Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 1 | mCCDc11 cells: a proper cell model to study lithium-NDI. (a) mCCDc11 cells were grown to confluence, treated for the indicated times (in hours) with 1 nM dDAVP, and subjected to AQP2 immunoblotting or, after blotting, stained with coomassie blue. Non-glycosylated (29 kDa) and complex-glycosylated (40–45 kDa) forms of AQP2, and an a-specific band of 35 kDa, are detected. (b) mCCDc11 cells grown as previously described were treated for 96 h with 1 nM dDAVP, and for the last 24 or 48 h, in the absence () or presence of 1 mM lithium at the basolateral side and 1 or with 10 mM lithium at the apical side. Cells were lysed and immunoblotted for AQP2. Blots were also stained with coomassie blue. Molecular masses (in kDa) are indicated on the left. The signals for non-glycosylated and complex-glycosylated AQP2 were densitometrically quantified and normalized for coomassie blue staining. Mean values of normalized AQP2 expression per condition are given as the percentage of control (±s.e.m.) and were determined from three independent filters per condition. Significant differences (Po0.05) from control () are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Western Blot, Staining, Expressing, Control

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 2 | ENaC blockers reduce lithium-induced AQP2 downregulation in mCCDc11 cells. (a) Confluent mCCDc11 monolayers were treated for 96 h with 1 nM dDAVP and incubated for the last 48 h in the absence () or presence ( þ ) of lithium and/or with 10 mM amiloride as indicated. At the basolateral and apical side, 1 and 10 mM lithium were used, respectively. (b) Confluent mCCDc11 monolayers were treated described earlier with 10 mM lithium with/without 10 mM benzamil (Li þ Ben) at the apical side for the last 24 h. (c) mCCDc11cells were grown as previously described and treated for the last 12 h in medium containing a lower sodium chloride concentration at the apical side only, with or without lithium (indicated). (a–c) Cells were lysed and immunoblotted for AQP2. Molecular masses (in kDa) are indicated on the left. Semiquantification of the AQP2 signals, normalization, and statistical analysis were carried out as described in the Figure 1 caption. Mean values were determined from three independent filters per condition. Significant differences (Po0.05) are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Incubation, Concentration Assay

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 4 | Effects of lithium on GSK3b. Confluent mpkCCDcl4 monolayers were treated for 96 h with 1 nM dDAVP. (a) Cells were treated with 1 mM lithium at the basolateral side and 10 mM lithium at the apical side, or with 20 and 1 mM zinc at both sides for the last 48 h and subjected to AQP2, GSK3b, and phospho-GSK3b(Ser9) immunoblotting or, after blotting, stained with coomassie blue. (b) mpkCCDcl4 cells were treated with a specific GSK3-inhibitor (BIO-Acetoxime) for the last 48 h and subjected to AQP2 immunoblotting. Concentrations are expressed in nanomolar. (c) Cells were incubated for the last 48 h in the absence or presence of lithium with or without 10 mM amiloride as indicated. At the basolateral and apical side, 1 and 10 mM lithium were used, respectively. Molecular masses (in kDa) are indicated on the left. Semiquantification, normalization, and statistical analysis were carried out as described in the legend of Figure 1. Significant differences (Po0.05) from control are indicated by an asterisk.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Western Blot, Staining, Incubation, Control

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 5 | Amiloride prevents effects of lithium on AQP2 and H-ATPase expressions in lithium-NDI rats. Wistar rats were fed a normal diet (; n ¼ 6), a diet containing lithium (Li; n ¼ 6), or a diet containing lithium and amiloride (Li þ Am; n ¼ 7). After 4 weeks, one kidney was divided in the cortex, outer medulla, and inner medulla segments and solubilized. An equal amount of protein of the cortex of each rat was immunoblotted for AQP2, H-ATPase, or tubulin (indicated). Molecular masses (in kDa) are indicated on the left.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques:

