MPM imaging conditions in a kidney of a MWF rat in vivo.

<p>(A) Image of a superficial glomerulus (g) and a distal tubule (dt) in the kidney of a MWF rat. Proximal tubules (pt) showed green autofluorescence. Intra- and extraglomerular capillaries (arrows) were visualized with Texas Red-labeled 70 kDa dextran. The glomerulus was located directly underneath the renal capsule allowing high resolution imaging. (B) Histology of a MWF rat kidney section with many superficial glomeruli (arrows).</p

z-stack of the postcentral gyrus of the mouse brain in vivo.

<p>(A)–(E) show blood vessels (arrows), stained by Texas Red 70 kDa Dextran in 5, 60, 120, 200 and 300 µm underneath the brain surface. Internal detectors were used with maintained settings for gain and offset. a, left column: Without laser adjustment all blood vessels within the 300 µm range were visible, but appeared darker and blurry below a depth of 200 µm. b, right column: the same region acquired with adjustment of the laser power. In contrast to the liver and kidney a much deeper penetration into the tissue was possible.</p

Depth of glomeruli in 10-week-old adult mice.

<p>Box-plot shows mean±25% (boxes) and the 5–95% percentile (whiskers). (A) Box-plot of all ten strains arranged according to the 25% percentile. (B) Box-plot of the peer group (129, CB6F1, C3H/N, NMRI, SJL, DBA/2, FVB, CD-1) vs. Bl/6 and BALB/c.</p

Depth of glomeruli in 4-week-old juvenile mice.

<p>Box-plot shows mean±25% (boxes) and the 5–95% percentile (whiskers). (A) Box-plot of all ten strains arranged according to the 25% percentile. (B) Box-plot of the peer group (129, CB6F1, NMRI, SJL, FVB, CD-1) vs. Bl/6, BALB/c, C3H/N and DBA/2.</p

AngII-evoked Ca²⁺ response is mediated by PLC/IP3 pathway in porcine RPE cells.

<p>Application of AngII for 80 seconds (bars) caused transient Ca²⁺response in pRPE cells. <i>A:</i> Bath application of 100 nM AngII (bars) together with 10 µM U73122 (gray shadow), a phospholipase C (PLC) blocker abolished AngII-evoked Ca²⁺signal in pRPE cells. On the left panels of Figures A and C, it is shown the effect of AngII application under control conditions. In the same cell, after washing out first AngII application (until [Ca²⁺]_I returned back to basal levels) it was further perfused 100 nM of AngII in the presence of U73122 (right panel in A) or xest C (right panel in C). The wash out is not shown and the x-axis is interrupted accordingly between the panels. Note that application of U73122 alone led to a slight increase in intracellular free Ca²⁺. <i>B</i>: summary of data from experiments shown in <i>A</i>. <i>C</i>: Co-application of 100 nM AngII (bar) with the IP3 blocker xestospongin C (xest C) (gray shadow) at 10 µM reduced AngII-evoked Ca²⁺response in pRPE cells. <i>D</i>: summary of data from experiments shown in <i>C</i>. Bars in Fig. 2B and D represent means ± SEM for AngII-evoked Ca²⁺responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars). *<i>p</i><0.05, (***; ###) <i>p</i><0.0001; repeated measures ANOVA. n = number of cells from 4 (<i>B</i>) or 5 (<i>D</i>) independent experiments.</p

Lack of Atrap reduces AngII-mediated Ca²⁺ signaling in mouse RPE cells.

<p>RT-PCR from freshly isolated mouse <i>Atrap^+/+</i>RPE cells shows expression of both AT1 receptor paralogs (AT1R 1A and AT1R 1B; 565 bp) (<i>A</i>) and Atrap; 194 bp (<i>B</i>). βactin mRNA (540 bp) from retinal and kidney tissues served as control. <i>C</i>: Western Blot analysis indicates the expression of both AT1R and Atrap in mouse RPE cells. <i>D</i>: Immunostaining of <i>Atrap</i>^+/+and <i>Atrap</i>^−/− mouse retinas using antibodies against AT1R (arrows, left panels) and Atrap (arrows, right panels). Insets represent an enlarged confocal area around the arrows, indicating basolateral localization of AT1R (arrowheads, upper left panel) and Atrap (arrowheads, upper right panel) at both the basolateral and the apical side. Differential Interference Contrast (DIC) image illustrates the retinal layers as outer plexiform layer (OPL), outer nuclear layer (ONL) and retinal pigment epithelium (RPE). <i>E</i>: Traces show transient Ca²⁺response from <i>Atrap</i>^+/+(mean, white line; SEM in black) and <i>Atrap</i>^−/− (mean, black line; SEM in gray) RPE cells upon 80 seconds AngII (100 nM) stimulation (bars). AngII-evoked Ca²⁺response was smaller in <i>Atrap</i>^−/− mouse cells than that from <i>Atrap</i>^+/+. <i>F</i>: Summary of data from experiments shown in <i>E</i>. Bars in Fig. 3F represent means ± SEM for AngII-evoked responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars). (*; #) <i>p</i><0.05; ANOVA analysis. n = number of cells from 15 independent experiments.</p

TRPV2 channels is present in porcine RPE cells.

