Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells

Abstract

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Fig. 1.

Effect of gene deletion on plasma selenoproteins and selenium. A: mice were injected with a tracer dose of ⁷⁵SeO₃²⁻, and plasma was obtained 24 h later. The Gpx3^−/− mouse had been fed a diet supplemented with 0.25 mg selenium/kg, and the other 3 mice had been fed a diet supplemented with 1 mg selenium/kg. Plasma samples (0.5 μl) from C57BL/6 (Sepp1^+/+/Gpx3^+/+), Sepp1^−/−, Gpx3^−/−, and Sepp1^−/−/Gpx3^−/− mice were subjected to SDS-PAGE on a 10% acrylamide gel, and the dried gel was exposed to Kodak XAR film for 3 days before being developed. B: plasma from C57BL/6 mice and Sepp1^−/−/Gpx3^−/− mice was obtained 4 wk after weaning. The C57BL/6 mice (n = 4) had been fed the diet supplemented with 0.25 mg selenium/kg, and the Sepp1^−/−/Gpx3^−/− mice (n = 6) had been fed the diet supplemented with 1.0 mg selenium/kg for 2 wk followed by the diet supplemented with 0.25 mg selenium/kg for 2 wk. The half-brackets indicate 1 SD, and the 2 values were significantly different (P < 0.001) by Student's t-test.

Fig. 2.

Plasma (A–C) and tissue (D–G) selenium markers in Gpx3^−/− (■) and Gpx3^+/+ (☐) mice fed selenium-deficient and selenium-supplemented diets. Values are means, n = 5, and the half-brackets indicate 1 SD. The Gpx3^−/− values in A that are marked with crosses are different from the selenium-deficient value (P < 0.05) but not from one another by ANOVA. Pairs marked by asterisks in panels B–G are significantly different (P < 0.05) by Student's t-test.

Fig. 3.

Gpx3 in kidney centrifugal fractions. A: Western blot after SDS-PAGE of 600-g supernatants (40 μg protein/lane) of 10% homogenates of the kidneys of 3 Gpx3^−/− (lanes 1–3) and 3 Gpx3^+/+ (lanes 5–7) mice. Plasma (1 μl) from a Gpx3^+/+ mouse is in lane 4 as a positive control. B: Western blot after SDS-PAGE of the 600-g pellets (40 μg protein/lane) of the same kidneys. In B, lane 4 did not contain a sample. C: assessments of Gpx3 after SDS-PAGE (NuPAGE gels) of kidney homogenates (Gpx3^+/+ in lanes 1 and 4, Gpx3^−/− in lanes 2 and 5; each lane is loaded with homogenate from 12.5 μg of kidney), 1 μg purified plasma Gpx3 (lanes 3 and 6), and 0.5 μl ⁷⁵Se-labeled plasma (lanes 7 and 8). Lanes 1–3 were stained with Coomassie blue for protein loading. Lanes 4–6 and 8 were immunostained with anti-Gpx3. Lane 7 is an autoradiogram.

Fig. 4.

Kidney homogenates from Gpx3^+/+ mice fed a diet supplemented with 0.25 mg selenium/kg were prepared in either 0.15 M NaCl-containing (lanes 1–6) or 1 M NaCl-containing (lanes 7–12) homogenizing buffer. Samples were prepared in homogenizing buffer alone (lanes 1, 2, 7, and 8), homogenizing buffer containing 20 mM DTT (lanes 3, 4, 9, and 10), or homogenizing buffer containing 20 mM DTT and 0.25% Triton X-100 (lanes 5, 6, 11, and 12). Homogenates were centrifuged, and the supernatant (S) and pellet (P) fractions were analyzed for Gpx3 content by Western blot analysis. Gpx3 fractionated to the pellet (P) fraction in all 0.15 M NaCl homogenates. In contrast, Gpx3 was released to the supernatant (S) fraction in all homogenates prepared with 1 M NaCl. Each lane represents the P and S fractions from 62 μg of intact kidney.

Fig. 5.

Immunohistochemical localization of Gpx3 in the kidney. Matched phase-contrast and immunofluorescence images of anti-Gpx3-stained (red) cryosections of Gpx3^+/+ and Gpx3^−/− kidneys. A and B: outer cortex of a Gpx3^+/+ kidney. Intense staining is detected at the perimeter of the proximal tubules (PT) and of the urinary pole (arrowheads) of the glomerulus (G). Less intense staining is detected at the perimeter of the distal tubules (DT). C and D: outer medulla of a Gpx3^+/+ kidney. The Gpx3 staining terminates abruptly at the junction of the outer and inner stripes (arrows), where the PT terminate and transition into descending thin limbs. E and F: no Gpx3 expression is detected in the cortex of a Gpx3^−/− kidney.

Fig. 6.

Tissue overlay analysis of Gpx3^−/− kidney demonstrating Gpx3 binding (red) to the perimeter of cortical tubules. A: control experiment showing an absence of Gpx3 staining in Gpx3^−/− kidneys. B and C: cryosections of Gpx3^−/− kidneys incubated with Gpx3 before immunostaining with anti-Gpx3. B: cortical tubules show specific staining at their perimeter. C: Gpx3 binding to the terminal portion of PT in the outer medulla where they abruptly terminate (arrows) and transition to the descending thin limbs. Medullary tubules located below this junction (arrows) show no Gpx3 binding.

Fig. 7.

Gpx3 in kidney basement membranes. A: phase-contrast photomicrograph of kidney basement membrane-enriched fraction showing phase lucent sheetlike structures. Insets show that these structures are intensely immunostained (red) by anti-collagen IV (α-Col IV) and anti-laminin (α-laminin). B and C: matched phase-contrast and fluorescence images of basement membrane from Gpx3^+/+kidney immunostained with anti-Gpx3 (red). D and E: phase-contrast and fluorescence images of Gpx3^−/− kidney basement membrane immunostained with anti-Gpx3. No Gpx3 is detected. F and G: phase-contrast and fluorescence images of Gpx3^−/− kidney basement membrane that had been incubated with purified Gpx3 before immunostaining with anti-Gpx3. Gpx3 binding to the basement membrane is evident.