Journal: Kidney international

Article Title: Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

doi: 10.1038/ki.2009.91

Figure Lengend Snippet: Figure 6 | Amiloride prevents cell conversion in lithium-NDI rats. (a) Of the rats described in the Figure 5 caption, one kidney was removed and fixed. Cryosections were prepared and incubated with rabbit H-ATPase (green) and guinea pig AQP2 (red) antibodies, followed by Alexa-488-conjugated goat-anti-rabbit and Alexa-594-conjugated goat anti-guinea pig antibodies. TOTO-3 (blue) was used to counterstain the sections. Images were produced with confocal laser scanning microscopy. Bars ¼ 10 mm. (b) Of 45 defined areas of the kidney cortex of each control (n ¼ 6), lithium (n ¼ 6), and lithium þ amiloride (n ¼ 7) rats, cells positive for AQP2 or H-ATPase were counted and expressed as the ratio of principal and intercalated cells (±s.e.m.) (total cells control (): 1244; Li: 1393; Li þ Am: 2064). Significant differences *Po0.05.

Article Snippet: Polyacrylamide gel electrophoresis, blotting, and blocking of the PVDF membranes were carried out as described.42 The membranes were incubated for 16 h with affinitypurified rabbit AQP2 antibodies (1:3000 dilution),43 affinitypurified rabbit anti-v1 H-ATPase antibodies (1:2000-dilution; gift from Dr S Nielsen, Denmark), rabbit anti-Ser9-GSK3b (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA), mouse antiGSK3b (1:5000 dilution; BD Transduction Laboratories, San Jose, CA, USA), or with mouse anti-tubulin antibodies (1:100,000 dilution; gift from Dr Kreis, Switzerland) in Tris-buffered saline Tween-20 supplemented with 1% non-fat dried milk.

Techniques: Incubation, Produced, Confocal Laser Scanning Microscopy, Control

Journal: Kidney360

Article Title: A Novel Human Distal Tubuloid-on-a-Chip Model for Investigating Sodium and Water Transport Mechanisms

doi: 10.34067/KID.0000000992

Figure Lengend Snippet: Expression profile of distal nephron markers in differentiated tubuloids. (A) Left: Schematic illustration of DNS highlighting the TAL of the loop of Henle (TAL; SLC12A1/NKCC2) and CD (SCNN1a/ENaC, AQP2) markers. Right: qPCR analysis showing segment-specific gene expression across four donors after differentiation (fold change relative to basal medium, log scale, Mean±SD). Samples from five chips per donor were pooled to create each replicate, with three independent samples per condition for each of the four donors. (B) Immunofluorescence analysis of tubuloid cultures (20× magnification). Upper panels show full-length tubules; lower panels show magnified regions (dotted boxes). Staining shows expression of ZO-1 (tight junctions), ECAD (distal tubule), NKCC2 (TAL), and AQP2 (CD), all counterstained with DNA (representative images, n =2–3). (C) Comparative gene expression analysis between 3D (OrganoPlate) and 2D (tubuloids grown on static 24-well plates) culture conditions across four donors (fold change, n =3). Comparisons made within each donor due to variability in baseline expression. All qPCR data were normalized to the housekeeping gene GAPDH and expressed as fold change relative to the corresponding 2D static culture condition for each donor. 2D, two-dimensional; CD, collecting duct; DCT, Distal convoluted tubule; DNS, distal nephron segments; ECAD, E-cadherin; ENaC, epithelial sodium channel; PT, proximal tubule; TAL, thick ascending limb.

Article Snippet: , AQP2 , Santa Cruz Biotechnology , Rabbit , — , Sc-515770 , 1:300.