<p><i>A:</i> RT-PCR using mRNA from cultured porcine (pRPE) cells. mRNA from retinal and lung tissues were used as control. Porcine RPE robustly expressed TRPV2 (343 bp). <i>B</i>: <i>left panel,</i> 15 µM cannabidiol (CBD) was applied for a period of 10 min (bars) where it caused a reversible Ca²⁺response in a pRPE cell. <i>Right panel:</i> Bath application of 15 µM CBD (bar) together with 100 µM SKF96365, a TRPV channel inhibitor (gray shadow) reduced the CBD-evoked Ca²⁺signal in another pRPE cell. <i>C</i>: summary of data from experiments shown in <i>B</i>. <i>D</i>: Application of AngII for 80 seconds (bars) caused transient Ca²⁺response in a pRPE cell. In the same cell, after washing out AngII until [Ca²⁺]_I returned back to resting levels, AngII-mediated Ca²⁺signal was prevented by application of 100 µM SKF96365 (gray shadow). Note the wash out is not shown and the x-axis is interrupted accordingly between the panels. <i>E</i>: summary of data from experiments shown in <i>D</i>. Bars in Fig. 4C and E represent respectively means ± SEM for CBD- or AngII-evoked Ca²⁺responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars). (**; ##) <i>p</i><0.001, (***; ###) <i>p</i><0.0001; repeated measures ANOVA. n = number of cells from 5 (<i>C</i>) or 6 (<i>E</i>) independent experiments.</p

Angiotensin-2-Mediated Ca²⁺ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

<div><p>Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca²⁺inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca²⁺response. RPE cells from Atrap^−/− mice showed smaller AngII-evoked Ca²⁺peak (by 22%) and loss of sustained Ca²⁺elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca²⁺-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca²⁺-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca²⁺transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca²⁺transients in the RPE by releasing Ca²⁺from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca²⁺elevation.</p> </div

AngII-evoked Ca²⁺ response is mediated by TRPV2-dependent Ca²⁺ influx in porcine RPE cells.

<p><i>A</i>: Western blot analysis indicated successful suppression of TRPV2 channel by RNAi. Transfection with mock RNA served as control. <i>B</i>: Densitometry analysis of each lane in the western blot shown in <i>A</i>. TRPV2- RNAi treated cells (green) showed a down-regulation of TRPV2 protein in about 55% compared to mock transfected cells (gray). TRPV2 bands in each group (mock and RNAi) were normalized to actin. Bars represent means ± SEM; * <i>p</i><0.05, paired <i>t</i> test. Densitometry analysis results from 3 different blots; n = 3. <i>C</i>: Representative Fura 2-AM loaded cells (in red) and cells transfected with either TRPV2-RNAi or mock RNA- bound to Alexa 488 fluorescent dye (green dots) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049624#s2" target="_blank">methods</a>). Scale bar, 30 µm. <i>D</i>: TRPV2-RNAi treated cells (green trace) showed a quick reduction (within 60 s, arrow) of AngII-evoked Ca²⁺(bar) elevation when compared to non-transfected (black trace) or mock-transfected cells (gray trace). Application of AngII at 100 nM for 80 seconds (bar) evokes similar Ca²⁺responses in mock (gray trace) or non-transfected (black trace) porcine RPE cells. Transfection with mock RNA (gray trace) did not alter the sustained AngII-evoked Ca²⁺elevation (delayed recovery phase) as TRPV2-RNAi did. Sixty seconds after the maximum AngII-elicited calcium response is shown by arrows. <i>E</i>: Summary of data from experiments shown in <i>D</i>. Black (for non-transfected cells), green (for TRPV2-RNAi) and gray (for mock) transfected cells. Bars represent means ± SEM of the difference between AngII-evoked Ca²⁺signal at the peak and 60 seconds after the peak respectively. (**) <i>p</i><0.001; repeated measures ANOVA. n.s = not significant. n = number of cells from 26–41 (<i>E</i>) independent experiments. <i>A:</i> Application of AngII (100 nM) for 80 seconds (bars) caused transient Ca²⁺response in pRPE cells. Bath application of 100 nM AngII (bars) produced a Ca²⁺response that was not abolished by co-application of 10 µM U73343 (gray shadow), the inactive analog of the phospholipase C (PLC) blocker U73122. <i>B</i>: summary of data from experiments shown in <i>A.</i> Bars in Fig. 2B represent means ± SEM for AngII-evoked Ca²⁺responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars). (*; #) <i>p</i><0.05, ** <i>p</i><0.0001; repeated measures ANOVA. n = number of cells from 9 independent experiments.</p

Porcine RPE cells constitute a suitable model to study AngII-evoked Ca²⁺

<p><b>response. </b><i>A</i>: Freshly isolated porcine RPE (pRPE) in culture formed a pigmented monolayer. Scale bar, 40 µm. <i>B</i>: RT-PCR using mRNA from cultured pRPE cells expressed both Atrap (182 bp) and AT1R (203 bp), GAPDH (340 bp) was used as control. <i>C</i>: Bath application of AngII at 100 nM for 80 seconds (bars) evoked Ca²⁺responses in a pRPE cell. In the same cell, after washing out 100 nM AngII (until [Ca²⁺]_I returned back to resting levels) further application of AngII led to a second rise in [Ca²⁺]_i. <i>D</i>: Summary of data from experiments shown in <i>C</i>. <i>E</i>: Losartan at 10 µM inhibited AngII-evoked Ca²⁺responses in pRPE cells. On the left panel it is shown the effect of AngII application under control conditions. In the same cell, after washing out AngII (until [Ca²⁺]_I returned back to resting levels) AngII was applied in the presence of losartan (right panel). The wash out is not shown and the x-axis is interrupted accordingly between the panels. Note that losartan itself led to an increase in intracellular free Ca²⁺. <i>F</i>: Summary of data from experiments shown in <i>E</i>. Bars in Fig. 1D and F represent means ± SEM for AngII-evoked Ca²⁺responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars). * <i>p</i><0.05, (***; ###) <i>p</i><0.0001; repeated measures ANOVA. n = number of cells from 4 (<i>D</i>) or 7 (<i>F</i>) independent experiments.</p