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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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Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with 35S labeled probes for CXCR7 or CXCR4. (A) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (B,B′,C,C′) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a 35S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (D,D′) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (E,F) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (G) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (H) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.
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pone-0042814-g002: Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with 35S labeled probes for CXCR7 or CXCR4. (A) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (B,B′,C,C′) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a 35S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (D,D′) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (E,F) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (G) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (H) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.

Mentions: We then focussed on the nephrogenic zone and forming glomeruli. To distinguish ureteric buds from mesenchymal derivates, we detected transcripts of the transcription factor WT1 (Wilms' tumor protein 1), which labels mesenchyme and its derivates during the process of mesenchymal-to-epithelial transition: cap mesenchymal cells, pretubular mesenchymal aggregates as well as epithelial cells of renal vesicles, comma- and S-shaped bodies, capillary-loop nephrons, and the mature glomeruli [20]. By dual in situ hybridization with a radiolabeled probe for CXCR4 or CXCR7 mRNA (Figure 2: detected as grains), respectively, and a DIG labeled probe for WT1 mRNA (color), we identified CXCR7 and CXCR4 transcripts in WT1 negative ureteric buds. Early ureteric bud tips, which were associated with cap mesenchyme/pretubular aggregates, strongly expressed the CXCR4 and CXCR7 transcripts (Figure 2A,D′: eub). Late ureteric bud tips (Figure 2A,D′: lub), which were juxtaposed to renal vesicles, showed weaker detection signals for CXCR4 and CXCR7 antisense mRNA. CXCR4 and CXCR7 genes were expressed in WT1 positive cap mesenchymal structures as well (Figure 2A,D′: cm) however with a much more stronger expression of CXCR4 mRNA. CXCR7 labeling increased during mesenchymal-to-epithelial transition from faint at the cap mesenchyme stage to strong at the pretubular stage (Figure 2A: cm, pa). After epithelialization, CXCR7 expression remained high and was identified in renal vesicles, comma-shaped bodies, S-shaped bodies, and capillary-loop glomeruli (Figure 2A–C; Table 1). In contrast, CXCR4 labeling was faint in the ‘early’ cap mesenchyme (Figure 2D,D′: cm at eub), strong in ‘late’ cap mesenchyme (Figure 2D,D′: cm at lub) and pretubular aggregates but absent in epithelialized renal vesicles and S-shaped bodies (Figure 2D,D′: rv, sb). The vascular clefts of these S-shaped bodies, which are invaded by endothelial and mesangial cells, were heavily labeled for CXCR4 riboprobe (Figure 2D′: arrows). In addition, we detected strong CXCR4 signals in putative arterioles (Figure 1F: arrows; Figure 2D′: arrowhead).


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)

Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with 35S labeled probes for CXCR7 or CXCR4. (A) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (B,B′,C,C′) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a 35S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (D,D′) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (E,F) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (G) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (H) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.
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Related In: Results  -  Collection