Techniques: Expressing, Gene Expression, Immunofluorescence, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Effect of calyculin on total aquaporin 2 (AQP2) and pSAQP2 abundance in rat kidney inner medulla. Rat inner medullas were harvested after 30-min incubation with or without calyculin (5 μM) and analyzed by Western blot for total AQP2 and pS-AQP2 (arrows) as follows: (A) total AQP2, (B) pS256-AQP2, (C) pS261-AQP2, (D) pS264-AQP2, and (E) pS269-AQP2. Left: representative Western blots. The combined densitometry of total AQP2 and pSAQP2 (n = 8, means ± SE) is shown in the right graphs, respectively. Open bars, control; filled bars, calyculin treated. *P < 0.05, **P < 0.01.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: AQP2 expression in the cell membrane in rat kidney inner medulla. Rat inner medullas were incubated in calyculin (5 μM) for 30 min immediately after being harvested, and an inner medullary collecting duct (IMCD) suspension was prepared. The suspended IMCDs were then biotinylated, and the biotinylated protein pool was analyzed by Western blot for biotin-AQP2 (arrows) as follows: (A) AQP2, (B) pS256-AQP2, (C) pS264-AQP2, and (D) pre-bead lysis sample. Left: representative Western blots. Combined densitometry of the biotinylated samples is shown in the right graphs (n = 8, means ± SE). Open bars, control; filled bars, calyculin treated. **P < 0.01.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Expressing, Incubation, Western Blot, Lysis

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Effect of tacrolimus on total AQP2 and pS-AQP2 abundance in rat kidney inner medulla. Rat inner medullas were harvested after 30-min incubation with or without tacrolimus (5 μM) and analyzed by Western blot for total AQP2 and pS-AQP2 (arrows) as follows: (A) total AQP2, (B) pS256-AQP2, (C) pS261-AQP2, (D) pS264-AQP2, and (E) pS269-AQP2. Left: representative Western blots. The combined densitometry of total AQP2 and pS-AQP2 (n = 8, means ± SE) is shown in the right graphs, respectively. Open bars, control; filled bars, calyculin treated. *P < 0.05.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: AQP2 expression in the cell membrane in rat kidney inner medulla. Rat inner medullas were incubated in tacrolimus (5 μM) for 30 min immediately after being harvested, and an IMCD suspension was prepared. The suspended IMCDs were then biotinylated, and the biotinylated protein pool was analyzed by Western blot for biotin-AQP2 (arrows) as follows: (A) AQP2 and (B) pre-bead lysis sample. Left: representative Western blots. Combined densitometry of the biotinylated samples is shown in the right graphs (n = 8, means ± SE). Open bars, control; filled bars, calyculin treated.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Expressing, Incubation, Western Blot, Lysis

