Deficits in sialylation impair podocyte maturation

Abstract

The role of sialylation in kidney biology is not fully understood. The synthesis of sialoglycoconjugates, which form the outermost structures of animal cells, requires CMP-sialic acid, which is a product of the nuclear enzyme CMAS. We used a knock-in strategy to create a mouse with point mutations in the canonical nuclear localization signal of CMAS, which relocated the enzyme to the cytoplasm of transfected cells without affecting its activity. Although insufficient to prevent nuclear entry in mice, the mutation led to a drastically reduced concentration of nuclear-expressed enzyme. Mice homozygous for the mutation died from kidney failure within 72 hours after birth. The Cmas(nls) mouse exhibited podocyte foot process effacement, absence of slit diaphragms, and massive proteinuria, recapitulating features of nephrin-knockout mice and of patients with Finnish-type congenital nephrotic syndrome. Although the Cmas(nls) mouse displayed normal sialylation in all organs including kidney, a critical shortage of CMP-sialic acid prevented sialylation of nephrin and podocalyxin in the maturing podocyte where it is required during the formation of foot processes. Accordingly, the sialylation defects progressed with time and paralleled the morphologic changes. In summary, sialylation is critical during the development of the glomerular filtration barrier and required for the proper function of nephrin. Whether altered sialylation impairs nephrin function in human disease requires further study.

Figure 1.

Targeted mutagenesis of CMAS. (A) The NLS (K¹⁹⁸RPRR) in exon 4 of Cmas was targeted by mutations marked in blue (nls). A neomycin resistance (neo) cassette flanked by loxP sites (triangles) was inserted into intron 4 and a diphtheria-toxin (DT) cassette was inserted into intron 3. Correct homologous recombination was detected by 5′ outside and 3′ outside primers combined with neo primer pairs. A 1-kb fragment generated by inside primers was used as a control. (B) PCR products from targeting vector (TV), targeted allele (TA) from Cmas^{nls neo} embryonic stem cells, and wild-type (wt) embryonic stem cells and DNA marker. (C) P0.5 homozygous Cmas^nls mutants were indistinguishable from wt with respect to gross anatomy and coat color. nls refers to the mutant Cmas^nls allele lacking the neo after CRE-mediated excision.

Figure 2.

Cmas^nls mice develop massive proteinuria. (A) The protein/creatinine ratio was determined in individual samples at P0.5 (n=5) and P1.5 (n=5) of each genotype. (B) Silver staining (upper panel) and albumin immunoblot (lower panel) of urine from P1.5 wt (n=2), nls/+ (n=2), and nls/nls (n=3) individual mice.

Figure 3.

Destruction of the glomerular filtration barrier in nls/nls mice. (A, B, I and J) Hematoxylin and eosin–stained paraffin sections of renal cortex from (A and I) wt and (B and J) nls/nls mice. Whereas morphologic changes are not obvious at P0.5 in nls/nls mice (B), renal corpuscles with enlarged urinary space (white arrow) and dilated tubules with cast formation in the lumen (black arrow) are evident at P1.5 (J). (C, D, K, and L) Low-power TEM of renal corpuscles from (C) wt and (D) homozygous mutant at P0.5 and from (K) wt and (L) nls/nls mice at P1.5 showing the glomerular filtration barrier. No differences are obvious in immature podocytes at P0.5 in nls/nls mice (D), but dramatically effaced FPs are observed at P1.5 (L). (E–H and M–P) TEM of glomerular filtration barrier of wt (E and G) and homozygous mutants (F and H) at P0.5 showing regular slit membranes (asterisk) and FPs in wt (G), but either immature or malformed FPs with apically located slit-like junctions (black dots) in mutants (H). At P1.5, FPs with mature slit diaphragms (asterisk) are evident in wt (M and O) but immature FPs with occluding junctions or ladder-like structures (arrowhead) are observed in nls/nls mice (N and P). TEM, transmission electron microscopy; C, capillary; E, endothelial cell; P, podocyte.

Figure 4.

