Journal: Kidney Diseases

Article Title: Loss of Pten in Renal Tubular Cells Leads to Water Retention by Upregulating AQP2

doi: 10.1159/000528010

Figure Lengend Snippet: AQP2 was upregulated and concentrated in the apical plasma membrane of collecting ducts cells in the kidney of Pten Δ/Δ mice. a The expression of Aqp2 mRNA in the kidneys of Pten fl/fl and Pten Δ/Δ mice. b Western blots analysis of AQP2 and pS256-AQP2 levels in the kidney medulla lysates of Pten fl/fl and Pten Δ/Δ mice. c IHC staining of AQP2 in collecting ducts of Pten fl/fl and Pten Δ/Δ mice. Bars = 100 μm. d Higher magnification of IHC staining showed different distribution pattern of AQP2 in the collecting ducts cells of Pten fl/fl and Pten Δ/Δ mice. e Immunofluorescence staining of AQP2 (green) in collecting ducts of Pten fl/fl and Pten Δ/Δ mice. Higher magnifications of the selected areas are shown on the right.

Article Snippet: After blocked in 5% skim milk, the membranes were incubated with primary antibodies: anti-PTEN (9188L, CST, 1:2,000), anti-AQP2 (AQP-002, Alomone, 1:2,000), anti-pSer256-AQP2 (bs-12507R, Bioss, 1:500), anti-AKT (4691S, CST, 1:2,000), anti-pSer473-AKT (4060S, CST, 1:2,000), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000) at 4°C overnight.

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: Kidney Diseases

Article Title: Loss of Pten in Renal Tubular Cells Leads to Water Retention by Upregulating AQP2

doi: 10.1159/000528010

Figure Lengend Snippet: PTEN regulated AQP2 by dephosphorylating p-AKT. a IMCD cells were transfected with control shRNA (shControl) or Pten -specific shRNA (shPTEN), respectively. Relative expression level of Aqp2 genes in the shPTEN group is higher than that in the shControl group. b Western blots analysis of PTEN, AQP2, and p-AQP2 protein levels in shControl and shPTEN groups. c Western blots analysis of AKT and p-AKT proteins in kidney medulla lysates of Pten fl/fl and Pten Δ/Δ mice ( n = 3 for each group). IMCD3-shPTEN cells were treated with LY ( d ) or MK ( f ) for 24 h and cell lysates of different groups were subjected to immunoblotting to detect the protein levels of AKT, p-AKT, AQP2, and p-AQP2 ( n = 3-4 for each group). e, g Expression of membrane AQP2 (m-AQP2) and p-AQP2 (m-p-AQP2) in different groups of cells ( n = 3 for each group). ns p > 0.05, * p < 0.05, ** p < 0.02, *** p < 0.001.

Article Snippet: After blocked in 5% skim milk, the membranes were incubated with primary antibodies: anti-PTEN (9188L, CST, 1:2,000), anti-AQP2 (AQP-002, Alomone, 1:2,000), anti-pSer256-AQP2 (bs-12507R, Bioss, 1:500), anti-AKT (4691S, CST, 1:2,000), anti-pSer473-AKT (4060S, CST, 1:2,000), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000) at 4°C overnight.

Techniques: Transfection, shRNA, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Purinergic Activation of Ca 2 + -Permeable TRPV4 Channels Is Essential for Mechano-Sensitivity in the Aldosterone-Sensitive Distal Nephron

doi: 10.1371/journal.pone.0022824

Figure Lengend Snippet: ( A ) The average time course of [Ca 2+ ] i changes in individual cells of ASDN in response to 10 min application of 10 µM ATP (shown with bar on the top). ( B ) The average time course of relative changes in ΔF 340 /F 380 ratio in response to consecutive 2 min ATP applications (shown with bars). ( C ) Histogram of the magnitudes of ATP-induced ΔF 340 /F 380 peak values of single cells in the ASDN. ( D ) The average time courses of elevations of [Ca 2+ ] i in response to ATP for individual cells of CCD and CNT (similar to that shown in ). ( E ) A representative fluorescent micrograph of AQP2 expression (pseudocolor red) within a split-opened area of ASDN. The examples of AQP2-negative (intercalated) cells are indicated with a white asterisk. Nuclear DAPI staining is also shown (pseudocolor blue). ( F ) Summary graph of [Ca 2+ ] i comparison for AQP2-positive (PC) and AQP2-negative (IC) cells similar to that shown in 2F in the resting condition (baseline) and during stimulation with 10 µM ATP (both peak and plateau values are reported).