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Immunostaining for AQP2 and pSAQP2 in rat kidney inner medullas. Representative images (original magnification ×400) of immunostaining of rat inner medullary samples of control group (top), calyculin-treated group (middle), and tacrolimus-treated group (bottom) with AQP2, pS256-AQP2, pS261-AQP2, and pS264-AQP2. The apical membrane abundance of AQP2, pS256-AQP2, and pS264-AQP2 is increased in the calyculin- and tacrolimus-treated groups compared with the control group samples.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: AQP2 abundance in PKA knockout cells and its rescue by PKA. All observations were made in the presence of dDAVP (0.1 nM continuously). (A, D, and G) Western blots for PKA-Cα, PKA-Cβ, and AQP2 are shown for 12 control clones versus 12 PKA-Cα knockout clones (A), 13 control clones versus 11 PKA-Cβ knockout clones (D), and 12 control clones versus 12 PKA dKO (G). Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 10 μg; Coomassie, 7 μg. G, glycosylated; nG, nonglycosylated. (B, C, E, F, and H) Band density quantification of the respective immunoblots using beeswarm plus boxplot visualization. Each point is a quantification of a single lane. The heavy horizontal lines represent the median. Band density for AQP2 is summed for glycosylated and nonglycosylated bands. Band densities were normalized by the mean of respective control observations. (I) Immunofluorescence images showing that clusters of PKA dKO cells transfected with PKA-Cα or PKA-Cβ plasmids express AQP2 protein. DAPI labeling shows that the cells are confluent. (Scale bars, 30 μm.) (J and K) Western blot (J) and quantification (K) of protein abundance in transfected cells (n = 3, mean ± SD, *P < 0.05). Values are normalized by band density of AQP2 in vasopressin-treated control cells and expressed as a percentage. Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 60 μg; Coomassie, 7 μg.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Knock-Out, Western Blot, Clone Assay, Immunofluorescence, Transfection, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: RNA-seq–based transcriptomic and SILAC-based quantitative proteomic analysis of PKA dKO versus control cells. (A) Distribution of RNA-seq reads across gene bodies of selected genes for one PKA dKO/control pair: Aqp2 (decreased), Prkaca (decreased), Prkacb (unchanged), Avpr2 (unchanged), Marcks (increased), and Rhcg (increased). Reading direction: blue, left to right; red, right to left. The vertical axis shows binned read counts normalized by total read number (read counts per 107). (B) Volcano plot for 10,190 transcripts expressed in control and PKA dKO cells, quantified for three pairs of PKA dKO vs. control clones. Red points are transcripts with FDR <0.05. Labeled transcripts are discussed in the text. (C) MS1 spectra of representative β-actin, PKA-Cα, PKA-Cβ, and AQP2 peptides showing peaks for control cells (labeled with heavy amino acids) and PKA dKO cells (labeled with light amino acids). Dashed brackets indicate the expected (but not observed) peak locations in the PKA dKO cells (light). The vertical axis shows peptide ion intensity, and the horizontal axis shows the mass-to-charge ratio (m/z). Each peptide has several m/z peaks due to the presence of natural isotopes. (D) The distribution of protein abundance changes for 7,647 proteins in PKA dKO versus control cells (n = 3 pairs). The horizontal axis shows the median log2 (dKO/Ctrl) over the three determinations. Vertical dashed lines indicate mean and ±2 SD (mean, −0.02; SD, 0.87). (E) Volcano plot for the 7,647 proteins quantified in all three pairs of samples. Red points indicate proteins with FDR <0.05. Proteins with FDR <0.05 and absolute value of log2 (dKO/Ctrl) >2 are labeled with the official gene symbol. (F) Correlation between changes in transcript abundance (RNA-seq) and protein abundance (SILAC) in PKA dKO versus control cells. Genes with FDR <0.05 in both analyses are labeled with the official gene symbol. The blue line shows linear regression (with ±SE in gray) calculated from all data.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: RNA Sequencing Assay, Clone Assay, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of aquaporin-2 in PKA dKO versus control mpkCCD cells. (A) AQP2 membrane-spanning topology. AQP2 has six transmembrane domains. A cluster of four vasopressin-dependent phosphorylation sites is present within the terminal 16 amino acids in the C-terminal tail. P, phosphorylation site. (B) The vasopressin-regulated phosphorylation sites are shown. Sequences surrounding Ser256, Ser264, and Ser269 are compatible with phosphorylation by basophilic protein kinases. Ser261 has a proline in position +1 and is presumably phosphorylated by a member of the MAPK family. Vasopressin decreases phosphorylation of Ser261 and increases phosphorylation at the other three sites. (C) Effect of PKA dKO on AQP2 phosphorylation levels. Both control and PKA dKO cells were transfected with AQP2, grown on a solid substratum for 24 h, and then treated with the adenylyl cyclase activator forskolin for 30 min (n = 3). Western blotting was done with phospho-specific antibodies recognizing each of the four phosphorylation sites (Upper) and quantified by densitometry (Lower). The bar graphs show normalized abundances as mean ± SD. Total AQP2 was quantified with a non–phospho-specific AQP2 antibody. (D) Low-power immunofluorescence images of total and phosphorylated AQP2 in PKA dKO and control cells. Cells were transfected with AQP2 and grown on a permeable support without dDAVP for 4 d. Subsequently, cells were stimulated with 0.1 nM dDAVP for 30 min. (Scale bars, 50 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Transfection, Western Blot, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of AQP2 in PKA single-KO cells. PKA single-KO cells were grown on membrane supports in the presence of dDAVP (0.1 nM) to assure high levels of endogenous AQP2. dDAVP was withdrawn for a 2-h incubation and then was readded at 0.1 nM dDAVP for 30 min. Loading volume was adjusted to be the same total AQP2 to allow direct comparison of phosphorylation. n = 3, mean ± SD. (A) PKA-Cα single-knockout cells. (B) PKA-Cβ single-knockout cells. Bars represent normalized band densities (normalized by mean value for control cells in the absence of dDAVP).