Nuclear but reduced expression of CMAS^nls does not affect the general sialylation in Cmas^nls kidney homogenates. (A) CMAS staining with S59 in P0.5 kidney sections. Mature glomeruli (black arrows) of the inner cortex are marked. Both CMAS and Cmas^nls are localized in the nuclear compartment at single cell level. (B) Immunostaining of CMAS with S56 in cytosolic (c) and nuclear (n) fractions of kidney homogenates at P1.5. Actin expression as internal standard. (C) Total and protein-bound Sia were quantified in kidney homogenates of P1.5 mice by the DMB-HPLC method. Values represent means ± SD of six animals for each genotype. N-acetylneuraminic acid (Neu5Ac, black bars) and N-glycolylneuraminic acid (Neu5Gc, gray bars) were separately assessed.

Figure 5.

Spatial reduction of sialylation in nls/nls glomeruli. (A) Staining of terminal Sia with LFA in P1.5 kidney sections before (−) and after neuraminidase (Neu) treatment. To visualize even weak staining, the tissues were not counterstained. Neu-treated sections are devoid of Sia and serve as negative controls for the LFA staining. Lower lanes represent a larger magnification of the inner rim of the cortex. (B) Staining of terminal galactosyl-β-1,3-N-acetylgalactosamine with PNA in P1.5 kidney sections. Sialylation inhibits PNA binding, whereas Neu treatment uncovers the recognition structure.

Figure 6.

Kidney-specific sialylation analysis of distinct proteins in nls/nls mice. (A) Podocalyxin immunoblot of total lysates from P0.5 and P1.5 organs of all genotypes before (−) and after (+) removal of sialic acids by neuraminidase (Neu) treatment. (B) Immunostaining of podocalyxin in paraffin-embedded kidney sections of wt and nls/nls mice analyzed by light microscopy (upper panel) and fluorescence staining of podocalyxin (lower panel, green) in P1.5 wt and nls/nls kidney paraffin sections analyzed by confocal microscopy. MGs were stained positive for podocalyxin, whereas ureteric buds, renal vesicles, or comma-shaped bodies remain unstained. (C) Nephrin immunoblot of total kidney lysates at P0.5 and P1.5 before (−) and after (+) Neu treatment. (D) Immunostaining of nephrin in paraffin-embedded kidney sections of wt and nls/nls mice analyzed by light microscopy (upper panel), fluorescence staining of nephrin in red (middle panel), and fluorescence double staining (bottom panel) of podocalyxin (green)/nephrin (red) in P1.5 wt and nls/nls kidney paraffin sections analyzed by confocal microscopy. Selected areas are shown with larger magnification in white boxes. Nephrin dots are marked with white arrows. Erythrocytes are stained unspecifically. (E) β1-integrin immunoblot of P0.5 and P1.5 untreated (−) or Neu-treated (+) kidney lysates. Actin is the loading control. Us, enlarged urinary space.

Figure 7.

Quality of nephrin and podocalyxin sialylation influences downstream targets in nls/nls mice. (A) Lectin analysis of nephrin and podocalyxin sialylation. Nephrin and podocalyxin were immunoprecipitated from wt kidney homogenates and analyzed by Western blotting before and after neuraminidase (Neu) treatment (upper panels). α2,6-linked Sia were detected on both proteins with SNA, whereas α2,3-linked Sia was only visible on podocalyxin (MAA staining). PNA detects terminal galactosyl (β-1,3) N-acetylgalactosamine (lower panels). Fetuin served as a positive control for lectin staining. (B) Nephrin phosphorylation and (C) expression of ezrin and phosphorylated ezrin were analyzed in P1.5 kidney homogenates of the indicated genotypes by SDS-PAGE and Western blotting. Cross-reaction with phosphorylated moesin (asterisk) was observed. Actin and GAPDH were used as loading standards. IP, nephrin immunoprecipitate; co, beads without antibody-load served as negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SNA, S. nigra agglutinin; MAA, M. amurensis agglutinin.