Article Snippet: After washing with PBS (3 times for 5 min) the samples were incubated for 1.5 hr at RT in dark with anti-aquaporin 2 labeled with ATTO-550 (1∶100 dilution; Alomone labs) in 1% serum+0.1% Triton in PBS.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: Western blots were performed on lysates of LLC-PK1 cells stably expressing AQP2 and stained with specific phospho-AQP2 antibodies. Phosphoserine band intensities were quantified and normalized to their respective total AQP2 loading controls. Erlotinib increased S256 phosphorylation similar to VP, and this effect was significantly decreased in cells pre-treated with the RSK inhibitor BI-D1870. Data were analyzed using a Student t-test, and expressed as mean ± SD. (n=5)

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Western Blot, Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: Erlotinib-induced AQP2 membrane accumulation in LLC-PK1 cells is blocked by RSK inhibition. As expected, both VP and erlotinib (Erl) induced an increase in AQP2 membrane accumulation (top row). When pre-treated with RSK inhibitor, BI-D1870, erlotinib-induced AQP2 membrane accumulation is inhibited, and AQP2 is mostly in the cytoplasm (bottom right panel), similar to controls (CT). BI-D1870 does not inhibit VP-induced AQP2 membrane accumulation (bottom middle panel) and BI-D1870 alone does not cause visible changes in AQP2 localization (bottom right panel). These images are representative of 4 independent experiments (n=4)

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: RSK protein abundance is significantly reduced after cells were incubated with siRNA specific for RSK protein (siRSK, top panel). When incubated with scrambled siRNA, increased phosphorylation of S256 is seen with VP and erlotinib treatment (bottom). The phosphorylation of S256 AQP2 induced by erlotinib is significantly reduced in cells incubated with siRSK, but not in VP-treated cells. This is representative of 5 independent experiments (n=5). After incubating cells with scrambled or siRSK for 48 hours, cells were exposed to VP or erlotinib. In cells incubated with scrambled siRNA (top row), VP and erlotinib increased AQP2 membrane accumulation. In cells incubated with siRSK, VP continued to induce AQP2 membrane accumulation, but erlotinib no longer induced membrane accumulation of AQP2. These images are representative of 3 independent experiments (n = 3).

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Incubation

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: Immunohistochemistry shows RSK (green, left column) and AQP2 (red, middle column) in cortex (top row), medulla (middle row) and papilla (bottom row). Merged panels are shown in the right column. RSK was heavily expressed in principal cells in the collecting ducts in the medulla and papilla, co-localized with AQP2 in red. Expression of RSK is to a lesser degree in the cortex. The insets in the left column show the lack of specific RSK staining in the presence of a blocking peptide. The insets in the middle column show the expected AQP2 (red) staining in tissues double stained after application of RSK blocking peptide. These images are a representative of three independent experiments on three different animals. (n=3)

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Immunohistochemistry, Expressing, Staining, Blocking Assay

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: Kidney slices were pre-treated with the RSK inhibitor, BI-D1870, followed by erlotinib treatment for 30 min. Apical membrane accumulation of AQP2 is noted in kidney slices treated with VP and erlotinib (Erl), but pre-treatment with BI-D1870 abolishes Erl-induced apical membrane accumulation as AQP2 instead remains diffusely distributed in the cytoplasm of principal cells. These images are representative of 4 independent experiments from 4 different animals (n=4)

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: A C-terminal tail (∼7kDa) construct of AQP2 was incubated with RSK and radioisotope 32 P in vitro . As a positive control, the AQP2 c-tail was incubated with recombinant protein kinase A (PKA), and as expected our assay demonstrated phosphorylation of the AQP2 c-tail. A serine to alanine mutation behaves functionally as a permanently dephosphorylated residue, and recombinant AQP2 c-tail with this S256A mutation failed to show phosphorylation by PKA. H89, a non-specific PKA inhibitor, prevents the ability of PKA to phosphorylate AQP2 c-tail (left three lanes). RSK (right three lanes) can also directly phosphorylate the AQP2 c-tail, similar to PKA, and cannot phosphorylate the c-tail S256A mutation. BI-D1870 inhibits the ability of RSK to phosphorylate AQP2 c-tail. This image is a representative of three independent experiments (n=3).

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Construct, Incubation, In Vitro, Positive Control, Recombinant, Mutagenesis

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: Phosphoinositide-dependent kinase 1 (PDK1) activation increases AQP2 membrane accumulation. PS210, a PDK1 activator, caused AQP2 membrane accumulation as also seen with erlotinib or vasopressin (top row). Membrane accumulation of AQP2 induced by either erlotinib or PS210 is decreased by the PDK1 inhibitor GSK-470 (middle row). The non-specific kinase inhibitor H89 decreased membrane trafficking induced by PS210 in addition to membrane trafficking in vasopressin and erlotinib treated cells (bottom row).

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib

doi: 10.1101/2022.12.06.519384

Figure Lengend Snippet: RSK knockout cells do not show increased AQP2 S256 phosphorylation in response to erlotinib. An RSK knockout in the LLC-AQP2 cell line (RSK KO) was created using CRISPR/Cas9 and compared to regular LLC-AQP2 cells (W2) with intact RSK. Knockout was verified by western blot with clone 3 showing successful knockout. Cells were treated with erlotinib (20μM), or VP (10nM) for 30 or 15 min respectively. Western blots were performed on lysates of cells and stained with specific phospho-AQP2 antibodies. Both cell lines showed increased AQP2 phosphorylation in response to the positive control vasopressin, while only the cells with intact RSK demonstrated increased AQP2 S256 in response to erlotinib. This result was repeated 5 times in duplicate. Mean ± SD, N=5, ** P <.01, **** P <.0001).