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Incubation, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: PKA signaling mapped to functional effects of vasopressin. (A) Direct PKA targets and their physiological and functional effects. (B–H) PKA-regulated signaling network in MAP kinase signaling (B), decreased apoptosis (C), Aqp2 gene transcription (D), actin dynamics (E), AQP2 phosphorylation (F), exocytosis (G), AQP2 endocytosis (H), and AQP2 protein stability (H). Data sources are given at https://hpcwebapps.cit.nih.gov/ESBL/PKANetwork/.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Role of PKA in nuclear translocation of transcriptional regulators, histone acetylation, actin polymerization, and apical membrane trafficking of AQP2. (A) Nuclear translocation of transcriptional regulators in response to vasopressin. Western blot of nuclear and cytoplasmic extracts of various transcriptional regulators (Left). Densitometric analysis showing mean and SD (Right). CE, cytoplasmic extract; NE, nuclear extract. (B) Distribution of ChIP-seq reads across gene bodies of selected genes for vehicle- or dDAVP-treated cells. Green boxes highlight two genomic regions in which increases were observable. (C) Confocal projection x–y (Top) and x–z (Bottom thin panels) images showing changes in actin polymerization in response to vasopressin. Alexa-594 phalloidin staining in cells treated with vehicle or dDAVP. (Scale bars, 10 μm.) (D) Vasopressin-dependent AQP2 trafficking to the apical plasma membrane in control and PKA dKO cells. Confocal x–y (Top) and x–z (Bottom thin panels) images of cells treated with vehicle or dDAVP using anti-AQP2 antibody (green). DAPI-stained nuclei, blue. (Scale bars, 10 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Translocation Assay, Western Blot, ChIP-sequencing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Materials

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Recombinant, Transfection, Electron Microscopy, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Purification, Chromatin Immunoprecipitation, Magnetic Beads, Expressing, Plasmid Preparation, Sequencing, CRISPR, Software, Microscopy

Journal: Physiological Reports

Article Title: TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium

doi: 10.14814/phy2.15641

Figure Lengend Snippet: TRPV4 activity regulates subcellular AQP2 distribution in cystic cells of PCK453 rats. (a) Representative confocal images showing AQP2 (pseudocolor red) distribution in kidney sections of PCK453 rats fed regular (0.9%K + ), high KCl (5%K + ), and high KB/C (5%K + , bicarbonate: citrate as 4:1) diets for 1 month. Nuclear Dapi staining is shown with pseudocolor blue. Areas with cystic (1) and non‐dilated collecting duct (2) are shown below at higher magnification. The averaged intensities of AQP2‐reporting fluorescent signals around the apical area in cystic (b) and non‐dilated collecting duct (c) cells from the conditions in (a). For each individual cell the fluorescent signals were normalized to their corresponding maximal value.

Article Snippet: Sections were incubated overnight at +4°C with anti‐AQP2‐ATTO Fluor‐550 (1:200, Alomone Labs; Cat. # AQP2‐002‐AO) and anti‐TRPV4 (1:500, Alomone labs; Cat.#.

Techniques: Activity Assay, Staining

Journal: Physiological Reports

Article Title: TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium

doi: 10.14814/phy2.15641

Figure Lengend Snippet: TRPV4 activity is inversely related to cAMP levels in cystic cells of PCK453 rats. Summary graph comparing dispersion (decrease by 50% from maximum) of AQP2‐reporting signal in non‐dilated collecting duct versus cystic cells in kidney sections of PCK453 rats fed regular (0.9%K + ), high KCl (5%K + ), and high KB/C (5%K + , bicarbonate: citrate as 4:1) diets for 1 month. Bars and whiskers represent SE and SD, respectively. Mean and median values are denoted with lines. Numbers of each experimental groups are shown below. Kidney sections from at least 4 different animals were used for each tested group. *Significant changes ( p < 0.05, one‐way ANOVA with post hoc Tukey test) between groups shown with brackets on the top.

Article Snippet: Sections were incubated overnight at +4°C with anti‐AQP2‐ATTO Fluor‐550 (1:200, Alomone Labs; Cat. # AQP2‐002‐AO) and anti‐TRPV4 (1:500, Alomone labs; Cat.#.

Techniques: Activity Assay, Dispersion