Subcellular localization of glucocorticoid receptor protein in the human kidney glomerulus

Subcellular localization of glucocorticoid receptor protein in the human kidney glomerulus.BackgroundThe detailed mechanisms of glucocorticoid action in idiopathic nephrotic syndrome and progressive glomerulonephritides have not been clearly elucidated. The pharmacological actions of glucocorticoids are mediated by their binding to an intracellular protein, the glucocorticoid receptor (GR). The determination of GR localization in normal glomerular cells is essential to elucidate the mechanisms of glucocorticoid action in various glomerular diseases.MethodsWe carried out an immunoblot examination using antihuman GR-specific antibody and homogenates of isolated normal human glomeruli and mesangial cells in culture. Immunohistochemical examinations were also performed on normal human kidney specimens at light and electron microscopic levels. The nuclear translocation of GRs elicited by ligand binding was further investigated by confocal laser-scanning microscopic inspection of freshly isolated glomeruli and mesangial cells cultured with dexamethasone.ResultsAn immunoblot examination demonstrated the presence of a 94 kDa protein, a molecular weight consistent with that of GRs, in the homogenates of glomeruli and cultured mesangial cells. By light microscopic examination, GRs were strongly detected in the nucleus and moderately in the cytoplasm of all glomerular cells, parietal and visceral epithelial cells, endothelial cells, and mesangial cells. By electron microscopic examination, the nuclear GRs of all glomerular cells were found to be diffusely distributed in the euchromatin. Additionally, the immunofluorescence intensities of nuclear GRs in isolated glomeruli and mesangial cells in culture became more intense by the addition of dexamethasone.ConclusionsOur findings suggest that all subsets of human glomerular cells definitely express the GR protein, which potentially undergoes translocation by glucocorticoids

The renormalization of the effective Lagrangian with spontaneous symmetry breaking: the SU(2) case

We study the renormalization of the nonlinear effective SU(2) Lagrangian up to $O(p^4)$ with spontaneous symmetry breaking. The Stueckelberg transformation, the background field gauge, the Schwinger proper time and heat kernel method, and the covariant short distance expansion technology, guarantee the gauge covariance and incooperate the Ward indentities in our calculations. The renormalization group equations of the effective couplings are derived and analyzed. We find that the difference between the results gotten from the direct method and the renormalization group equation method can be quite large when the Higgs scalar is far below its decoupling limit.Comment: ReVTeX, 12 figures, 22 pages, some bugs are kicked off from programs, numerical analysis is renew

Expression of SLC2A9 isoforms in the kidney and their localization in polarized epithelial cells.

BACKGROUND: Many genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Isoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type. CONCLUSIONS/SIGNIFICANCE: SLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking

Effect of N-terminal deletion of SLC2A9-L near the transmembrane side.

<p>GFP-tagged Long_30-50del (A), in which amino acids from 30th to 50th were deleted, or GFP-tagged Long_25-50del (B), in which amino acids from 25th to 50th were deleted from SLC2A9-L were expressed in MDCK cells. These constructs were visualized in green. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Conforcal images of xy and z axes were obtained and the magnification is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084996#pone-0084996-g003" target="_blank">Fig. 3</a>.</p

SLC2A9 expression in human kidney.

<p>SLC2A9-S (A,D) or SLC2A9-L (G,J) was stained together with fluorescein-Lotus Tetragonolobus lectin (LTL; green) (B,H) or rhodamine - Dolichos Biflorus agglutinin (DBA; red) (E,K). Overlay images staining with nucleus (blue) and with phase contrast images were shown in C, F, I and L. The scale bar of 30 µm was shown in L.</p

Characterization of isoform specific SLC2A9 antibodies.

<p>30 µg of COS cell lysate transfected with mock or SLC2A9s were separated by SDS-PAGE and Western blotting was performed with anti-SLC2A9-S (A), antigen pre-absorbed anti- SLC2A9-S (B), anti-SLC2A9-L (C), or antigen pre-absorbed anti-SLC2A9-L (D) antibodies. COS cells were transfected with GFP tagged SLC2A9-S (E and K) or SLC2A9-L (H and N) and immunofluorescence was performed with anti-SLC2A9-S (F and I) or anti-SLC2A9-L (L and O) antibodies. Overlay images were shown in G, J, M and P. The scale bar of 40 µm was shown in K.</p

Amino acids sequences of SLC2A9 N-termini of 2 wild types and mutant constructs.

<p>A. Two isoforms, SLC2A9-S and SLC2A9-L, have different N-termini of 21 and 50 amino acids, respectively. B, C. Deleted or mutated constructs of SLC2A9-S (B) and -L (C) were shown.</p

Localization of N-terminal deletion constructs of SLC2A9-S.

<p>GFP-tagged Short_16AAdel construct (green), which was deleted with N-terminal 16 amino acids from SLC2A9-S, was transfected into MDCK cells. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Conforcal images of xy and z axes were obtained and the magnification is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084996#pone-0084996-g003" target="_blank">Fig. 3</a>.</p

Effect of N-terminal deletion or exchange of SLC2A9-L.

<p>Long_25AAdel (A) or Long_30AAdel (B) constructs, in which N-terminal 25 or 30 amino acids were deleted from SLC2A9-L, respectively, Long_27-34A (C), in which amino acids from 27^th and 31st were changed to alanines, or Long_27-31del (D), in which 5 amino acids from 27^th and 31^st were deleted, were transfected into MDCK cells. These GFP-tagged constructs were visualized in green. Cell surface of the apical or basolateral membrane was biotinylated and visualized in red. Confocal images of xy and z axes were obtained and the magnification is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084996#pone-0084996-g003" target="_blank">Fig. 3</a>.</p