Changes in glomerular parietal epithelial cells in mouse kidneys with advanced age

Abstract

Kidney aging is accompanied by characteristic changes in the glomerulus, but little is known about the effect of aging on glomerular parietal epithelial cells (PECs), nor if the characteristic glomerular changes in humans and rats also occur in very old mice. Accordingly, a descriptive analysis was undertaken in 27-mo-old C57B6 mice, considered advanced age. PEC density was significantly lower in older mice compared with young mice (aged 3 mo), and the decrease was more pronounced in juxtamedullary glomeruli compared with outer cortical glomeruli. In addition to segmental and global glomerulosclerosis in older mice, staining for matrix proteins collagen type IV and heparan sulfate proteoglycan were markedly increased in Bowman's capsules of older mouse glomeruli, consistent with increased extracellular matrix production by PECs. De novo staining for CD44, a marker of activated and profibrotic PECs, was significantly increased in aged glomeruli. CD44 staining was more pronounced in the juxtamedullary region and colocalized with phosphorylated ERK. Additionally, a subset of aged PECs de novo expressed the epithelial-to-mesenchymal transition markers α-smooth muscle and vimentin, with no changes in epithelial-to-mesenchymal transition markers E-cadherin and β-catenin. The mural cell markers neural/glial antigen 2, PDGF receptor-β, and CD146 as well as Notch 3 were also substantially increased in aged PECs. These data show that mice can be used to better understand the aging kidney and that PECs undergo substantial changes, especially in juxtamedullary glomeruli, that may participate in the overall decline in glomerular structure and function with advancing age.

Keywords: CD146; CD44; Notch 3; collagen type IV; epithelial-to-mesenchymal transition; extracellular signal-regulated kinase; glomerulosclerosis; glomerulus; heparan sulfate proteoglycan; neural/glial antigen 2; platelet-derived growth factor receptor-β; podocyte; vimentin; α-smooth muscle actin.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Glomerulosclerosis increases in mice with advanced age. Periodic acid-Schiff (PAS) staining was used to identify and quantitate glomerular scarring in young and old mice. Scale bars are provided. A: low-power magnification (×100) of PAS staining of a 27-mo-old (27m) mouse. The dashed line demarcates the juxtamedullary (JM) region on the left from the outer cortical (OC) region on the right. Arrows indicate the kidney capsule. *An obsolescent glomerulus. B: example of an aged glomerulus with segmental glomerulosclerosis. C: example of an aged glomerulus with global glomerulosclerosis. D: the percentage of glomeruli with segmental glomerulosclerosis increased in aged mice in both the OC and JM compared with young mice. Within the group of mice with advanced age, segmental glomerulosclerosis was more marked in JM glomeruli. E: the percentage of glomeruli with global glomerulosclerosis in JM glomeruli increased in aged mice compared with young mice. n.s., not significant; n/a, statistical analysis was not applicable as means equalled zero.

Fig. 2.

Podocyte number and density decrease in mice with advanced age. Podocytes were identified by p57 staining (brown nuclear stain; examples indicated by arrowheads) in tissue counterstained with PAS from young and old mice at 3 mo (3m) and 27 mo of age, respectively. Representative images were taken under the same magnification (×400). Scale bars are provided. A–D: examples of p57 staining from the young OC (A), young JM (B), old OC (C), and old JM (D). E: in young and old mice, the glomerular tuft area was higher in JM glomeruli compared with OC glomeruli for their given age. The glomerular tuft area was higher in older mice for OC and JM glomeruli compared with young mice. F: in young mice, the absolute number of podocytes, as measured by p57-stained cells per glomerular cross section, was higher in JM glomeruli. In aged mice, p57 staining was significantly reduced in both OC and JM glomeruli. G: unbiased stereology showing that compared with young mice aged 3 mo, podocyte density (number of podocytes/tuft area) was lower in mice with advanced age at 27 mo in both OC and JM glomeruli. Within the group of old mice, podocyte density was lower in JM glomeruli compared with OC glomeruli.

Fig. 3.

