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CXC chemokine receptor 7 (CXCR7) regulates CXCR4 protein expression and capillary tuft development in mouse kidney.

Haege S, Einer C, Thiele S, Mueller W, Nietzsche S, Lupp A, Mackay F, Schulz S, Stumm R - PLoS ONE (2012)

Bottom Line: Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme.We established that there is a similar glomerular pathology in CXCR7 and CXCR4 embryos.Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany. sammy.haege@mti.uni-jena.de

ABSTRACT

Background: The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/principal findings: We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 mice was associated with mesangial cell clumping.

Conclusions/significance: We established that there is a similar glomerular pathology in CXCR7 and CXCR4 embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

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Comparison of glomerular tuft abnormalities in E16.5 CXCR7 and CXCR4 knockout mice.(A,B) Immunostainings of the CXCR4 protein in an E16.5 wildtype and a CXCR7 deficient littermate show decreased CXCR4-immunoreactivity in the glomerular tuft of the CXCR7 mutant (glomerular tufts are encircled). (C–F) PAS stained E16.5 kidney sections comparing glomeruli of wildtype (C, E) and mutant littermates (D,F). Note severe dilation of capillaries (right glomerulus in D) and a single ballooned capillary (left glomerulus in D) in the CXCR4 mutant. The glomerulus in the CXCR7 mutant (F) is similarly dilated as compared with the corresponding wildtype (E) but abnormalities within the capillary tuft are not as severe as in the CXCR4-mutant (D). (G,H) Micrographs of E16.5 glomeruli immunostained for the mesangium marker PDGFRβ demonstrate mesangium agglutination in a CXCR7 knockout as compared with a wildtype littermate. Dotted lines encircle glomerular tufts. (I) The perimeter of the mesangial tree is reduced in CXCR7 knockout mice as compared with wildtype littermates (p<0.05; Mann-Whitney U test). Data represent mean values ±SEM as percentage of the wildtype group. Scale bars represent 10 µm.
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pone-0042814-g006: Comparison of glomerular tuft abnormalities in E16.5 CXCR7 and CXCR4 knockout mice.(A,B) Immunostainings of the CXCR4 protein in an E16.5 wildtype and a CXCR7 deficient littermate show decreased CXCR4-immunoreactivity in the glomerular tuft of the CXCR7 mutant (glomerular tufts are encircled). (C–F) PAS stained E16.5 kidney sections comparing glomeruli of wildtype (C, E) and mutant littermates (D,F). Note severe dilation of capillaries (right glomerulus in D) and a single ballooned capillary (left glomerulus in D) in the CXCR4 mutant. The glomerulus in the CXCR7 mutant (F) is similarly dilated as compared with the corresponding wildtype (E) but abnormalities within the capillary tuft are not as severe as in the CXCR4-mutant (D). (G,H) Micrographs of E16.5 glomeruli immunostained for the mesangium marker PDGFRβ demonstrate mesangium agglutination in a CXCR7 knockout as compared with a wildtype littermate. Dotted lines encircle glomerular tufts. (I) The perimeter of the mesangial tree is reduced in CXCR7 knockout mice as compared with wildtype littermates (p<0.05; Mann-Whitney U test). Data represent mean values ±SEM as percentage of the wildtype group. Scale bars represent 10 µm.

Mentions: Similar to the situation in the nephrogenic zone we found that CXCR4 immunoreactivity was diminished by around 60% in the glomerular tuft of CXCR7 mutants (Figure 5C,D: arrowheads; Figure 6A,B). Because CXCR4 is required for glomerular tuft development [8] we then focussed on a potential glomerular phenotype in CXCR7−/− mice and reexamined the known phenotype in CXCR4−/− mice for comparison. This showed that more than two third of all scored glomeruli contained dilated capillaries in both CXCR4 and CXCR7 mutants (Figure 6D and 6F). However, dilation of the capillaries was less severe in CXCR7−/− than in CXCR4−/− mice. In detail, 13.5% of all monitored CXCR7 glomeruli displayed a severe ballooning phenotype of the glomerular capillaries (37.2% in CXCR4−/−) and 59.6% of the CXCR7 deficient glomeruli a mild phenotype (37.2% in CXCR4−/−). To check possible defects in mesangium of the CXCR7 mutants, we performed immunostaining with the mesangial marker PDGFRβ and observed clumping of the mesangium in the mutants (Figure 6H). The perimeter of the PDGFRβ-positive area as an index for this agglutination phenotype demonstrated a clear difference between knockout and wildtype littermates (Figure 6I, p<0.05). However no alterations in total podocin and nephrin immunoreactivity were observed between the glomeruli of wildtype and CXCR7−/− embryos at E16.5. This observation was confirmed by electron microscopical analysis showing no morphological abnormalities of CXCR7−/− podocytes and foot processes (Figure 7C,D: pod). Endothelial cells though seemed to be detached from the glomerular basement membrane (Figure 7D: arrows).


