Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria

Abstract

Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.

Figure 1 |. The cartoon depicts a simplified scheme of the assembly and extension of HS-GAG chains on a PG core protein.

The initial step in the assembly of the HS-GAG chain (and common to all HS and chondroitin sulfate-bearing PGs) is the assembly of a linker tetrasaccharide (glucuronosyl-β 1,3-galactosyl-β 1,3-galactosyl-β 1,4 xylose) attached to a serine residue present in the proteoglycan core protein. Each carbohydrate residue in the linkage region is assembled by a specific glycosyltransferase. In the case of HS-GAG, following the assembly of the linkage region, an N-acetyl glucosamine residue (α-GlcNAC) is the next carbohydrate residue attached to the linkage region by the enzyme EXTL3. Once assembled, the pentasaccharide is recognized by heparan sulfate copolymerase, EXT1/EXT2. The HS copolymerase extends the nascent GAG chain by the sequential addition of a glucuronic acid (UA) and N-acetyl glucosamine (GLN), which are derived from UDP precursors, forming a repetitive copolymer of these subunits (GlcNAcα1–4GlcAβ1–4)_n. For the sake of simplification, the subsequent events in the process, such as deacetylation, sulfation, and uronic acid epimerization, which occur during HS chain elongation are not depicted.

Figure 2 |. The results of a PCR-based genotype screen for the presence of Ext1^fl/fl alleles and the 2.5P-Cre transgene.

Amplification of the wild-type Ext1 allele (Ext1^wt) yields a product of 360 bp, whereas the Ext1^floxed allele yields a larger 460-bp product. The 2.5P-Cre product is 268 bp. The boxed-in area of the gel represents an animal with the PEXTKO genotype.

Figure 3 |. The micrographs of tissue sections from control (a, c, e, g) or PEXTKO animals (b, d, f, h) stained with hematoxylin and eosin.

The age-matched animals were 1 (a, b), 3 (c, d), 8 (e, f), and 12 (g, h) months of age. Overall there were no profound differences in either glomerular or tubular structure between control and PEXTKO kidney at 1 and 3 months of age; no differences among the control animals were seen at any age examined. At 8 months of age, there were very apparent changes in the proximal tubule epithelial cells in the PEXTKO animals (f, arrows), the cells of which had large vacuoles present in the cytoplasm. These changes persisted in older PEXTKO animals (h).

Figure 4 |. The micrographs are of tissue sections of kidney immunostained with antibodies against HS-GAGs (antibody HS4C3, a–f) or chondroitin sulfate GAGs (CS-GAGs, antibody 3B3, g, h).

(a–f) Deconvolved images resolving a single focal plane, (b, d, f) glomeruli from PEXTKO animals of 1, 3, and 8 months of age respectively. (a, c, e) Age-matched control animals. (g, h) Glomeruli from 3-month-old control and PEXTKO animals, respectively. In control glomeruli (a, c, e), the HS4C3 antibody immunostains the pericapillary (arrows) and perimesangial GBM as well as the mesangial matrix (M). In PEXTKO glomeruli (b, d, f), HS4C3 stains primarily the mesangial matrix (M), with significantly reduced staining in the pericapillary GBM. The glomeruli in (g) (control) and (h) (PEXTKO) show similar patterns of mesangial staining (M) with no GBM staining, indicating that CS-GAGs were not substituted on GBM proteoglycan core proteins as a default mechanism. (i) The results of morphometric measures of glomerular area in control and PEXTKO animals. At both ages examined, the glomeruli from PEXTKO animals were hypertrophic compared to age-matched control animals (*P<0.0001); and glomerular hypertrophy gradually increased over time (**P<0.006).

Figure 5 |. The micrographs are of tissue sections immunostained with antibodies against either perlecan (antibody A7L6; a, b, e, f, i, j) and agrin (antibody K-17; c, d, g, h, k, l).

Glomeruli from control (a, c, e, g, i, k) and PEXTKO animals (b, d, f, h, j, l) are presented in an age-matched manner identical to Figure 1. To show staining intensity differences in the agrin-stained glomeruli, the exposure for each image pair was set to the specimen with the brightest intensity, and both control and PEXTKO images were subsequently imaged at the same exposure setting. For both proteoglycan core protein species, there is no visible change in the pattern of distribution within the glomerulus. The distribution of perlecan staining in both control and PEXTKO animals is identical, being strongest in the mesangial matrix and weaker staining seen in the glomerular capillary wall. In contrast, at each age (1, 3, and 8 months) the intensity for agrin staining in the glomeruli from PEXTKO animals is greater than that seen in the control animals.

