Ca²⁺-imaging in aldosterone-sensitive distal nephron.

<p>Representative micrographs of split-opened cortical collecting duct (top raw) and connecting tubules (bottom raw) after loading with Fura-2 taken with bright-field illumination (left column), 380 nm excitation (middle column), and the merged image (right column).</p

ATP increases [Ca²⁺]_i in a PLC-dependent manner.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the absence and presence of a PLC inhibitor, U73122 (black bar). (<b>B</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after PLC inhibition. * - significant decrease versus ATP. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the absence and presence of a PLA inhibitor, AACOCF3 (black bar). (<b>D</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after PLA inhibition. * - significant decrease versus ATP.</p

Extracellular and intracellular Ca²⁺ sources account for ATP-evoked [Ca²⁺]_i elevations.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with a gray bar on the top) for individual cells of ASDN in the absence and presence of a Ca²⁺-pump SERCA inhibitor, thapsigargin (black bar). (<b>B</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after SERCA inhibition. * - significant decrease versus ATP. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the control and in Ca²⁺-free extracellular media (black bar). (<b>D</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after extracellular Ca²⁺ removal. * - significant decrease versus ATP.</p

Disruption of purinergic signaling compromises mechano-sensitive responses in ASDN cells.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min of hypotonic (hypo) media application (shown with bar on the top) for individual cells of ASDN isolated from wild type (wt, black) and P2Y2 −/− (light gray) mice. (<b>B</b>) Summary graph of the ΔF₃₄₀/F₃₈₀ peak changes in response to hypotonic media application for wild type and P2Y2 −/− mice. * - significant decrease versus hypo WT. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to elevated flow (shown with bar on the top) for individual cells of ASDN from wild type (black) and P2Y2 −/− (light gray) mice. (<b>D</b>) Summary graph of the magnitudes of high flow-induced Ca²⁺ spikes for wild type and P2Y2 −/− mice. * - significant decrease versus flow WT.</p

TRPV4 is critical for the ATP-induced Ca²⁺-plateau.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP application (shown with bar on the top) for individual cells of ASDN from wild type (black) and TRPV4 −/− (gray) mice. (<b>B</b>) Summary graph of relative contributions of TRPV4 and TRPCs in ATP-induced [Ca²⁺]_i response. For this, the values of ΔF₃₄₀/F₃₈₀ during second ATP application from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022824#pone-0022824-g007" target="_blank">Figures 7A, B</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022824#pone-0022824-g008" target="_blank">8A</a> were subtracted from the corresponding values during the first ATP application.</p

Genetic deletion of P2Y2 receptors abolishes Ca²⁺ transients in response to purinergic stimulation of ASDN cells.

<p>The average time course of changes in absolute [Ca²⁺]_i values in response to 2 min application of 10 µM ATP (<b>A</b>) and 10 µM UTP (<b>B</b>) for individual cells of ASDN from wild type (black) and P2Y2 −/− (light gray) mice, respectively. ATP and UTP application is shown as a bar on the top of each graph.</p

Immunohistochemical staining of SK3 for thick ascending limb tubules.

<p>Sagital section (5 µm) of WT mouse kidney showing staining for Tamm-Horsefall protein (THP, red), a marker of TAL cells, and SK3 (green). <b>Panels A, C, E, and G</b> are low magnification images showing THP staining of TAL structrues (<b>A</b>), SK3 labeling of the same structures (C), and a merged image (E). As shown at higher resolution for one of the tubules (inset from A), THP strongly stains the luminal border of the TAL (<b>B</b> and <b>F</b>) with SK3 also showing strong labeling of the luminal border and, to a variable degree, the abluminal border (<b>D</b> and <b>F</b>). The merged image (<b>F</b>) clearly identifies SK3 staining in the TAL cells. Panel <b>H</b> is a magnified view of a proximal tubule (PT), located left of the TAL in <b>A</b>. The PT showed minimal staining for SK3, although light staining was apparent along the luminal brush border. Scale bar is 10 µm.</p

SK3 expression in WT mouse kidney.

<p><b>A.</b> RT-PCR analysis using whole kidney mRNA extracts revealed prominent bands of the appropriate size on agarose gels for both SK3 (473 bp) and BKα (318 bp), demonstrating expression of both of these channels in the kidney. SK3 primers were selected to cross the exon 2 and exon 3 borders to rule out amplification of intron sequences from genomic DNA. The electropherogram for SK3 is shown with both nucleotide sequences (NT) and amino acid sequences (AA) indicated for the segment across the exon border region, demonstrating that the PCR product does not originate from genomic DNA. 100-bp marker standards are shown (Lane M). <b>B.</b> Western blot of WT mouse kidney-SK3. SK3 protein is expressed as a single band near 90 kD in mouse kidney. SK3 blocking peptide (SK3-BP) was used as a control to verify antibody specificity which, as shown, abolished binding of the anti-SK3 antibody (right lane). Alpha-tubulin expression was used as a loading control (lower panel).</p

Immunohistochemical staining for SK3 and aquaporin-2 (AQP2) in WT mouse kidney sections.

<p>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 (<b>A.</b> AQP2, red), a marker of the collecting ducts, SK3 (<b>B.</b> SK3, green), and a merger of both channels (<b>C.</b> 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). <b>Middle Pannel (D–F):</b> 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). <b>Bottom Panel (G–H):</b> 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.</p

Antibodies and markers used for immunohistochemistry.

<p>Antibodies and markers used for immunohistochemistry.</p