Renal water and sodium handling during rUII and URP infusion.

<p>Effect of exogenous rUII (6 pmol. min^-1. 100 g bwt^-1, solid bars, n = 7 per strain), URP (6 pmol. min^-1. 100 g bwt^-1, hatched bars, n = 5 per strain) or vehicle (0.154 mol/L NaCl, open bars, n = 6 per strain) infusion on urine flow rate (A-B), sodium excretion rate (C-D) and fractional sodium excretion (E-F) in anaesthetised 4-5 week-old WKY rats (left column) and pre-hypertensive SHRs (right column). Data shown are mean ± SEM for the baseline control period and the final 15 mins of the treatment period when effects were maximal. * P < 0.05 compared with vehicle-treated rats, Dunnett’s test.</p

Systemic and renal haemodynamics during rUII and URP infusion.

<p>Effect of exogenous rUII (6 pmol. min^-1. 100 g bwt^-1, solid bars, n = 7 per strain), URP (6 pmol. min^-1. 100 g bwt^-1, hatched bars, n = 5 per strain) or vehicle (0.154 mol/L NaCl, open bars, n = 6 per strain) infusion on mean arterial pressure (A-B), effective renal blood flow (C-D) and glomerular filtration rate (E-F) in anaesthetised 4-5 week-old WKY rats (left column) and pre-hypertensive SHRs (right column). Data shown are mean ± SEM for the baseline control period and the final 15 mins of the treatment period when effects were maximal. * P < 0.05, ** P < 0.01 compared with vehicle-treated rats, Dunnett’s test.</p

Immunolocalisation of urotensin system proteins in the pre-hypertensive SHR and WKY rat kidney.

<p>UII (A-F) and UT (G-L) proteins in the kidneys of 4-5-week-old WKY rats and SHRs. All images are representative and captured at x40 magnification (scale bar 50 µm). Images in the right hand column (C, F, I and L) represent typical negative control sections where primary antibody was omitted. UII-immunoreactivity in both the WKY rat (A) and the SHR (D) cortex was diffuse in distal (arrowheads) and proximal (black arrows) tubules, with no UII-immunoreactivity in glomeruli (red arrow, A). UII-immunoreactivity in both the WKY rat (B) and the SHR (E) medulla was located principally in the collecting ducts (arrowheads), with some immunoreactivity in the loops of Henle (arrows). In both the WKY rat (G) and the SHR (J) cortex UT-immunoreactivity was more intense in the distal (arrowheads) compared to proximal (black arrows) tubules, with UT-immunoreactivity absent in the SHR proximal tubules (black arrow, J); there was also some immunoreactivity in the glomeruli (red arrows). UT-immunoreactivity in both the WJY rat (H) and the SHR (K) medulla was localised to collecting ducts (arrowheads), with little immunoreactivity in the loops of Henle (arrows). </p

Histology of explants cultured for six days.

<p>Representative images for 3–5 organs in each condition. All sections counterstained with hematoxylin (blue nuclei). <b>A–D.</b> Note lack of cysts in control (<i>Vehicle</i>) explants and the progressively greater size (a typical cyst is boxed in each image) and extent of cysts per organs exposed to 470 nM dexamethasone (<i>Dex</i>)-alone, 100 µM 8-Br-cAMP (<i>cAMP</i>)-only, or both chemicals. <b>E–H.</b> Brown color indicates immunoreactivity to megalin antibody. Note patchy reactivity of cyst epithelia in organs exposed to 470 nM dexamethasone-alone, 100 µM 8-Br-cAMP-alone or both chemicals. <b>I–L.</b> Uromodulin immunoreactivity (brown); note that cysts are not labeled. <b>M–P.</b> Immunostaining with antibody to calbindin-28. Most cyst epithelia are unreactive (M, O and P) but a small subset of cysts in cAMP-exposed organs were positive and one such is depicted in O′. <b>Q–T.</b> These sections were probed with <i>Dolichos biflorus</i> lectin. Note that the lectin prominently labeled non-dilated tubules between cysts; in addition, the great majority of cysts did not bind the lectin. In (R), an asterisk indicates a rare, labeled cyst. Bar in A–D is 500 µm, and bar for other frames is 50 µm.</p

Whole mounts of E13 metanephroi cultured for six days.

