Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption
Ikhui Kho, Ekaterina P. Demina, Xuefang Pan, Irene Londono, Christopher W. Cairo, Luisa Sturiale, Angelo Palmigiano, Angela Messina, Domenico Garozzo, Roth-Visal Ung, Fabrice Mac-Way, Éric Bonneil, Pierre Thibault, Mathieu Lemaire, Carlos R. Morales, Alexey V. Pshezhetsky
Ikhui Kho, Ekaterina P. Demina, Xuefang Pan, Irene Londono, Christopher W. Cairo, Luisa Sturiale, Angelo Palmigiano, Angela Messina, Domenico Garozzo, Roth-Visal Ung, Fabrice Mac-Way, Éric Bonneil, Pierre Thibault, Mathieu Lemaire, Carlos R. Morales, Alexey V. Pshezhetsky
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Research Article Genetics Nephrology

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption

Abstract

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

Authors

Ikhui Kho, Ekaterina P. Demina, Xuefang Pan, Irene Londono, Christopher W. Cairo, Luisa Sturiale, Angelo Palmigiano, Angela Messina, Domenico Garozzo, Roth-Visal Ung, Fabrice Mac-Way, Éric Bonneil, Pierre Thibault, Mathieu Lemaire, Carlos R. Morales, Alexey V. Pshezhetsky

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Figure 2

Neu1ΔEx3 and Neu1Cx3cr1ΔEx3 homozygous mice show deficiency of NEU1 activity and increased lysosomal biogenesis in kidney tissues.

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Neu1ΔEx3 and Neu1Cx3cr1ΔEx3 homozygous mice show deficiency of NEU1 act...
(A) Total neuraminidase and NEU1 enzyme activity were measured in the tissue homogenates of 4-month-old WT, Neu1ΔEx3, and Neu1Cx3cr1ΔEx3 homozygous male and female mice, using fluorogenic substrate, 4-MU NANA, in the absence and in the presence of the NEU3/NEU4 inhibitor, C9-4BPT-DANA. Residual NEU1 activity was reduced to below detection levels in all studied tissues, except for the brain, where the NEU1 activity was reduced in Neu1ΔEx3 but not in Neu1Cx3cr1ΔEx3 mice. P values were calculated using 1-way ANOVA with Dunnett’s post hoc test. (B) mRNA levels of Neu1, Neu2, Neu3, and Neu4 were measured in the kidneys of 3 mice per genotype using quantitative reverse transcription PCR (RT-PCR). (C and D) Elevated levels of lysosomal β-galactosidase and β-hexosaminidase activities, characteristic of increased lysosomal biogenesis, were found in all studied tissues of Neu1ΔEx3 mice as well as in the kidney, spleen, lungs, and brain of female Neu1Cx3cr1ΔEx3 mice and showed a trend toward an increase in the tissues of males. P values were calculated using 1-way ANOVA with Tukey’s post hoc test. All graphs show individual data, means, and SD of experiments performed using tissues from 5 mice per genotype. (E) Increased levels of lysosomal proteins in kidney of Neu1ΔEx3 mice. Bar graph shows exclusive unique peptide counts for 15 most abundant lysosomal proteins. Proteomic analyses were performed using kidney protein extracts from 3 mice per sex per genotype. P values for the exclusive unique peptide counts were calculated using 2-way ANOVA with Holm-Šídák post hoc test. (F) Immunohistochemical analysis shows increased TFEB levels (shown in green) in the nuclei of endothelial cells in proximal tubules of Neu1ΔEx3 mice. DAPI (blue) was used as nuclear counterstain. Bar graph shows quantification (individual data, means, and SD, n = 3) of TFEB/DAPI-labeled areas by ImageJ software (NIH). P values were calculated by unpaired 2-tailed t test.