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pone-0042814-g002: Differential expression of CXCR7 and CXCR4 in nephrogenic mesenchyme, ureteric bud, and forming glomeruli.(A–D) A digoxigenin (DIG) labeled WT1 antisense probe was used as a marker for mesenchymal and mesenchyme derived nephrogenic structures at E14.5 and co-hybridized with 35S labeled probes for CXCR7 or CXCR4. (A) Strong CXCR7 signals (black grains) are present in a T-shaped WT1 negative early ureteric bud tip (eub) which is associated with a CXCR7/WT1 co-positive pretubular aggregate (pa; black grains/brown staining). Weaker CXCR7 expression is found in a late ureteric bud tip (lub) which is associated with CXCR7/WT1 co-positive renal vesicles (rv). Note weak CXCR7 mRNA expression in the cap mesenchyme (cm) and strong CXCR7 gene activity above the cm (brown staining) at the renal capsule (rc). (B,B′,C,C′) Bright- and darkfield views of a comma-shaped body (cb in B) and S-shaped body (sb in C) after hybridization with a DIG labeled WT1 probe and a 35S labeled CXCR7 riboprobe (black grains in B,C; white grains in B′,C′). Both WT1 positive structures exhibit clear CXCR7 antisense mRNA signals. (D,D′) Bright- and darkfield micrographs showing WT1-positive renal tissue (D) and radiosignals of CXCR4 riboprobe (D′). Strong CXCR4 gene expression is detected in a WT1 negative early ureteric bud tip (eub in D, dotted line in D′). Weak CXCR4 labeling is seen in a late ureteric bud tip (lub) which is associated with a WT1 positive/CXCR4 negative renal vesicle (rv). Note that CXCR4 mRNA is also present in WT1 positive cap mesenchyme (cm). WT1 positive epithelial cells of S-Shaped bodies display no CXCR4 mRNA expression. The vascular cleft of S-shaped bodies (arrows in D′) as well as putative arterioles (arrowhead in D′) are CXCR4 positive. (E,F) GFP immunostaining in sections from BAC transgenic mice expressing EGFP under the control of the CXCR7 promotor (G) or CXCR4 promotor (H). Calbindin was co-stained as an ureteric bud marker. (G) CXCR7-GFP is highly expressed in the renal capsule (rc) as well as in comma- and S-shaped bodies (cb, sb) associated with a calbindin positive late ureteric bud (lub). CXCR7-GFP is weak expressed in cap mesenchyme (cm) and not present in the late ureteric bud. (H) Strong CXCR4-GFP signals are detected only in cap mesenchyme (cm). All scale bars equal 20 µm.
Mentions: We then focussed on the nephrogenic zone and forming glomeruli. To distinguish ureteric buds from mesenchymal derivates, we detected transcripts of the transcription factor WT1 (Wilms' tumor protein 1), which labels mesenchyme and its derivates during the process of mesenchymal-to-epithelial transition: cap mesenchymal cells, pretubular mesenchymal aggregates as well as epithelial cells of renal vesicles, comma- and S-shaped bodies, capillary-loop nephrons, and the mature glomeruli [20]. By dual in situ hybridization with a radiolabeled probe for CXCR4 or CXCR7 mRNA (Figure 2: detected as grains), respectively, and a DIG labeled probe for WT1 mRNA (color), we identified CXCR7 and CXCR4 transcripts in WT1 negative ureteric buds. Early ureteric bud tips, which were associated with cap mesenchyme/pretubular aggregates, strongly expressed the CXCR4 and CXCR7 transcripts (Figure 2A,D′: eub). Late ureteric bud tips (Figure 2A,D′: lub), which were juxtaposed to renal vesicles, showed weaker detection signals for CXCR4 and CXCR7 antisense mRNA. CXCR4 and CXCR7 genes were expressed in WT1 positive cap mesenchymal structures as well (Figure 2A,D′: cm) however with a much more stronger expression of CXCR4 mRNA. CXCR7 labeling increased during mesenchymal-to-epithelial transition from faint at the cap mesenchyme stage to strong at the pretubular stage (Figure 2A: cm, pa). After epithelialization, CXCR7 expression remained high and was identified in renal vesicles, comma-shaped bodies, S-shaped bodies, and capillary-loop glomeruli (Figure 2A–C; Table 1). In contrast, CXCR4 labeling was faint in the ‘early’ cap mesenchyme (Figure 2D,D′: cm at eub), strong in ‘late’ cap mesenchyme (Figure 2D,D′: cm at lub) and pretubular aggregates but absent in epithelialized renal vesicles and S-shaped bodies (Figure 2D,D′: rv, sb). The vascular clefts of these S-shaped bodies, which are invaded by endothelial and mesangial cells, were heavily labeled for CXCR4 riboprobe (Figure 2D′: arrows). In addition, we detected strong CXCR4 signals in putative arterioles (Figure 1F: arrows; Figure 2D′: arrowhead).

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