CXC chemokine receptor 7 (CXCR7) regulates CXCR4 protein expression and capillary tuft development in mouse kidney.

Haege S, Einer C, Thiele S, Mueller W, Nietzsche S, Lupp A, Mackay F, Schulz S, Stumm R - PLoS ONE (2012)

Comparison of glomerular tuft abnormalities in E16.5 CXCR7 and CXCR4 knockout mice.(A,B) Immunostainings of the CXCR4 protein in an E16.5 wildtype and a CXCR7 deficient littermate show decreased CXCR4-immunoreactivity in the glomerular tuft of the CXCR7 mutant (glomerular tufts are encircled). (C–F) PAS stained E16.5 kidney sections comparing glomeruli of wildtype (C, E) and mutant littermates (D,F). Note severe dilation of capillaries (right glomerulus in D) and a single ballooned capillary (left glomerulus in D) in the CXCR4 mutant. The glomerulus in the CXCR7 mutant (F) is similarly dilated as compared with the corresponding wildtype (E) but abnormalities within the capillary tuft are not as severe as in the CXCR4-mutant (D). (G,H) Micrographs of E16.5 glomeruli immunostained for the mesangium marker PDGFRβ demonstrate mesangium agglutination in a CXCR7 knockout as compared with a wildtype littermate. Dotted lines encircle glomerular tufts. (I) The perimeter of the mesangial tree is reduced in CXCR7 knockout mice as compared with wildtype littermates (p<0.05; Mann-Whitney U test). Data represent mean values ±SEM as percentage of the wildtype group. Scale bars represent 10 µm.
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pone-0042814-g006: Comparison of glomerular tuft abnormalities in E16.5 CXCR7 and CXCR4 knockout mice.(A,B) Immunostainings of the CXCR4 protein in an E16.5 wildtype and a CXCR7 deficient littermate show decreased CXCR4-immunoreactivity in the glomerular tuft of the CXCR7 mutant (glomerular tufts are encircled). (C–F) PAS stained E16.5 kidney sections comparing glomeruli of wildtype (C, E) and mutant littermates (D,F). Note severe dilation of capillaries (right glomerulus in D) and a single ballooned capillary (left glomerulus in D) in the CXCR4 mutant. The glomerulus in the CXCR7 mutant (F) is similarly dilated as compared with the corresponding wildtype (E) but abnormalities within the capillary tuft are not as severe as in the CXCR4-mutant (D). (G,H) Micrographs of E16.5 glomeruli immunostained for the mesangium marker PDGFRβ demonstrate mesangium agglutination in a CXCR7 knockout as compared with a wildtype littermate. Dotted lines encircle glomerular tufts. (I) The perimeter of the mesangial tree is reduced in CXCR7 knockout mice as compared with wildtype littermates (p<0.05; Mann-Whitney U test). Data represent mean values ±SEM as percentage of the wildtype group. Scale bars represent 10 µm.
Mentions: Similar to the situation in the nephrogenic zone we found that CXCR4 immunoreactivity was diminished by around 60% in the glomerular tuft of CXCR7 mutants (Figure 5C,D: arrowheads; Figure 6A,B). Because CXCR4 is required for glomerular tuft development [8] we then focussed on a potential glomerular phenotype in CXCR7−/− mice and reexamined the known phenotype in CXCR4−/− mice for comparison. This showed that more than two third of all scored glomeruli contained dilated capillaries in both CXCR4 and CXCR7 mutants (Figure 6D and 6F). However, dilation of the capillaries was less severe in CXCR7−/− than in CXCR4−/− mice. In detail, 13.5% of all monitored CXCR7 glomeruli displayed a severe ballooning phenotype of the glomerular capillaries (37.2% in CXCR4−/−) and 59.6% of the CXCR7 deficient glomeruli a mild phenotype (37.2% in CXCR4−/−). To check possible defects in mesangium of the CXCR7 mutants, we performed immunostaining with the mesangial marker PDGFRβ and observed clumping of the mesangium in the mutants (Figure 6H). The perimeter of the PDGFRβ-positive area as an index for this agglutination phenotype demonstrated a clear difference between knockout and wildtype littermates (Figure 6I, p<0.05). However no alterations in total podocin and nephrin immunoreactivity were observed between the glomeruli of wildtype and CXCR7−/− embryos at E16.5. This observation was confirmed by electron microscopical analysis showing no morphological abnormalities of CXCR7−/− podocytes and foot processes (Figure 7C,D: pod). Endothelial cells though seemed to be detached from the glomerular basement membrane (Figure 7D: arrows).

Bottom Line: Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme.We established that there is a similar glomerular pathology in CXCR7 and CXCR4 embryos.Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany. sammy.haege@mti.uni-jena.de

ABSTRACT

Background: The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/principal findings: We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 mice was associated with mesangial cell clumping.

Conclusions/significance: We established that there is a similar glomerular pathology in CXCR7 and CXCR4 embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

Show MeSH
Related in: MedlinePlus