The mutation (R823W) in PKD/Mhm(<i>cy/cy</i>) rats does not alter ANKS6-ANKS3 renal co-localisation.

<p>Immunohistochemical staining of renal cortex and medulla was performed in 3 week-old wild-type and PKD/Mhm(<i>cy/cy</i>) rats for ANKS3 (upper panel) and ANKS6 (lower panel).</p

<i>In vivo</i> LNA ASO-induced downregulation of<i>Anks3</i> expression in mouse kidney.

<p>(<b>A</b>) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (<b>B-D</b>) <i>Anks3</i> renal decreased expression does not affect expression of <i>Anks6</i>. Abundance of <i>Anks3</i> transcripts (<b>B</b>) and protein (<b>C</b>), and <i>Anks6</i> transcripts (<b>D</b>) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to <i>Actb</i> gene expression level. (<b>C</b>) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.</p

<i>Anks3</i> expression knock-down <i>in vivo</i> stimulates expression of genes involved in the vasopressin signaling pathway.

<p>(<b>A</b>-<b>B</b>) Renal mRNA expression of <i>Vit32</i>, <i>Aqp1</i>, <i>Aqp2</i> and <i>Aqp3</i> was evaluated by quantitative RT-PCR in mice four days (n = 3)(<b>A</b>) and twelve days (n = 5)(<b>B</b>) after the final injection of Locked Nucleic Acid modified AntiSense Oligonucleotides (LNA ASO). Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression level. (<b>C</b>) AQP2 expression in kidney tissues. Representative Western blots performed with protein extracts from 5 kidneys treated with scrambled (SCR) or ANKS3 ASO (top) and quantitative analysis of blots normalized to β-actin expression (bottom) show that AQP2 expression is significantly increased in mice twelve days after the final injection of ANKS3 ASO when compared to mice injected with SCR ASO. (<b>D</b>, <b>E</b>) Immunofluorescence staining of the papilla for AQP2 (<b>D</b>) and AVPR2 (<b>E</b>) show a dramatic increase of the proteins in kidneys of ANKS3 ASO treated mice twelve days after the final injection compared to SCR ASO controls. Scale bar: 300 μm. Data are means ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice injected with saline (Ctr)- or SCR ASO. *P<0.05; **P<0.01 significantly different to controls.</p

Identification of ANKS3, a partner of ANKS6.

<p>(<b>A</b>) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (<b>B</b>) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (<b>C</b>) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (<b>D</b>) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.</p

Induction of apoptosis and proliferation in <i>in vivo Anks3</i> knock-down mice.

<p>(<b>A</b>) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(<b>B</b>, <b>C</b>) Abundance of caspase 9 (<b>B</b>) and caspase 3 (<b>C</b>) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (<b>D</b>) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (<b>E</b>) <i>p53</i> mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.</p

Organ expression, physiological interaction and renal location of ANKS3 and ANKS6 in the mouse.

<p>ANKS3 and ANKS6 are strongly expressed in the mouse kidney.<i>Anks3</i> (<b>A</b>) and <i>Anks6</i> (<b>B</b>) mRNA expression in kidney, liver, pancreas and spleen was evaluated by quantitative RT-PCR. Data are shown as means ± SEM. Quantification of cDNA from 3 mice was normalized to <i>Actb</i> gene expression level. One-way ANOVA was applied to assess statistical significance. P<0.0001 significantly different to renal expression of <i>Anks3</i> and <i>Anks6</i>. (<b>C</b>) Co-localisation of ANKS3 and ANKS6 in the kidney. Representative photomicrographs of 6 kidney sections. ANKS3 staining (revealed by Alexa Fluor 488 in green, left column) co-localises (merge staining in central column) with ANKS6 (right vertical column, revealed by Alexa Fluor 594 in red) in glomeruli (upper panel, scale bar: 90μm) and in tubules (lower panel, scale bar: 300μm. (<b>D</b>) Immunoprecipitation experiments performed with goat anti-ANKS6 antibody, goat anti-ANKS3 antibody or goat IgGs on protein lysates and followed by an immunoblot performed with rabbit anti-ANKS3 antibody (top) and rabbit anti-ANKS6 antibody (bottom). Note that ANKS6 antibody co-immunoprecipitates ANKS3 in kidney protein lysates.</p

Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

<div><p>Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.</p></div

Immunolocalization of GFAP (A) and nestin (B) in retinal slices.

<p>The slices were obtained from control (SD; <i>above</i>) and transgenic (TG) rats (<i>below</i>). Cell nuclei were labeled with Hoechst 33258 (<i>blue</i>). Note the age-dependent thinning of the outer nuclear layer (ONL) in the tissues of TG rats. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; NFL, nerve fiber layer; OPL, outer plexiform layer. Bars, 20 µm.</p

Age-dependent alterations in the immunolocalization of AQP1.

<p>Retinal slices were obtained from control (SD; <i>above</i>) and transgenic (TG) rats (<i>middle and below</i>). Cell nuclei were labeled with Hoechst 33258 (<i>blue</i>). The images <i>below</i> display co-immunolabeling of retinal slices against AQP1 (<i>green</i>) and GFAP (<i>red</i>). Double labeling of both proteins yielded a <i>yellow-orange</i> merge signal. <i>Arrows</i>, AQP1-positive amacrine cells. <i>Filled arrowheads</i>, AQP1-positive Müller cell fibers. <i>Unfilled arrowheads</i>, GFAP- and AQP1-positive glial processes that surround large vessels. *, large vessel. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 20 µm.</p

Retinal gene expression of GFAP, vimentin, nestin, Kir4.1, AQP1, and AQP4.

<p>The expression was determined in the neural retina of 3-months transgenic rats (n = 4) compared to the gene expression in the retina of age-matched control rats (n = 4). Significant difference <i>vs</i>. control: *<i>P</i><0.05.</p