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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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CXCR4 immunoreactivity in CXCR7 deficient kidneys.(A,B) In situ hybridizations with a radiolabeled CXCR4 probe show similar CXCR4 expression patterns and labeling intensities in the E14.5 kidney of a wildtype and a CXCR7 deficient mutant. (C,D) Immunostaining of the CXCR4 protein in E16.5 kidney sections of a wildtype and a CXCR7 mutant littermate show severely reduced immunoreactivity in the nephrogenic zone (nz), in glomerular tufts (arrowheads) but not in tubular structures (arrows) of the mutant. (E–H) Higher magnification pictures showing CXCR4 protein signals in wildtype and CXCR7 deficient nephrogenic zone (E,F) and tubular structures (G,H). In the cap mesenchyme (cm) of the CXCR7−/− embryo (F), CXCR4 immunoreactivity is heavily reduced to single protein signals allocated to the cytoplasm of the mesenchymal cells (arrows). CXCR4 protein signals at the apical site of tubular epithelial cells (ep) are similar in a CXCR7 wildtype (G) and a mutant littermate (H). (I) Measurement of CXCR4 mRNA-radiosignal positive area in the nephrogenic zone (see A,B: nz). (J) Measurement of CXCR4 immunoreactivity positive area in the nephrogenic zone (see C,D: nz) reveals severe downregulation of the CXCR4 protein in CXCR7−/− kidney sections (Mann-Whitney U test, p<0.001). Data represent mean values ±SEM as percentage of the wildtype group (I,J). (K) Western Blot showing CXCR4 protein expression from 22 pooled E14.5 kidneys of control and CXCR7 knockout embryos. Endogenous transferring receptor (TFR) was used as loading control. lum, lumen; rp, renal pelvis: ub, ureteric bud. Scale bars equal 100 µm (B), 200 µm (D) and 10 µm (F,H).
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pone-0042814-g005: CXCR4 immunoreactivity in CXCR7 deficient kidneys.(A,B) In situ hybridizations with a radiolabeled CXCR4 probe show similar CXCR4 expression patterns and labeling intensities in the E14.5 kidney of a wildtype and a CXCR7 deficient mutant. (C,D) Immunostaining of the CXCR4 protein in E16.5 kidney sections of a wildtype and a CXCR7 mutant littermate show severely reduced immunoreactivity in the nephrogenic zone (nz), in glomerular tufts (arrowheads) but not in tubular structures (arrows) of the mutant. (E–H) Higher magnification pictures showing CXCR4 protein signals in wildtype and CXCR7 deficient nephrogenic zone (E,F) and tubular structures (G,H). In the cap mesenchyme (cm) of the CXCR7−/− embryo (F), CXCR4 immunoreactivity is heavily reduced to single protein signals allocated to the cytoplasm of the mesenchymal cells (arrows). CXCR4 protein signals at the apical site of tubular epithelial cells (ep) are similar in a CXCR7 wildtype (G) and a mutant littermate (H). (I) Measurement of CXCR4 mRNA-radiosignal positive area in the nephrogenic zone (see A,B: nz). (J) Measurement of CXCR4 immunoreactivity positive area in the nephrogenic zone (see C,D: nz) reveals severe downregulation of the CXCR4 protein in CXCR7−/− kidney sections (Mann-Whitney U test, p<0.001). Data represent mean values ±SEM as percentage of the wildtype group (I,J). (K) Western Blot showing CXCR4 protein expression from 22 pooled E14.5 kidneys of control and CXCR7 knockout embryos. Endogenous transferring receptor (TFR) was used as loading control. lum, lumen; rp, renal pelvis: ub, ureteric bud. Scale bars equal 100 µm (B), 200 µm (D) and 10 µm (F,H).

Mentions: Because CXCR7 has been shown to influence CXCL12/CXCR4 dependent histogenesis in brain [11], [21] and heart [18], [19], [22], we then analysed the CXCR4 mRNA pattern in CXCR7−/− and CXCR7+/+ littermates. This showed that neither the overall pattern of CXCR4 mRNA expression nor the CXCR4 mRNA positive area was changed in the CXCR7 mutants (Figure 5A,B). As we previously established that CXCR7 is required to maintain sufficient amounts of CXCR4 receptor protein to sustain CXCL12/CXCR4 dependent signaling in developing neurons [11] we examined CXCR4 immunoreactivity in the kidney of CXCR7−/− embryos. Here we found that the protein signal intensity was dramatically reduced by around 75% in the nephrogenic zone (nz) as compared with control littermates (CXCR4 positive area in nz: CXCR7+/+, 14.08%±1.16; CXCR7−/−, 3.21%±0.97; p<0.001) (Figure 5C,D: nz, Figure 5J). This observation was verified by Western Blot analysis of E14.5 kidneys (Figure 5K). The CXCR4 mRNA positive area however was not altered in the nephrogenic zone of both cohorts (CXCR7+/+, 12.93%±0.89; CXCR7−/−, 12.54%±0.86) (Figure 5A,B: nz, Figure 5I), indicating that CXCR4 protein but not mRNA was down-regulated in the CXCR7 deficient cap mesenchyme (Figure 5E,F: cm). This conclusion was confirmed by the observation that CXCR4 protein signals in the wildtype could be allocated to the plasma membrane (Figure 5E: arrowheads) and those in the CXCR7 knockout to the cytoplasm of the mesenchymal cells (Figure 5F: arrows). In contrast to the nephrogenic zone, CXCR4 protein (and CXCR4 mRNA) was not diminished in the epithelium of renal tubules and collecting ducts in CXCR7 kidneys (Figure 5C,D: arrows, Figure 5G,H).


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)

CXCR4 immunoreactivity in CXCR7 deficient kidneys.(A,B) In situ hybridizations with a radiolabeled CXCR4 probe show similar CXCR4 expression patterns and labeling intensities in the E14.5 kidney of a wildtype and a CXCR7 deficient mutant. (C,D) Immunostaining of the CXCR4 protein in E16.5 kidney sections of a wildtype and a CXCR7 mutant littermate show severely reduced immunoreactivity in the nephrogenic zone (nz), in glomerular tufts (arrowheads) but not in tubular structures (arrows) of the mutant. (E–H) Higher magnification pictures showing CXCR4 protein signals in wildtype and CXCR7 deficient nephrogenic zone (E,F) and tubular structures (G,H). In the cap mesenchyme (cm) of the CXCR7−/− embryo (F), CXCR4 immunoreactivity is heavily reduced to single protein signals allocated to the cytoplasm of the mesenchymal cells (arrows). CXCR4 protein signals at the apical site of tubular epithelial cells (ep) are similar in a CXCR7 wildtype (G) and a mutant littermate (H). (I) Measurement of CXCR4 mRNA-radiosignal positive area in the nephrogenic zone (see A,B: nz). (J) Measurement of CXCR4 immunoreactivity positive area in the nephrogenic zone (see C,D: nz) reveals severe downregulation of the CXCR4 protein in CXCR7−/− kidney sections (Mann-Whitney U test, p<0.001). Data represent mean values ±SEM as percentage of the wildtype group (I,J). (K) Western Blot showing CXCR4 protein expression from 22 pooled E14.5 kidneys of control and CXCR7 knockout embryos. Endogenous transferring receptor (TFR) was used as loading control. lum, lumen; rp, renal pelvis: ub, ureteric bud. Scale bars equal 100 µm (B), 200 µm (D) and 10 µm (F,H).
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pone-0042814-g005: CXCR4 immunoreactivity in CXCR7 deficient kidneys.(A,B) In situ hybridizations with a radiolabeled CXCR4 probe show similar CXCR4 expression patterns and labeling intensities in the E14.5 kidney of a wildtype and a CXCR7 deficient mutant. (C,D) Immunostaining of the CXCR4 protein in E16.5 kidney sections of a wildtype and a CXCR7 mutant littermate show severely reduced immunoreactivity in the nephrogenic zone (nz), in glomerular tufts (arrowheads) but not in tubular structures (arrows) of the mutant. (E–H) Higher magnification pictures showing CXCR4 protein signals in wildtype and CXCR7 deficient nephrogenic zone (E,F) and tubular structures (G,H). In the cap mesenchyme (cm) of the CXCR7−/− embryo (F), CXCR4 immunoreactivity is heavily reduced to single protein signals allocated to the cytoplasm of the mesenchymal cells (arrows). CXCR4 protein signals at the apical site of tubular epithelial cells (ep) are similar in a CXCR7 wildtype (G) and a mutant littermate (H). (I) Measurement of CXCR4 mRNA-radiosignal positive area in the nephrogenic zone (see A,B: nz). (J) Measurement of CXCR4 immunoreactivity positive area in the nephrogenic zone (see C,D: nz) reveals severe downregulation of the CXCR4 protein in CXCR7−/− kidney sections (Mann-Whitney U test, p<0.001). Data represent mean values ±SEM as percentage of the wildtype group (I,J). (K) Western Blot showing CXCR4 protein expression from 22 pooled E14.5 kidneys of control and CXCR7 knockout embryos. Endogenous transferring receptor (TFR) was used as loading control. lum, lumen; rp, renal pelvis: ub, ureteric bud. Scale bars equal 100 µm (B), 200 µm (D) and 10 µm (F,H).
Mentions: Because CXCR7 has been shown to influence CXCL12/CXCR4 dependent histogenesis in brain [11], [21] and heart [18], [19], [22], we then analysed the CXCR4 mRNA pattern in CXCR7−/− and CXCR7+/+ littermates. This showed that neither the overall pattern of CXCR4 mRNA expression nor the CXCR4 mRNA positive area was changed in the CXCR7 mutants (Figure 5A,B). As we previously established that CXCR7 is required to maintain sufficient amounts of CXCR4 receptor protein to sustain CXCL12/CXCR4 dependent signaling in developing neurons [11] we examined CXCR4 immunoreactivity in the kidney of CXCR7−/− embryos. Here we found that the protein signal intensity was dramatically reduced by around 75% in the nephrogenic zone (nz) as compared with control littermates (CXCR4 positive area in nz: CXCR7+/+, 14.08%±1.16; CXCR7−/−, 3.21%±0.97; p<0.001) (Figure 5C,D: nz, Figure 5J). This observation was verified by Western Blot analysis of E14.5 kidneys (Figure 5K). The CXCR4 mRNA positive area however was not altered in the nephrogenic zone of both cohorts (CXCR7+/+, 12.93%±0.89; CXCR7−/−, 12.54%±0.86) (Figure 5A,B: nz, Figure 5I), indicating that CXCR4 protein but not mRNA was down-regulated in the CXCR7 deficient cap mesenchyme (Figure 5E,F: cm). This conclusion was confirmed by the observation that CXCR4 protein signals in the wildtype could be allocated to the plasma membrane (Figure 5E: arrowheads) and those in the CXCR7 knockout to the cytoplasm of the mesenchymal cells (Figure 5F: arrows). In contrast to the nephrogenic zone, CXCR4 protein (and CXCR4 mRNA) was not diminished in the epithelium of renal tubules and collecting ducts in CXCR7 kidneys (Figure 5C,D: arrows, Figure 5G,H).

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