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

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

Antibodies and markers used for immunohistochemistry.

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

Immunohistochemical staining of SK3 in the collecting duct.

<p>Section (5 µm) from WT mouse kidney showing staining for AQP2 (red), a marker of PCs in collecting duct, and SK3 (green). <b>Panels A, C, and E</b> are low magnification views of a cross-section through a CCD identified by AQP2 staining. <b>Panels B, D, and F</b> represent a magnified view of the inset area from <b>A</b> (yellow inset box). <b>Panel B</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 <b>D</b> and <b>F</b>, 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 (<b>Panel G</b>). <b>H</b>. 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.</p

Effect of TRPV4-mediated activation of SK3 channels on membrane potential, Vm.

<p><b>A.</b> Fluorescence image of a split-open CCD loaded with the voltage-sensitive fluorescence dye, DiSBAC₂(3), showing loading of all cells. The fluorescence intensity is an index of Vm and is presented as relative fluorescence units (RFU). <b>B.</b> Effect of 50 mM K⁺ (High K⁺) application on Vm of CCD cells showing the expected membrane depolarization (increased RFU). <b>C.</b> Effect of 300 nM apamin or 50 nM IbTX application on Vm in basal conditions showing little or no effect of either apamin (Apa) or IbTX in the basal state (TRPV4 not activated). <b>D.</b> Effect of TRPV4 activation with GSK101 (50 nM) leading to membrane hyperpolarization of Vm (decreased RFU), as expected for SK3 and BK activation. Subsequent application of either 300 nM apamin or 50 nM IbTX now induce a marked depolarization of Vm (increased RFU) demonstrating inhibition of SK3 and BK, respectively. <b>E.</b> Summary graph showing mean changes in Vm in basal conditions upon addition of High K⁺ (High K⁺, n = 44 cells), 300 nM apamin, or 50 nM IbTX (Left panel, Basal). Right panel (GSK101: TRPV4 Activation) shows the results after activation of TRPV4 (Ca²⁺ influx). Both apamin and IbTX now bring about a significant depolarization of Vm (*P<0.01 compared to Basal). The combine addition of both apamin and IbTX (Apa + IbTX) displays an enhanced depolarization compared to addition of apamin or IbTX alone (**P<0.01). The number in parentheses is the number of cells for each group (n).</p

Immunohistochemical staining of SK3 in the distal convoluted tubule (DCT).

<p>Mouse (WT) kidney section (5 µm) showing staining for the sodium-calcium exchanger (NCX, red), a marker of DCT, especially the later portion (DCT2), and SK3 (green). The heavy NCX staining of the upper portion of the tubule in <b>Panel A</b> (within yellow inset box) is consistent with the DCT2 segment with the weaker, more basolateral staining in the lower half of the tubule indicating this is the connecting tubule (CNT) (see text for details). Higher resolution image of the DCT2 (<b>D</b>) shows strong staining of SK3 along the luminal border with more variable, weaker staining along the abluminal border. The merged image clearly identifies SK3 staining of the DCT (<b>F</b>). In the CNT segment (<b>A.</b>, labeled CNT), SK3 staining was also apparent along the luminal border with abluminal staining appearing weaker. Scale bar is 10 µm.</p

Dot1a as indicated by H3m2K79 is expressed in glomerular cells.

<p>Representative immunofluorescence images showing di-methyl histone H3 K79 (H3m2K79) staining in a normal rat glomerulus. Boxed areas were amplified and shown at the bottom. H3m2K79, an indicator of Dot1a methyltransferase activity, was stained in red. Nuclei were visualized by DAPI staining. The merged images demonstrate the presence of H3m2K79 in the nuclei of some (arrowhead), but not all of cells in the glomerulus. Scale bar: 100 µm and 25 µm for the amplified boxed areas.</p

Spironolactone significantly improves glomerulosclerosis and tubulointerstitial injury in STZ-induced diabetic rats.

<p>(<b>A–B</b>) As in Fig. 1, representative renal histological images showing hematoxylin and eosin stain (<b>A</b>) and Masson’s Trichrome stain (<b>B</b>) of the rats in the three groups as indicated. (<b>C</b>) Whole kidney average score of tubulointerstitial injury based on Masson’s Trichrome stain. n = 8 rats/group. *P<0.05 vs. CT. #P<0.05 vs. STZ.</p

Spironolactone partially normalizes mRNA expression of Dot1a, Af9, and ET-1 in STZ-induced diabetic rats. (A)

<p>As in Fig. 1, representative agarose gel analyses of RT-PCR for expression of genes as indicated in the cortex of rats. (<b>B–D</b>) For each mRNA as indicated, the intensity of each corresponding band was quantified using ImageJ64, normalized to that of GAPDH or β-actin as shown in <b>A</b>, averaged within each group, and plotted, with the relative intensity in control (CT) set to 1. n = 8 rats/group. *P<0.05 vs. CT. #P<0.05 vs. STZ.</p