Polysialic acid regulates glomerular microvasculature formation by interaction with VEGF-A188 in mice

Abstract

Vascular endothelial growth factor A (VEGF-A) is a key signalling protein that stimulates blood vessel development and repair. Its tight control is essential for organ development and tissue homeostasis. However, the complex regulatory network for balanced bioavailability of VEGF-A is not fully understood. Here, we assessed the role of the glycocalyx component polysialic acid (polySia) for kidney development and its potential interactions with VEGF-A isoforms, in vitro and in vivo, using mouse models of polySia deficiency. PolySia acts as negative regulator of cell adhesion, but also may interact with extracellular components. In murine kidney, polySia was identified on nephron progenitor and endothelial cell subsets in developing nephrons with declining expression during maturation. Loss of polySia in Ncam^-/- mice revealed the neural cell adhesion molecule NCAM as major protein carrier. Both polysialyltransferase-negative and Ncam^-/- mice displayed impaired glomerular microvasculature development with reduced endothelial cell numbers, reminiscent to the phenotype of mice with impaired VEGF-A signalling. In vitro, immobilized polySia specifically interacted with the VEGF-A188 isoform demonstrating an isoform-specific direct interaction. Single cell RNA sequencing data analysis of newborn mouse kidneys implicated activation of VEGF-A-signalling in polysialyltransferase-positive endothelial cells. Consistently, loss of polySia resulted in diminished VEGFR2 activation in perinatal kidney and human endothelial cells. At transcriptional level, the expression of polysialyltransferases and known polySia carrier proteins is conserved in human developing kidney. Together, these data demonstrate a direct impact of polySia on VEGF-A signalling with the perspective that polysialylation could be a therapeutic target to ameliorate microvasculature repair after renal injury.

Keywords: Kidney development; Microvasculature; Polysialic acid; Sialic acid; VEGF-A.

Fig. 1

PolySia distribution in murine kidney. (A)N-Acetylneuraminic acid. (B) PolySia DP8 scheme. (C, D) PolySia immunohistochemistry with 735 antibody on paraffin-embedded kidney sections from wildtype mice at postnatal day (P)0.5. (C) An entire stained renal section is shown to appreciate signal distribution with enlarged micrographs of the medullary and cortical area. Strong 735 staining is visible in the nephrogenic zone (NZ) and in the medulla. (D) Enlarged micrograph of the NZ, where different stages of nephron development are visible. (E) PolySia immunohistochemistry with 735 antibody on paraffin-embedded kidney sections from wildtype mice at embryonic day (E)14.5. An entire stained renal section is shown to appreciate signal distribution with enlarged micrographs of different stages of nephron development. (F) 735 immunohistochemistry of renal sections from P7.5 and (G) P15.5 old wildtype mice. PolySia expression decreases over time and residual cortical polySia staining at P15.5 is restricted to parietal epithelial cells (Bowman capsule, arrows) and cell layer adjacent to blood vessels (arrowheads). (H) Western blot analysis of kidney homogenates from wildtype (WT) and St8sia2^−/− St8sia4^−/− (KO) mice at different postnatal time points. Protein concentrations of the samples were determined and 25 µg were loaded per lane. 735 staining shows decreasing signals in wildtype kidney over time and completely abolished signal in St8sia2^−/− St8sia4^−/− kidneys at shown time points. Actin staining served as loading control. (I) 735 immunohistochemistry on renal sections of St8sia2^−/− St8sia4^−/− mice at P0.5, (J) P7.5 and (K) P15.5. Representative micrographs of the cortical part are shown. CM, cap mesenchyme; RC, renal corpuscle (RC); SSB, S-shaped body; UBT, ureteric bud tip