Bowman's capsule (BC) length increases and glomerular parietal epithelial cell (PEC) density decreases in mice with advanced age. PECs were identified by paired box gene 2 (PAX2) staining (Brown nuclear stain; arrowheads show examples) in tissue counterstained with PAS. Representative images were taken under the same magnification (×400). Scale bars are provided. A–D: examples of PAX2 staining are shown from the OC of young mice aged 3 mo (A), JM of young mice aged 3 mo (B), OC of old mice aged 27 mo (C), and JM of old mice aged 27 mo (D). E: Bowman's capsule length was longer in JM glomeruli compared with OC glomeruli in both young and old mice. However, Bowman's capsule length was longer in older mice for both OC and JM glomeruli compared with young mice. F: in young and old mice, the absolute number of PECs, as measured by PAX2-stained cells per glomerular cross section, was higher in JM glomeruli compared with OC glomeruli and increased with age in both regions. G: unbiased stereology showing that compared with young mice, PEC density (number of PECs/Bowman's capsule length) was lower in mice with advanced age in both OC and JM glomeruli. The decrease was more pronounced in JM glomeruli.

Fig. 4.

Extracellular matrix protein immunostaining increases in Bowman's capsule in aged mice. Representative pictures were taken by confocal microscopy (magnification: ×400). Because sclerosis was mostly restricted to JM glomeruli, images from this region are shown. Glomeruli from 3- and 27-mo-old mice are indicated by dashed circles. Arrowheads show examples of positive staining. Scale bars are provided. A and B: heparan sulfate proteoglycan (HSPG). HSPG staining was detected in Bowman's capsule of young mice (A) and increased markedly in old mice (B). C and D: laminin. Laminin staining along Bowman's capsule was faint in young mice (C) and decreased slightly with age (D). E and F: collagen type IV. Collagen type IV staining, which was present in young animals along Bowman's capsule (E), increased substantially in old mice (F). Collagen type IV staining was also increased in the glomerular tuft of old animals (arrow).

Fig. 5.

CD44 immunostaining increases in aged PECs and colocalizes with phosphorylated (p-)ERK. A–D: CD44 staining (brown, cytoplasmic) and PAS counterstaining were performed in 3- and 27-mo–old mice. Representative pictures were taken under the same magnification (magnification: ×400). Scale bars are provided. A and B: CD44 staining was not detected in young mice aged 3 mo in the OC (A) or in the JM (B). C: in contrast, in mice aged 27 mo, considered as advanced age, CD44 staining was detected in cells lining Bowman's capsule in the OC (arrowheads). An occasional CD44-positive cell was detected in the capillary loops (arrow). D: CD44 staining was readily detected in cells lining Bowman's capsule in the JM of 27-mo-old mice (arrowheads). CD44-positive cells were detected in the capillary loops (arrows). E: the percentage of glomeruli with CD44-stained cells on Bowman's capsule increased in 27-mo-old aged mice compared with 3-mo-old young mice; the percentage was higher in JM glomeruli. F–H: confocal microscopy images of immunofluorescent staining in aged JM glomeruli (magnification: ×400). F and G: staining for CD44 (F; red; arrowheads show examples) and p-ERK (G; green; arrowheads show examples) increased in cells lining Bowman's capsule. H: CD44 and p-ERK colocalized (yellow).

Fig. 6.

Immunostaining for the epithelial-to-mesenchymal transition (EMT) markers α-smooth muscle actin (α-SMA) and vimentin increases in PECs in mice with advanced age. A–D: confocal microscopy (magnification: ×400) showing immunofluorescent double staining for α-SMA (green, arrowheads) and collagen type IV (red, arrows) in young and old mice. Collagen type IV staining was used to demarcate Bowman's capsule. Dashed circles indicate glomeruli. Scale bars are provided. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). A: α-SMA staining was not detected in OC glomeruli of young mice aged 3 mo. Dashed arrows indicate positive staining in the adjacent vasculature, which was used as an internal positive control. B: light staining for α-SMA was detected along the urinary side of Bowman's capsule (identified by collagen type IV staining) in young JM glomeruli. C: in contrast to young mice, α-SMA staining was readily detected in 27-mo-old OC glomeruli. D: α-SMA staining was markedly increased in cells lining Bowman's capsule, consistent with PECs (arrowheads). When quantitated, the percentage of glomeruli with α-SMA staining in cells lining Bowman's capsule was significantly higher in older OC and JM glomeruli. E–H: confocal microscopy (magnification: ×400) showing immunofluorescent double staining for vimentin (green, arrowheads) and collagen type IV (red, arrows) in young and old mice. E and F: vimentin staining was not detected in PECs in young OC glomeruli (E) and only occasionally in young JM glomeruli (F). G: in old OC glomeruli, vimentin staining (arrowheads) was present in cells along Bowman's capsule (arrows). H: in older JM glomeruli, vimentin staining was abundant along Bowman's capsule. I: percentage of glomeruli with positive α-SMA staining in cells lining Bowman's capsule J: the percentage of glomeruli with positive vimentin staining in cells lining Bowman's capsule was significantly higher in old OC and JM glomeruli compared with young mice.

