loading control GAPDH protein intensity and expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t -test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

Article Snippet: Antibody for adiponectin (26 kDa, 3553) was supplied by ProSci (Poway, CA, USA); antibodies for ADIPOR1 (43 kDa, sc-99183), ADIPOR2 (50 kDa, sc-46755), GAPDH (37 kDa, sc-365062), GLUT4 (53 kDa, sc-7938) and PPARG (53 kDa, sc-7196) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies for AKT (60 kDa, cs-2920), pAKT (60 kDa, cs-4060) and APPL1 (82 kDa, cs-3858) were supplied by Cell Signaling (Danvers, MA, USA).

Techniques:

Journal: bioRxiv

Article Title: Water and Electrolyte Homeostasis in a Mouse Model with Reduced ENaC Gamma Subunit Expression

doi: 10.1101/2021.01.26.428168

Figure Lengend Snippet: Immunoblots of kidney and lung tissue lysates were probed with antibodies against the β or γ subunit of ENaC, then stripped and probed with antibodies against GAPDH. Arrowheads on the left of each blot show molecular weight marker positions. Carrots on the right of each blot indicate approximate region taken for signal quantification. Dot plots show protein normalized to mean signal from γ +/+ mice. Squares represent males, circles represent females. Open or shaded symbols represent data from 129sv or C57BL/6 mice, respectively. γ subunit protein levels in γ mt/mt mouse kidneys were reduced from 100 ± 10 % (mean ± SD, N = 5 mice) to 8 ± 4 % (N = 6). ***: p < 0.0001 (two-tailed Student’s t-test). In lung, the γ subunit was reduced from 100 ± 43 % (N = 6) to 16 ± 4% (N = 4) **: p < 0.01 (two-tailed Student’s t-test). The kidney β subunit point estimate decreased from 100 ± 60 % (N = 6) to 62 ± 9% (N = 6), but the difference was not significant. Lung β subunit protein signal also did not differ significantly between groups: 100 ± 49 % (N = 6) for γ +/+ vs. 144 ± 132 % for γ mt/mt (N = 5).

Article Snippet: Primary antibodies included anti-γ subunit (StressMarq; catalogue number SPC-405; 0.5 μg/mL), anti-β subunit (StressMarq; catalogue number SMC-241; 1 μg/mL), and anti-GAPDH (ProteinTech, 0.1 μg/mL).

Techniques: Western Blot, Molecular Weight, Marker, Two Tailed Test