Fig. 2

Analysis of polySia-deficient murine kidney. (A) H&E staining of paraffin-embedded kidney sections from wildtype and St8sia2^−/− St8sia4^−/− mice (P15.5). Glomeruli in the renal cortex are framed. (B) Glomerular tuft areas measured in H&E stained kidney sections (P15.5) of n = 5 wildtype and mutant mice, respectively. Per section, all visible glomerular tufts were measured. Means ± SD and individual data points are depicted. Non-parametrical Kruskal–Wallis test (p = 0.0032) and Dunn’s multiple comparison indicated significant group differences (* p < 0.05). (C) Protein/creatinine ratios measured in urine from wildtype, St8sia2^−/− St8sia4^−/− and Ncam^−/− mice collected at different postnatal time points (P0.5, 7.5 and 15.5). Means ± SD and individual data points of n = 4–16 individuals are depicted. Nonparametric Mann–Whitney test indicated significant differences between wildtype and St8sia2^−/− St8sia4^−/− mice at P0.5, during the maturation of the blood filtration barrier (* p < 0.05, highlighted). For clarity reasons, significant differences during kidney maturation, between P0.5-P7.5 and P0.5-P15.5, for all three indicated genotypes are not indicated. No significant differences were detected between the genotypes for the time points P7.5 and P15.5. (D) Total cell numbers per glomerulus were assessed on kidney sections from wildtype, St8sia2^−/− St8sia4^−/− and Ncam^−/− mice (P15.5) by counting hematoxylin stained nuclei and WT-1, ERG, or GATA3 immunoreactive cells (see F, G and H) or hematoxylin-stained cell nuclei in H&E stained renal sections. N = 13 wildtype, N = 13 St8sia2^−/− St8sia4^−/− animals and N = 11 Ncam^−/− animals and per section 30 glomeruli in comparable areas of the tissues were analysed. Means ± SD and individual data points are depicted. Statistical significance was tested with a nonparametric Mann–Whitney-U test (* p < 0.05; **** p < 0.0001). (E) Scheme of a mature glomerulus composed of three different cell types in the glomerular tuft: podocytes (blue), endothelial cells (green) and mesangial cells (orange). (F–H) Immunohistological staining of nuclear localized glomerular cell type markers on paraffin-embedded kidney sections (wildtype and St8sia2^−/− St8sia4^−/−, P15.5): GATA3 staining for mesangial cells (F), WT1 staining for podocytes (G) and ERG staining for endothelial cells (H). Evaluation of cell numbers of (I) GATA3⁺ cells, (J) WT1⁺ cells and (K) ERG⁺ cells (** p < 0.01, nonparametric Mann–Whitney test) from immunohistological staining on paraffin-embedded kidney sections (P15.5) of wildtype and St8sia2^−/− St8sia4^−/− mice. (L-N) Analysis of renal sections from Ncam^−/− and wildtype mice at P15.5 regarding the evaluation of cell numbers of (L) GATA3⁺ cells, (M) WT1⁺ cells and (N) ERG⁺ cells (* p < 0.05, nonparametric Mann–Whitney test). n = 4–5 individuals were analysed per genotype and per section 30 glomeruli were selected for counting in equal areas of the section. Data is displayed as averages per mouse with means and standard deviation for the group

Fig. 3

VEGF-A isoform expression in murine kidney, interaction with polySia in vitro and spatial relationship between VEGFA and polySia during nephron development (A) Scheme of the exon structure and the three major murine pro-angiogenic VEGF-A isoforms 120, 164 and 188. VEGFR and heparan sulfate (HS) binding sites are indicated. 24 amino acid sequence of exon 6, which is only present in VEGF-A188 is displayed and basic residues are underlined. (B) Western blot analysis of wildtype (WT) and St8sia2^−/− St8sia4^−/− (KO) kidney homogenates prepared at different postnatal time points. Protein concentrations of the samples were determined and 25 µg were loaded per lane. Staining with a VEGF-A antibody results in multiple bands. Actin staining serves as loading control. (C, D) Binding analysis of (C) VEGF-A188 and (D) VEGF-A120 and -A164 with polySia of different chain length (average DP50 (avDP50) and DP24-30) in horizontal native PAGE. Bovine serum albumin (BSA) is shown as a negative control and histone as a known polySia interaction partner is shown as a positive control. Complex formation of histone with both polySia pools increased the negative charge of the protein reflected by a reversion of the migration direction in the native PAGE (pH = 8.1). (C) Complex formation of VEGF-A188 (pI = 9.25) with avDP50 and DP24-30 reverted the migration direction towards the anode ( +) in horizontal native PAGE (pH = 7.4), as observed for histone. (D) In contrast, the mobility of BSA (pI ~ 5) and the two VEGF isoforms -A120 (pI = 6.48) and -A164 (pI = 7.93) was not grossly affected by either long chain (avDP50) or shorter chain polySia (DP24-30). Both polySia pools did not increase the covered migration distance of the VEGF-A isoforms to the anode, indicating a lack of interaction with polySia. Native PAGE conditions allowing optimal migration in PAGE were used for all three proteins. ( +) anode, (-) cathode. (E) ELISA of VEGF-A isoforms binding to polySia. PolySia of avDP50, derived from E. coli K1 capsule polysaccharide, was immobilized on a plate. Mean and standard deviation calculated from three individual experiments are shown with horizontal axis in log scale. The dotted lines show a non-linear fit to the mean values of the individual isoforms. Binding is observed only for the VEGF-A isoform 188. (F) Protein-glycan interaction analysis by microscale thermophoresis. Binding curves illustrate the interaction of fluorescently labelled VEGF-A188 with polySia (DP ≥ 40) and heparan sulfate (HS) as positive control. Normalized thermophoresis fluorescence averaged from 3 to 5 independent experiments is plotted against ligand concentration and K_D values were calculated based on the Hill equation (K_D(polySia) = 66.1 ± 3.17 µM, K_D(HS) = 3.38 ± 0.77 µM). The error bars report the standard deviation. (G) Illustration of endothelial (precursor) cells migration into the S-shaped body of a developing nephron. Podocyte precursors (blue) secrete VEGF-A (red) and thereby attract VEGFR-2 (dark purple) expressing endothelial cells (green) into the vascular cleft of the S-shaped body. (H)Vegfa188 base scope assay was performed on paraffin-embedded kidney sections from newborn wildtype mice. Vegfa188 mRNA is expressed in developing (black arrowheads) and mature (green arrowheads) glomeruli in the renal cortex. Micrographs with higher magnification clearly show intense red staining in (I) developing nephrons (black arrowheads) and (J) mature glomeruli, in which the outer cell layer (podocytes) is intensively stained. (K) Immunohistochemical staining for polySia on renal section (wildtype P0.5) with 735 antibody. Intense polySia staining is visible in the centre of an S-shaped body (circled with dashed line). (L) Immunofluorescence co-staining for polySia and VEGF-A on murine kidney section (wildtype P0.5). PolySia (magenta) and VEGF-A (yellow) staining is visible in the developing glomerulus (S-shaped body) and overlapping signals are shown in white in the merged image. Cell nuclei are stained with DAPI (blue)