Fig. 7.

Immunofluorescent staining for neural/glial antigen 2 (NG2) increases in PECs in mice with advanced age. Confocal microscopy images (magnification: ×400) are shown of JM glomeruli from young mice aged 3 mo and older mice aged 27 mo. Glomeruli are indicated by dashed circles. Scale bars are provided. Nuclei were stained blue with DAPI. A and B: NG2 staining (magenta, arrowheads) was very faintly detected in cells lining Bowman's capsule in 3-mo-old mice. B: NG2 staining was substantially increased in old mice in cells along Bowman's capsule (arrowheads). C: NG2 staining was not detected when the IgG isotype control was substituted for the primary antibody. D: positive controls in kidney interstitial tissue. NG2 staining was detected in the interstitium in small vessel pericytes (dashed arrow) and in stromal cells of larger vessels (solid arrows; D1), which overlapped with α-SMA (D2, red) and PDGF receptor-β (PDGFRβ; D3, green) to create a white color when merged (D4).

Fig. 8.

Immunofluorescent staining for PDGFRβ increases in PECs in mice with advanced age. Confocal microscopy images (magnification: ×400) are shown of JM glomeruli from young mice aged 3 mo and old mice aged 27 mo. Glomeruli are indicated by dashed circles. Scale bars are provided. Nuclei were stained blue with DAPI. A: PDGFRβ staining (green) was detected in cells in the glomerular tuft in young mice in a mesangial distribution, but not along Bowman's capsule. B: in old mice, PDGFRβ was detected in cells lining Bowman's capsule (arrowheads). C: PDGFRβ was not detected when the primary antibody was substituted with an IgG isotype control. D: double staining for PDGFRβ (D1, green) and the mesangial cell marker α8-integrin (D2, red) and nuclei stain with DAPI (D3, blue); colocalization of PDGFRβ and α8-integrin was observed when the images were merged (D4, yellow, arrows). These results show that PDGFRβ staining increased in PECs in aged kidneys.

Fig. 9.

CD146 immunofluorescent staining increases in aged PECs. Confocal microscopy images (magnification: ×400) are shown of JM glomeruli from young mice aged 3 mo and old mice aged 27 mo. Glomeruli are indicated by dashed circles. Scale bars are provided. Nuclei were stained blue with DAPI. A: staining for CD146 (magenta) was detected in mesangial cells, a subset of cortical tubular cells, and pericytes (dashed arrow), but not in PECs. B: in 27-mo-old kidneys, CD146 staining was detected in PECs (arrowheads). C: CD146 staining was not detected when the primary antibody was substituted with an IgG isotype control. D: positive controls for CD146 in the kidney interstitium of the medullary region. CD146 stained perivascular cells (D1, arrows), which also stained for α-SMA (D2) and PDGFRβ (D3) and overlapped when merged (D4).

Fig. 10.

Notch 3 immunofluorescent staining is increased in PECs of aged mice. Confocal microscopy images (magnification: ×400) are shown of JM glomeruli from young mice aged 3 mo and old mice aged 27 mo. Glomeruli are indicated by dashed circles. Scale bars are provided. Nuclei were stained blue with DAPI. A: Notch 3 staining was faintly detected in cells lining Bowman's capsule in young animals (red, arrowhead). Dashed arrows show internal positive controls for Notch 3 in vessels. B: in old mice, Notch 3 staining was markedly increased in cells along Bowman's capsule (arrowheads). C: when Notch 3 antibody was substituted with the IgG isotype control, staining was not detected.

Fig. 11.

Schematic proposal for changes in glomerular PECs in mice with advanced age. In young mice, confluent PECs (blue color with green nuclei) attach to Bowman's capsule. With advanced age, there is a decrease in PEC density (most pronounced in JM glomeruli). Of the remaining PECs, a subset become activated and express CD44 (red) and are likely profibrotic. Another PEC subset undergoes EMT (purple), whereas another increases expression for markers typical of pericytes (orange). These events are temporally associated with, and may even be secondary to, enhanced ERK activation and Notch 3 expression in aged PECs. We speculate that with advanced age, the combination of PEC activation, possible EMT, and pericyte-myofibroblast acquisition leads to increased matrix production by PECs and contributes to glomerulosclerosis. These events, together with reduced PEC density, likely limit any potential progenitor capacity for PECs to function as podocyte progenitors in aging.