Figure 6 |. The electron micrographs show glomeruli from adult control animals (a, 2.5P-Cre+/Ext1^wt/wt) and PEXTKO (b, 2.5P-Cre+/Ext1^fl/fl) animals.

In control animals, the basic architecture of the glomeruli from these animals is normal, with no visible sign of foot process effacement or obvious BM ‘outpockets.’ The inset in the corner of each larger micrograph is a higher magnification of the area in the main micrograph outlined by the dashed line. (b) An electron micrograph of a glomerular capillary wall from a 1-month-old PEXTKO mouse. Within the picture are the cell bodies of two podocytes (P) having abnormal, effaced foot processes (arrows). On the apical surface of the podocytes, microvilli (area denoted by M) can be found. The GBM also shows irregularities, having numerous outpockets or ‘humps’ (arrowheads) on the podocyte side of the GBM. The inset shows an enlargement of the boxed-in area of the capillary wall. In this region, the BM has an abnormal appearance, resembling two separate BMs. The presence of the outpockets, along with the double BM, suggests the possibility that GBM fusion, a normal developmental process, has been delayed in this area of the glomerulus. The presence of a double BM has not been seen in older (>3 months) animals.

Figure 7 |. The electron micrographs map the changes in glomerular capillaries seen in adult (3 months) PEXTKO animals.

(a) Low magnification electron micrograph of several glomerular capillaries. The arrowheads in the picture denote areas where there are BM ‘outpockets’ in the capillary wall. Although in developing/maturing kidneys this type of morphology is associated with areas where active BM synthesis/splicing is occurring, it is also possible that these areas could be areas of basement membrane remodeling by podocytes. The arrows point to two abnormally attenuated primary processes of a podocyte. The boxed-in area of (a) is shown in higher magnification in (b). The apical surface of the podocyte shows the abnormal presence of microvilli (M) and the region of foot process in the basal side of the podocyte is severely effaced. Despite the presence of the ‘outpockets’ of BM material, in the normal thickness GBM the classic LRE-lamina densa (LD)-LRI organization is easily seen, suggesting that BM assembly may be essentially normal.

Figure 8 |. (a) Silver-stained gel of urinary proteins from representative age-matched (3 months) control and PEXTKO animals (collection interval 24 h).

At 3 months of age, there was no discernable difference in albumin between control and PEXTKO animals. At 8 months of age, a slight amount of albuminuria is present in the PEXTKO animals compared to control. In ELISA-based assays for urinary albumin, a slight increase in urinary albumin (b) was seen in PEXTKO animals at 2 and 8 months of age when compared to controls, albeit the differences did not reach statistical significance (n = 5 animals per group). Urinary creatinine levels (c), as measured by a commercially available plate assay, were comparable between both groups at both ages. The ratio of urinary albumin/creatinine (d) was increased in PEXTKO animals at both ages but the differences did not reach statistical significance.

Figure 9 |. The micrographs in (a, b) are of two adjacent glomerular capillary walls from control (a) and PEXTKO (b) animals (8 months of age shown) that were labeled with PEI, a probe for the presence of anionic sites in the GBM.

In control animals, PEI label can be found immediately subtending the podocyte foot processes and endothelial cells (black arrows) in the lamina rara externa (LRE) and lamina rara interna (LRI), respectively. The labeling ratio for PEI in these areas is approximately 2:1 (LRE:LRI). The amount of PEI labeling in the PEXTKO animal is significantly decreased (black arrows). The graph in (c) compares the number of PEI sites in the LRE and LRI from control and PEXTKO kidneys of 2 and 8 months of age. The data show a significant decrease in the amount of PEI label in the LRE (*P<0.0001) in both age groups. An age-related decrease in anionic sites in the LRE was also seen in control animals (^@P =.0004). In the LRI of PEXTO animals, a decrease in anionic sites was also seen at both 2 months (**P = 0.0004) and 8 months (^#P<0.0019) compared to controls. control 2 months, PEXTKO 2 months, control 8 months, PEXTKO 8 months.