Fig. 4

PolySia expression in distinct renal cell types and contribution of the polysialyltransferases ST2SIA2 and ST8SIA4 during postnatal renal development. (A) Evaluation of polysialyltransferase gene expression in different renal cell types (Leiden clusters) in P0 wildtype mice. Single cell RNAseq data (Accession no. GSM4648414) was obtained from Naganuma et al. (2021). LOH: loop of Henle, IC: interstitial cell, EC: endothelial cell, NP: nephron progenitor (describing cells of the cap mesenchyme and the nascent nephron), PT: proximal tubule, UB: ureteric bud tip, CD: collecting duct, POD: podocyte, BC: blood cell. (B) Normalized histograms of spectral flow cytometry analysis from kidney single cell suspensions of newborn wildtype and polySia-deficient (St8sia2^−/− St8sia4^−/−) mice. Cells were stained for polySia with the 735 antibody and renal cell type markers for interstitial cells (PDGFRβ), (C) endothelial cells (CD31) and (D) immune cells (CD45). (E) Immunofluorescence co-staining of NCAM (yellow), polySia (magenta) and the endothelial cell marker ERG (cyan) on renal section of newborn wildtype mice. Partial colocalisation is observed in cells of an S-Shaped body. Overlapping signals are coloured white in the merged image. The white arrowhead indicates a polySia-positive endothelial cell. (F–H) Spectral flow cytometry analyses of renal single cell suspensions from newborn wildtype and polysialyltransferase knockout mouse strains St8sia2^−/−, St8sia4^−/− and St8sia2^−/− St8sia4^−/−. Cell suspensions were stained for polySia with inactive endosialidase (iEndo) and for interstitial cells (PDGFRβ, F), endothelial cells (CD31, G) and immune cells (CD45, H) to discriminate between different cell types. The calculated median fluorescence intensities (MFI) are shown with each data point representing one biological replicate (n = 4 for St8sia4^−/−, n = 3 for others). Means and standard deviations are depicted. Non-parametrical Kruskal–Wallis test indicated significant differences (PDGFRβ, CD31: p < 0.0001, CD45: p = 0.0001). Uncorrected Dunn’s post hoc tests were applied and significant group differences are indicated (* p < 0.05, ** p < 0.01). Representative histograms for the results depicted in B-D are shown in Supplemental Figure S8G-I. (I) PolySia (735) staining on renal sections of newborn St8sia2^−/− and (J)St8sia4^−/− mice. Cortical areas of the tissue are depicted to appreciate polySia staining in the nephrogenic zone. Residual polySia signal in St8sia2^−/− kidney on interstitial cells is marked with arrowheads