<p><b>A.</b> A representative organ at Day 0 and the same organ at Day 6 for each condition. Bar is 500 µm. <b>B.</b> Enlarged images of a typical organ from control (<i>Vehicle</i>), 470 nM dexamethasone (<i>Dex</i>) alone, 100 µM 8-Br-cAMP (<i>cAMP</i>)-only, or co-treatment with both. Cysts appear in this dark field illumination as black circles or ovals. Bar is 500 µm. <b>C.</b> Percentages of total explant areas occupied by cysts. Each ♦ represents the value for a separate organ (n = 21 to 28 organs for each condition), with the bars indicating the group medians. Above each group is a letter; those designated by the same letter (e.g. “a”) are not significantly different from each other. In contrast, groups designated by different letters are significantly (P<0.05) different from each other (e.g. those marked “a” are different from all the other groups designated “b”, “c” and “d”).</p

Histology of rudiments exposed to low concentration of cystogenic chemicals.

<p><b>A–D.</b> After three days in culture, neither 47 nM dexamethasone (<i>Dex</i>) nor 2 µM Br-cAMP (<i>cAMP</i>) alone resulted in cyst formation; however, in combination, small cysts formed (asterisk in D). <b>E–H.</b> On day six of culture, neither 4.7 nM dexamethasone nor 1 µM Br-cAMP alone resulted in cyst formation; however, in combination, numerous cysts were noted (asterisks in H). Bar is 50 µm.</p

Histology of day three rudiments exposed to 100 µM 8-Br-cAMP (<i>cAMP</i>)-only.

<p><b>A and B.</b> Adjacent sections counterstained with hematoxylin. In A, several non-dilated tubules are stained brown (arrows), having bound <i>Dolichos biflorus</i> agglutinin; they most likely represent collecting ducts. The centre of each frame is dominated by a tubule which has a “U-turn”. Uromodulin was immunolocalised (brown color) in the undilated limb (indicated by the arrows in B). In contrast, epithelia in the adjacent limb, which is dilated (asterisk), were unreactive with the uromodulin antibody. The simplest deduction is that the dilatation is present in the descending limb of the loop of Henle. Bar is 100 µm.</p

F4/80 immunostaining of macrophages in rudiments at day 6 of culture.

<p>(<b>A</b>) Vehicle-only exposed organ. (<b>B</b>) Dexamethasone-only exposed organ. (<b>C</b>) 8-Br-cAMP-only exposed organ. (<b>D</b>) Organ exposed to both cystogens. Immunoreactive cells, presumed macrophages, are brown and some are indicted by arrows. Note that F4/80 macrophages were detected in all four conditions. In cystic explants, their numbers did not appear increased and, moreover, they were often located distant from cyst epithelia. Bar is 50 µm.</p

Histology of explants cultured for three days.

<p>Representative images are shown for 3–5 organs which have been examined in each condition. All sections counterstained with hematoxylin (blue nuclei). <b>A–D.</b> Note lack of cysts in control (<i>Vehicle</i>) and 470 nM dexamethasone (<i>Dex</i>)-only exposed organs, with plentiful dilated tubules and cysts (some of which are indicated by asterisks) in rudiments exposed to 100 µM 8-Br-cAMP (<i>cAMP</i>)-only, or co-treated with both chemicals. In these frames arrows point to glomeruli; note the “glomerulocystic” phenotype in explants exposed to both 8-Br-cAMP and glucocorticoid. <b>E–H.</b> Tubules and cysts reactive (brown) with antibody to aquaporin-1 are indicted by arrows. <b>I–L.</b> Brown colour indicates tubules immunoreactive with calbinin-28 antibody. Note that cyst epithelia are negative. Calbinin-28 was prominently detected in non-dilated tubules in all four conditions. <b>M–P.</b> Nuclei that are brown have reacted with phospho-histone antibody. Note the prominent proliferation within the stromal compartment in organs exposed to 470 nM dexamethasone, either alone (N) or with 8-Br-cAMP (P). In P, the arrow indicates a rare labeled nucleus in cyst epithelia. Bar is 50 µm.</p

Effects of forskolin on day three of culture.

<p>Higher power views of typical organs from vehicle (<b>A</b>), 470 nM dexamethasone alone (<b>B</b>), 10 µM forskolin-only (<b>C</b>), or co-treatment with both (<b>D</b>). Cystic structures appear as pale circles or ovals, and some of these are arrowed. Bar is 500 µm.</p