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Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome - PubMed

Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome

S Doublier et al. Am J Pathol. 2001 May.

Abstract

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

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Figures

**Figure 1.**
Immunofluorescence staining for nephrin in glomeruli of normal controls (A), and of patients with membranous GN (B, C, and D). Co-localization of nephrin (green) and IgG (red) was evaluated in glomeruli of membranous GN patients by double IF. C: Distribution of nephrin. The overlap of staining for nephrin and IgG results in a yellow staining of granular deposits in the merge (D). Original magnifications: ×400 (A and B); ×600 (C and D).

**Figure 2.**
Immunofluorescence staining for nephrin in glomeruli of minimal change GN (A and B), FSGS (C), and IgA GN (D). Original magnifications: ×400 (**A–D**).

**Figure 3.**
Semiquantitative analysis of nephrin expression in glomeruli of normal control; membranous GN; and minimal change GN, FSGS, and IgA GN patients. **, P < 0.001 *versus* control patients.

**Figure 4.**
Linear regression analysis between relative fluorescence intensity for nephrin staining and extent of proteinuria in all GN patients (membranous GN and minimal change GN, FSGS, and IgA GN).

**Figure 5.**
A: Expression of the GAPDH mRNA (**lane 2**) and the nephrin mRNA (**lane 3**) in GECs. **Lane 1:** Molecular weight marker (range, 200 to 2,000 bp). B: Analysis of the expression of nephrin by Western blot in nonconfluent GECs (**lane 2**) and in confluent GECs (**lane 3**). **Lane 1** shows an irrelevant antibody (anti-E-selectin) in GECs. **Lane 4:** The absence of nephrin in a renal tubular epithelial cell line.

**Figure 6.**
Immunofluorescence staining for nephrin on GECs incubated 60 minutes with vehicle alone (A), agIgG₄ (1 μg/ml) (B and C), disaggregated IgG₄ (1 μg/ml) (G), TNF-α (10 ng/ml) (H), or after the plasma membrane insertion of MAC (I). Within 24 hours of removal of TNF-α from the culture medium, nephrin was fully re-expressed on GECs (J). Reduction of fluorescence intensity and morphological evidences of nephrin redistribution after incubation with TNF-α were prevented by pre-incubation with sodium azide (K) and cytochalasin B (L). FITC-phalloidin staining of actin microfilaments on permeabilized GECs was performed after incubation for 60 minutes with vehicle alone (D) or agIgG₄ (1 μg/ml) (E and F). Double staining for nephrin (green) and for IgG (red) on GECs incubated for 60 minutes with agIgG₄ (1 μg/ml) showed focal co-localization on the cell surface (B, **inset**). Nuclei were stained with ethidium bromide (dilution 1/1,000). Original magnifications: ×600 (**A–L**); ×240 (**inset**).

**Figure 7.**
A: Semiquantitative analysis of nephrin expression on GECs incubated for 60 minutes with vehicle alone (control), agIgG₄ (1 μg/ml), TNF-α (10 ng/ml), puromycin (5 μg/ml), or after the plasma membrane insertion of MAC. **, P < 0.001 *versus* control. B: Dose effect of sodium azide pre-incubation (10⁻³ mol/L to 10⁻¹ mol/L) on immunofluorescence staining for nephrin on GECs incubated for 60 minutes with agIgG₄ (1 μg/ml) or TNF-α (10 ng/ml). C: Dose effect of cytochalasin B pre-incubation (5 μg/ml and 10 μg/ml) on immunofluorescence staining for nephrin on GECs incubated for 60 minutes with agIgG₄ (1 μg/ml) or TNF-α (10 ng/ml). Results are expressed as percent of control (GECs incubated for 60 minutes with vehicle alone).

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Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome - PubMed