Fig. 5

PolySia expression in Ncam^−/− kidney. (A) 735 immunohistology on renal sections of newborn Ncam^−/− mice. A representative segment of the cortical area is depicted with single positively stained cells (arrowheads). Three enlarged micrographs are shown to better appreciate stained cells. (B-D) Histograms of spectral flow cytometry analysis of single cell suspensions from P3 wildtype and Ncam^−/− kidneys. Cells were stained for polySia with inactive endosialidase (iEndo) and for renal cell type markers: (B) immune cells (CD45), (C) interstitial cells (PDGFRβ) and (D) endothelial cells (CD31). The gating strategy for the spectral flow cytometry analysis is the same as described before and depicted in Supplemental Figure S8A-E. (E) Contour plot of P0.5 kidney CD31-positive endothelial cells, that were analysed regarding NCAM and polySia expression from wildtype and Ncam^−/− mice. (F) Frequencies of the identified populations shown in (E) in wildtype and Ncam^−/− mice. Each data point represent one biological replicate. About 15% of renal endothelial cells express polySia and NCAM in wildtype mice, a frequency significantly reduced in Ncam^−/− mice (Nonparametric Mann–Whitney test, ** p < 0.01, N = 5 mice per genotype). In both genotypes, less than 5% of endothelial cells are polySia-positive but lack NCAM. (G) Evaluation of gene expression values of known polySia protein carriers in different renal cell types in P0 wildtype mice. Single cell RNAseq data (Accession no. GSM4648414) was obtained from Naganuma et al. (2021) [37]. LOH: loop of Henle, IC: interstitial cell, EC: endothelial cell, NP: nephron progenitor, PT: proximal tubule, UB: ureteric bud tip, CD: collecting duct, POD: podocyte, BC: blood cell. Ncam1 or NCAM1: neural cell adhesion molecule, Nrp2 or NRP2: neuropilin-2, Glg1 or GLG1: E-selectin ligand-1/Golgi Glycoprotein 1, Cadm1 or CADM1: SynCAM 1/Cell adhesion molecule 1

Fig. 6

Impact of polySia on VEGF-A mediated signalling and proposed model of polySia-VEGF-A interaction. (A) Differential gene expression analysis between St8sia4 + and St8sia4- endothelial cells extracted from the scRNAseq data set from Naganuma et al. (2021) from newborn mouse kidney. Volcano plot with -log₁₀ (adj. p-values) plotted against the log₂ (fold-change) is shown. Dotted line represents the significance level (adj. p-value < 0.05). Significantly regulated genes are labelled. St8sia4 is not displayed in the volcano plot for reasons of clarity. (B) Western blot analysis of kidney homogenates from wildtype (WT) and St8sia2^−/−St8sia4^−/− (KO) mice and different postnatal (P) time points. Expression of VEGFR2 and phosphorylation status of Tyr1175 of VEGFR2 is shown. Actin staining served as loading control. 25 µg total protein were loaded per lane. Representative immunoblot from n = 3 individuals is shown. (C) Western blot quantification of P0.5 kidney homogenates from wildtype and St8sia2^−/−St8sia4^−/− mice. Samples from three wildtype and St8sia2^−/−St8sia4^−/− mice were analysed pairwise on three individual Western blots. For each Western blot, signal ratios of phosphorylated VEGFR2 to total VEGFR2 protein were calculated and normalized to the wildtype. Data are shown as single values with means, and the standard deviation is shown for St8sia2^−/−St8sia4^−/−. (D) PolySia immunoblot of HUVECs treated w/o endosialidase F. Actin staining served as loading control. 10 µg total protein were loaded per lane. (E) Western blot analysis of HUVEC lysates w/o endosialidase F (Endo) treatment and w/o subsequent VEGF-A188 stimulation. PolySia was stained with 735 antibody. Expression of VEGFR2 and phosphorylation status of Tyr1175 of VEGFR2 are displayed. Actin staining served as loading control. 8 µg (735, VEGFR2-P) and 4 µg (VEGFR2, ACTIN) total protein were loaded per lane. A representative immunoblot from n = 3 independent experiments is shown. (F) Western blot quantification of HUVEC homogenates after treatment w/o endosialidase F and incubation with VEGF-A188. Samples from three independent experiments were analysed pairwise on three individual Western blots. For each Western blot, signal ratios of phosphorylated VEGFR2 to total VEGFR2 protein were calculated and normalized to the control (without endosialidase F treatment). Data are shown as single values with means, and the standard deviation is shown for endosialidase F treated cells. (G) PolySia (purple) on NCAM (grey) and/or other carrier protein(s) expressed on the endothelial cell surface interacts with VEGF-A (red) in close proximity to VEGFR2 (blue, with phosphorylation sites in yellow) and acts as a co-receptor to facilitate VEGF-A binding. PolySia-NCAM and VEGFR2 are in cis position. (H) PolySia-NCAM on nephron progenitor cells and/or interstitial cells are in trans position to VEGFR2 on endothelial cells. The polySia-VEGF-A interaction promotes the formation of a VEGF-A gradient. A combination of these two proposed models is most likely