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: Journal of the American Society of Nephrology : JASN

Article Title: Tubulointerstitial Nephritis with IgM-Positive Plasma Cells

doi: 10.1681/ASN.2016101074

Figure Lengend Snippet: The dual staining of CD3 and AQP2 shows tubulitis in the proximal tubules and collecting ducts. Representative light photomicrographs (patient 13) show that infiltrating CD3-positive T lymphocytes were diffusely distributed in the renal interstitium (A and B), among proximal tubular epithelial cells (proximal tubulitis) (arrowheads) (C), and among collecting duct epithelial cells (collecting duct tubulitis) (arrow) (D) (red: AQP2, brown: CD3). (A): Bar = 100 μm; (B): bar = 50 μm; (C and D): bar = 20 μm.

Article Snippet: Immunohistochemistry Rabbit anti-human IgG polyclonal antibody (Dako, Glostrup, Denmark), rabbit anti-human IgM polyclonal antibody (Dako), mouse anti-human CD138 monoclonal antibody (Dako), anti-human IgG4 monoclonal antibody (Zymed Laboratories, San Francisco, CA), anti-human κ monoclonal antibody (Nichrei, Tokyo, Japan), and anti-human λ monoclonal antibody (Nichrei) for staining plasma cells; rabbit anti-human CD3 monoclonal antibody (Nichrei) for staining T lymphocytes; mouse anti-human CD20 monoclonal antibody (Dako) for staining B lymphocytes; rabbit anti-human AQP2 polyclonal antibody (Atlas antibodies, Stockholm, Sweden) for staining CCD epithelial cells; rabbit anti-human ATP6V1E1 polyclonal antibody (Sigma-Aldrich, St. Louis, MO) for staining H + -ATPase in CCDs; mouse anti-human band 3 monoclonal antibody (Atlas antibodies) for staining AE-1 in CCDs; and rabbit anti-human ATP12A polyclonal antibody (Atlas antibodies) for staining H + , K + -ATPase in CCDs were used as primary antibodies.

Techniques: Staining

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Shown are representative Western blots (left hand panels) from IM tips or bases from rats after 14 days treatment with or without meloxicam (top) or ibuprofen (bottom) probed for total AQP2 (arrows) The top bracket shows 35–50 kDa glycosylated forms; the bottom arrow designates the 29 kDa unglycosylated AQP2. L.C. = loading control. The right hand panels provide combined densitometry from n = 12 rats per group. Bars = Mean ± SEM; *P<0.05 compared with control rats.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Perfusion fixed kidneys from control, meloxicam- and ibuprofen-treated mice were prepared for histology examination as described in the Methods section. AQP2 is denoted by the brown stain indicating positive peroxidase product. Slides were counterstained with hematoxylin to show nuclei. L indicates examples of the lumen of the collecting ducts. Arrows show the apical staining location of the AQP2. Magnification, 400x.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques: Staining

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Tissue samples were isolated from rat IM tip and base after 14 days treatment without or with meloxicam (100 mg/kg) and analyzed by Western blot for phosphorylated AQP2 (arrows) as follows: (A) p256AQP2, (B) p261AQP2, (C) p264AQP2 and (D) p269AQP2. Left panels provide representative Western blots. The top bracket shows 35–50 kDa glycosylated forms; the bottom arrow designates the 29 kDa unglycosylated AQP2. L.C. = loading control. Right bar graphs show phosphoAQP2 combined densitometry from n = 12 rats per group. All proteins were normalized to loading controls. White bars, control; black bars, meloxicam treated. Bars = Mean ± SEM, n = 12 rats per group; *P<0.05 compared with control rats.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques: Isolation, Western Blot

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Bar graphs show the ratio of p256AQP2, p261AQP2, p264AQP2 and p269AQP2 to total AQP2. White bars, control in IM tip; gray bars, meloxicam treated in IM tip; white and oblique line bars, control in IM base; gray and oblique line bars, meloxicam treated in IM base. All proteins were normalized to loading controls. Bars = Mean ± SEM, n = 12 rats per group; *P<0.05 compared with control rats.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Tissue samples were isolated from rat IM tip and base after 14 days treatment without or with ibuprofen (5 mg/kg). and analyzed by Western blot for phosphorylated AQP2 (arrows) as follows: (A) p256AQP2, (B) p261AQP2, (C) p264AQP2 and (D) p269AQP2. Left panels provide representative Western blots. The top bracket shows 35–50 kDa glycosylated forms; the bottom arrow designates the 29 kDa unglycosylated AQP2. L.C. = loading control. Right bar graphs show phosphoAQP2 combined densitometry from n = 12 rats per group. All proteins were normalized to loading controls. White bars, control; black bars, ibuprofen treated. Bars = Mean ± SEM; *P<0.05 compared with control rats.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques: Isolation, Western Blot

Journal: PLoS ONE

Article Title: NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

doi: 10.1371/journal.pone.0141714

Figure Lengend Snippet: Bar graphs show the ratio of p256AQP2, p261AQP2, p264AQP2 and p269AQP2 to total AQP2. White bars, control in IM tip; gray bars, ibuprofen treated in IM tip; white and oblique line bars, control in IM base; gray and oblique line bars, ibuprofen treated in IM base. All proteins were normalized to loading controls. Bars = Mean ± SEM, n = 12 rats per group; *P<0.05 compared with control rats.

Article Snippet: PVDF membranes were blocked for 60 min with 5% of blotting grade nonfat dry milk (Bio-Rad, Hercules, CA) before overnight incubation with primary antibodies: our total AQP2 and UT-A1, p 256 -AQP2 (Biorbyt, Burlington, NC), p 261 -AQP2 (Avivasysbio, San Diego, CA), p 264 -AQP2 (Thermo Fisher Scientific), and p 269 -AQP2 (Thermo Fisher Scientific).

Techniques: