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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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CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with 35S- labeled probes for CXCR7, CXCR4, and CXCL12. (A,B) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (C) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with 35S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (D) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (E) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (F) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (G) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).
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pone-0042814-g003: CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with 35S- labeled probes for CXCR7, CXCR4, and CXCL12. (A,B) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (C) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with 35S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (D) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (E) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (F) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (G) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).

Mentions: Consequently, we were interested to clarify the spatial relationship between CXCR7, CXCR4 and CXCL12 in mature glomeruli. Highpower analysis of hybridized sections subjected to liquid emulsion coating and counterstaining with cresyl violet revealed expression of CXCR7 and CXCL12 in the visceral epithelium of the glomerulus and that of CXCR4 and CXCL12 in the center of the glomerular tuft (Figure 3). To confirm CXCR7 expression in the visceral blade we then combined immunofluorescence for GFP and the podocyte markers podocin (Figure 3G) and nephrin (Figure S1) in E16.5 kidney sections from CXCR7-GFP transgenic mice. Examination by confocal microscopy provided evidence that the CXCR7 gene was transcribed in podocytes. Dual labeling of the CXCR4 receptor protein and CXCR7-GFP showed CXCR4 positive cells in the glomerular tuft flanking the CXCR7 expressing podocytes (Figure 3F: arrow). Absence of CXCR4 from podocytes was confirmed by dual in situ hybridization for CXCR4 mRNA and WT1 mRNA which is selectively expressed in podocytes of the mature glomerulus (Figure 3D). By applying the same approach for CXCL12, we identified CXCL12 mRNA both in WT1-positive podocytes (Figure 3E: WT1) and the WT1 negative capillary loop region (Figure 3E: arrowhead). Thus, CXCR4 and CXCR7 were strictly segregated in mature E16.5 glomeruli whereas CXCL12 was reminiscent of both CXCR4 and CXCR7.


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)

CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with 35S- labeled probes for CXCR7, CXCR4, and CXCL12. (A,B) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (C) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with 35S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (D) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (E) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (F) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (G) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).
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Related In: Results  -  Collection

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pone-0042814-g003: CXCR7, CXCR4, and CXCL12 expression in mature glomeruli.(A–C) Brightfield micrographs of cresyl violet stained mature glomeruli (E16.5) after hybridization with 35S- labeled probes for CXCR7, CXCR4, and CXCL12. (A,B) Signals for CXCR7 are restricted to the podocyte layer whereas CXCR4 is restricted to the center of the glomerulus. (C) CXCL12 is detected both in the podocytes and in the area of the vascular pole (vp, arrowhead). (D–E) Mature glomeruli after dual in situ hybridization with 35S labeled probes for CXCR4 (D), CXCL12 (E), and a DIG-labeled probe for podocyte marker WT1 (D,E). (D) The darkfield micrograph reveals CXCR4 labeling (white signals) close to the vascular pole but not in WT1 stained podocytes of the visceral blade of Bowman's capsule. (E) The brightfield image shows labeling for CXCL12 mRNA (black grains) in the WT1 positive podocyte layer and in the WT1 negative area of the vascular pole (vp, arrowhead). (F–G) Confocal images of dual immunofluorescent stainings for GFP/CXCR4 (F) and GFP/podocin (G) in E16.5 BAC transgenic mice expressing EGFP under the control of the CXCR7 promoter. (F) CXCR4 immunoreactivity is present in the glomerular tuft (arrow), presumptive arterioles (arrowheads), and at the luminar membrane of tubular epithelial cells (asteriscs). Some tubules are co-positive for CXCR7-GFP and CXCR4 (filled asteristics), others display exclusively CXCR4 protein signals (open asteristics). In the glomerulus, signals for CXCR4 and CXCR7-GFP do not overlap. (G) Podocytes labeled by the selective marker podocin (red) are CXCR7-GFP positive. Scale bars represent 10 µm (C,G′″) and 20 µm (F).
Mentions: Consequently, we were interested to clarify the spatial relationship between CXCR7, CXCR4 and CXCL12 in mature glomeruli. Highpower analysis of hybridized sections subjected to liquid emulsion coating and counterstaining with cresyl violet revealed expression of CXCR7 and CXCL12 in the visceral epithelium of the glomerulus and that of CXCR4 and CXCL12 in the center of the glomerular tuft (Figure 3). To confirm CXCR7 expression in the visceral blade we then combined immunofluorescence for GFP and the podocyte markers podocin (Figure 3G) and nephrin (Figure S1) in E16.5 kidney sections from CXCR7-GFP transgenic mice. Examination by confocal microscopy provided evidence that the CXCR7 gene was transcribed in podocytes. Dual labeling of the CXCR4 receptor protein and CXCR7-GFP showed CXCR4 positive cells in the glomerular tuft flanking the CXCR7 expressing podocytes (Figure 3F: arrow). Absence of CXCR4 from podocytes was confirmed by dual in situ hybridization for CXCR4 mRNA and WT1 mRNA which is selectively expressed in podocytes of the mature glomerulus (Figure 3D). By applying the same approach for CXCL12, we identified CXCL12 mRNA both in WT1-positive podocytes (Figure 3E: WT1) and the WT1 negative capillary loop region (Figure 3E: arrowhead). Thus, CXCR4 and CXCR7 were strictly segregated in mature E16.5 glomeruli whereas CXCL12 was reminiscent of both CXCR4 and CXCR7.

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