Article Snippet: However, the sections were incubated with rabbit anti–AQP2 antibody (1 μ g/ml, Alomone Labs, Jerusalem, Israel) overnight, and subsequent incubation with donkey anti–rabbit IgG conjugated to Alexa-488 (6 μ g/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature.

Techniques: Knock-Out, CRISPR, Western Blot, Staining, Positive Control

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Antibodies and markers used for immunohistochemistry.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Immunohistochemistry, Plasmid Preparation

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Top Panel (A–C): A low-magnification transverse section (5 µm) of the mouse kidney is shown. Discrete labeling is shown for staining for aquaporin-2 ( A. AQP2, red), a marker of the collecting ducts, SK3 ( B. SK3, green), and a merger of both channels ( C. Merge, yellow-organge for co-localization of AQP2 and SK3). Labeling is apparent for SK3 in both the cortex (label C) and medullary (label M) (dashed line shows cortical-medullary demarcation). Middle Pannel (D–F): Magnified view of the yellow inset box from A. SK3 co-localizes with all AQP2-postive tubules as show by the yellow-orange images (F., asterisk). SK3 staining is also apparent in AQP2-negative structures including other tubular structures (F., arrows) and smaller secondary structures (possibly vascular structures, F., arrow heads). Bottom Panel (G–H): Magnified view of staining in the presence of SK3 blocking peptide. All SK3 staining is abolished demonstrating specificity of our anti-SK3 antibody. Scale bar is 50 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Labeling, Staining, Marker, Blocking Assay

Journal: PLoS ONE

Article Title: Emerging Role of the Calcium-Activated, Small Conductance, SK3 K + Channel in Distal Tubule Function: Regulation by TRPV4

doi: 10.1371/journal.pone.0095149

Figure Lengend Snippet: Section (5 µm) from WT mouse kidney showing staining for AQP2 (red), a marker of PCs in collecting duct, and SK3 (green). Panels A, C, and E are low magnification views of a cross-section through a CCD identified by AQP2 staining. Panels B, D, and F represent a magnified view of the inset area from A (yellow inset box). Panel B shows strong AQP2 staining along the luminal border of PCs (5–6 cells), but not of the ICs (2 cells without staining). As shown in D and F , strong staining of SK3 is evident along the luminal border of all cells, both PCs and ICs. Variable, but weak staining, is also apparent along the abluminal border of some cells. However, the staining is most pronounced along the luminal border for both PCs and ICs, although typically stronger in PCs, as indicated by the SK3 fluorescence line intensity profiles across (luminal to abluminal direction) two cells identified as PC and IC ( Panel G ). H . Relative mean intensity profiles (± SEM) across the cells from all sections showing the maximal values across the luminal border (Apical) and abluminal border (Basal) and the minimal values within the cytoplasm (Cytosol). The mean values are given for both PCs (n = 37) and ICs (n = 12) from all sections analyzed. The maximal luminal intensity is much greater than the abluminal intensity (*P<0.02) indicating dominant expression at the luminal border. Scale bar is 10 µm.

Article Snippet: Anti-AQP2 ATTO-550 , 1∶200 , Rabbit , Alomone.

Techniques: Staining, Marker, Fluorescence, Expressing

Journal: Cells

Article Title: Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days

doi: 10.3390/cells11111822

Figure Lengend Snippet: Tubule and collecting duct staining depicts healthy kidneys. Control kidney, cannulated but not perfused. LTL (green), KSP (red) double positive depict proximal tubules. E-cadherin (red), double positive with LTL depicts distal tubules. AQP2 (green) depict collecting ducts. Kidneys perfused with Renal Epithelial Medium for 4, 7, 9 days demonstrated healthy tubules and collecting ducts. Kidneys perfused with 10%FBS/DMEM for 4, 9 days demonstrate healthy tubules and collecting ducts. Day 9 morphology is intact. Block arrows = LTL, Arrow = KSP or E-cadherin. Bar = 45 µm.

Article Snippet: Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel).

Techniques: Staining, Blocking Assay

Journal: Cells

Article Title: Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days

doi: 10.3390/cells11111822

Figure Lengend Snippet: Tubule and collecting duct staining depicts healthy kidneys. Control kidney, cannulated but not perfused. LTL (green), KSP (red) double positive depict proximal tubules. E-cadherin (red), double positive with LTL depicts distal tubules. AQP2 (green) depict collecting ducts. Kidneys perfused with Renal Epithelial Medium for 4, 7, 9 days demonstrated healthy tubules and collecting ducts. Kidneys perfused with 10%FBS/DMEM for 4, 9 days demonstrate healthy tubules and collecting ducts. Day 9 morphology is intact. Block arrows = LTL, Arrow = KSP or E-cadherin. Bar = 45 µm.

Article Snippet: Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel).

Techniques: Staining, Blocking Assay