A schematic view of V-ATPase subunits.

<p>The exonic structure of all V-ATPase subunits is shown; untranslated regions are shown in light grey while protein coding regions are black. The intronic distances have been divided by 15 for display purposes. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009531#pone-0009531-t001" target="_blank">Table 1</a> for further information on subunits.</p

Characterizing the Interactions of Organic Nanoparticles with Renal Epithelial Cells <i>in Vivo</i>

Nanotechnology approaches are actively being pursued for drug delivery, novel diagnostics, implantable devices, and consumer products. While considerable research has been performed on the effects of these materials on targeted tumor or phagocytic cells, relatively little is known about their effects on renal cells. This becomes critical for supersmall nanoparticles (<10 nm), designed to be renally excreted. The active endocytic machinery of kidney proximal tubules avidly internalizes filtered proteins, which may also be the case for filtered nanoparticles. To test whether such interactions affect kidney function, we injected mice with either 5 nm dextran-based nanoparticles (DNP) that are similar in composition to FDA-approved materials or poly(amido amine) dendrimer nanoparticles (PNP) of comparable size. These fluorescently tagged nanoparticles were both filtered and internalized by renal tubular epithelial cells in a dose- and time-dependent fashion. The biological effects were quantitated by immunocytochemistry, measuring kidney injury markers and performing functional tests. DNP administration resulted in a dose-dependent increase in urinary output, while cellular albumin endocytosis was increased. The expression of megalin, a receptor involved in albumin uptake, was also increased, but AQP1 expression was unaffected. The effects after PNP administration were similar but additionally resulted in increased clathrin expression and increased endocytosis of dextran. We conclude that there are no major detrimental renal effects of DNP on overall kidney function, but changes in endocytosis-mediating protein expression do occur. These studies provide a framework for the testing of additional nanoparticle preparations as they become available

Dissolution of the AQP2 perinuclear patch after cold block release.

<p>Cells were cold blocked for 120 minutes to form the perinuclear patch after which the cold block was released by returning the cells to 37°C. The perinuclear patch rapidly disintegrated over 30 minutes of cold block release as the AQP2 mutants (except for AQP2-S256D which remained on the membrane) redistributed throughout the cytosol and membrane. Scale bar = 20 µm.</p

Quantified formation of AQP2 perinuclear patch after cold block at 20°C in cells expressing various AQP2 phosphorylation mutants.

<p>The time course of the development of the AQP2 perinuclear patch was quantified by measuring mean pixel intensity of the patch using IPlab software. The results are presented as the increase in mean patch fluorescence value starting from 0 minutes of cold block. The overall accumulation of the perinuclear patch reached a maximal density after cold block for 150 minutes with all mutants. The experiment was repeated in triplicate, N for each mean is ≥24. Bars represent standard error.</p

Association of AQP2 and cellular compartment markers during the dynamic process of cold block and cold block release.

<p>The association of subcellular compartment markers with AQP2 during the cold block and subsequent release was investigated. GM130, clathrin, EEA1, and HSP/HSC70, were co-stained with AQP2 (A, B, C, D). Panel A, GM130 staining also overlaps with AQP2 in the perinuclear patch but not after release of the cold block. In panel B, clathrin appeared to colocalize mostly with AQP2 during the development of the perinuclear patch and redistribution after cold block release. Panel C, EEA staining was colocalized with AQP2 at the early stage of endocytosis, and was partially associated with the perinuclear patch. After CBR at 37°C, a few large EEA/AQP2 positive structures appeared, but most AQP2 was not associated with EEA. Panel D, HSP/HSC70 partially colocalized with AQP2 during the formation of the perinuclear patch, but not during AQP2 redistribution after cold block release. Scale bar = 20 µm.</p

Formation of AQP2 perinuclear patch after cold block at 20°C in cells expressing various AQP2 phosphorylation mutants.

<p>After pretreatment with cycloheximide for one hour, cells underwent cold block at 20°C for up to 150 minutes. Cells were fixed and subjected to staining with anti-c-myc antibody to detect AQP2. During 120 minutes of cold block the development of the perinuclear AQP2 patch is evident in all cells expressing AQP2 variants except for AQP2-S256D, which remains mainly on the cell membrane (LLC 256D). Scale bar = 20 µm.</p

Quantified dissolution of the AQP2 perinuclear patch after cold block release.

<p>To quantify the rate of AQP2 redistribution a time course analysis of the dissolution of the patch was assayed by immunofluorescence staining and images were quantified using the IPlab software. The results are presented as the decrease from the maximum mean patch fluorescence value after release of cold block. The experiment was repeated in triplicate N for each mean is > = 24. Bars represent standard error.</p

Home-cage tests assessing behavior of wild-type and B1-deficient female mice in investigating urine of heterozygous (Atp6v1b1+/−) male mouse urine.

<p>Water presentations revealed no significant difference between wild type and B1-deficient mice or between presentations. Both urine presentations showed that wild-type mice spent significantly more time investigating the urine of the opposite sex than Atp6v1b1−/− mice (n = 6). Data are shown as mean ± SEM (**, p<0.01).</p

Reduced odor-evoked freezing by a predator odor.

<p>(A) A schematic diagram of the behavioral arena is depicted. (B) The percentage of time freezing in adult male B1-deficient mice (“KO”, n = 7) in the presence of the predator odor trimethyl-thiazoline (TMT) is less than half the time recorded in wild-type mice (“WT”, n = 6). Data are shown as mean ± SEM (p = 0.02). (C) Increased investigation of TMT in the V-ATPase B1-deficient mice: The number of individual sniffs of TMT is increased in adult male B1-null mice (“KO”, black bar) relative to wild-type mice (“WT”, gray bar). Data is shown as mean ± SEM (p = 0.05) and confirm the blunted response to TMT in B1-deficient mice compared to their wild type counterparts.</p

Dual immunofluorescence labeling for the V-ATPase B1 subunit isoform (red) and cytokeratin-18 (CK-18, green) in the olfactory mucosa of an adult male mouse decalcified with Cal-Ex.

<p>As also shown in the previous figure for the A subunit, B1 localizes to the apical membrane microvilli of SCs and to the basolateral membrane of a subset of OE cells (A and D, arrows). Interestingly, these cells also exhibit basolateral membrane staining for CK-18 (B and E, arrows). The merge panels (C and F) confirm the co-expression of the V-ATPase B1 subunit and CK-18. Bar = 20 µm.</p