Ablation of the renal stroma defines its critical role in nephron progenitor and vasculature patterning

Abstract

The renal stroma is an embryonic cell population located in the cortex that provides a structural framework as well as a source of endothelial progenitors for the developing kidney. The exact role of the renal stroma in normal kidney development hasn't been clearly defined. However, previous studies have shown that the genetic deletion of Foxd1, a renal stroma specific gene, leads to severe kidney malformations confirming the importance of stroma in normal kidney development. This study further investigates the role of renal stroma by ablating Foxd1-derived stroma cells themselves and observing the response of the remaining cell populations. A Foxd1cre (renal stroma specific) mouse was crossed with a diphtheria toxin mouse (DTA) to specifically induce apoptosis in stromal cells. Histological examination of kidneys at embryonic day 13.5-18.5 showed a lack of stromal tissue, mispatterning of renal structures, and dysplastic and/or fused horseshoe kidneys. Immunofluorescence staining of nephron progenitors, vasculature, ureteric epithelium, differentiated nephron progenitors, and vascular supportive cells revealed that mutants had thickened nephron progenitor caps, cortical regions devoid of nephron progenitors, aberrant vessel patterning and thickening, ureteric branching defects and migration of differentiated nephron structures into the medulla. The similarities between the renal deformities caused by Foxd1 genetic knockout and Foxd1DTA mouse models reveal the importance of Foxd1 in mediating and maintaining the functional integrity of the renal stroma.

Figure 1. Apoptosis is up-regulated in the metanephric mesenchyme of Foxd1DTA mutants.

A–C: Control E11.5 kidney showing apoptotic cells in relation to the developing mesenchyme and ureteric bud. A. Apoptotic cells are observed in the common nephric duct (arrow) however very few are seen throughout the metanephric mesenchyme (arrowhead). B–C. representative activated Caspase 3 staining showing very few apoptotic cells in the metanephric mesenchyme (dotted lines) or ureteric bud (UB) of controls as marked by Pax2 staining (C). D–F. Control E11.5 kidney showing abundant apoptotic cells via both apoptosis assay (D) and activated Caspase 3 (E) throughout the metanephric mesenchyme (arrowheads) as marked by Pax2 staining (F). Scale bar = 100 µm.

Figure 2. Renal stroma ablation causes morphological defects throughout development.

A–B: E11.5 H&E staining shows no renal abnormalities in Foxd1DTA mutants (B) compared to controls (A). C–D: E13.5 H&E staining highlights the absence of the renal stroma in the outer cortex and interdigitating between the caps (black arrows) in mutants (D) compared to controls (C). Early signs of irregular thickening and expansion of the nephron progenitor caps are also present (white arrow). E–G: E16.5 Foxd1DTA mutant kidneys (F,G) are smaller than controls (E) and have severe structural abnormalities such as dysplastic (F) and fused horseshoe (G) kidneys. E′, F′,G′: Close up images of cortical regions of controls (E′) and mutants (F′,G′) show the absence of stroma between renal structures, especially in the spaces between the nephron progenitor caps (black arrows). Furthermore in mutants, the renal structures lack an organized structure in the cortex compared to controls (white arrows). Scale bar = A–B:50 µm; C–D:100 µm; E–G:400 µm; E′–G′:100 µm.

Figure 3. The stroma of Foxd1DTA mutants is mispatterned.

A–F: E13.5 control (A–C) and Foxd1DTA (D–F) kidney sections stained with renal stromal markers. E13.5 kidneys stained with Meis1/2 (A and D), Tenascin (B and E) and PDGFRB (C and F) show that mutant samples have disorganization with a lack of stromal tissue interdigitating between the nephron progenitor units. G–L: E16.5 control (G–I) and Foxd1DTA (J–L) kidney sections stained with renal stromal markers. The lack of organization is again apparent, with a thickened capsule (concave arrow) and lack of interdigitation (arrow). Low power PDGFRB images show a fused kidney and the lack of stromal organization (I and L). Scale bars A–H and J–K = 100 µm, I and L = 200 µm.

Figure 4. Foxd1DTA mutants have thickened and widened nephron progenitor caps.

A–B: E13.5 Six2 staining reveals that mutant kidneys (B) have thicker nephron progenitors and widened progenitor caps (arrow) compared to controls (A). C–F: At E18.5, Six2 staining shows the nephron progenitor caps were thicker in the mutants (D) compared to controls (C). It is also apparent that the nephron progenitor caps experience a widening, with some mutant caps being almost twice the size (D) of the control caps (C). E–H: Amphiphysin staining reveals similar nephron progenitor thickening and disorganization of the nephron progenitors that remains apparent at E18.5 (H). I–L: Pax2 staining of the nephron progenitors confirms nephron progenitor thickening at E13.5 in mutants (J) compared to controls (I). At E18.5 the large and unorganized nephron progenitor caps are apparent in mutants (L). Scale bar = A–B:100 µm; C–F:25 µm.

Figure 5. Foxd1DTA mutants have cortical regions devoid of nephron progenitor caps and ureteric branching defects.

A–B: Six2 immunofluorescence staining showed that mutant kidneys (B) had gaps in Six2 expression in the renal cortex compared to the consistent line of nephron progenitor caps in controls (A). C–D: DBA staining shows an decrease in the number of ureteric branch tips in mutants (D) compared to controls. (C). E–H: Representative images of Pan-Cytokeratin (Pan-CK) staining at E16.5. Low power images show the lack of organization in the ureteric branching in mutants (F) compared to controls (E). Higher power images show that mutant ureteric epithelium (H) in some cases fails to branch (arrow), while in other disorganized branching is seen (concave arrow). Scale bar = A–B and G–H:100 µm, C–F:400 µm.

Figure 6. Ureteric tip markers are mis-expressed in Foxd1DTA mutants.

A–H: Ret expression in Foxd1DTA mutants compared to controls. At E13.5 Ret expression is confined to the ureteric tips in controls (A and E) however in the mutants it can be seen extending down into the ureteric trunk (B and F). At E16.5 Ret expression can similarly be seen extending beyond the tips in mutants (D and H) while controls are confined to the tips (C and G). I–P: Wnt11 expression in Foxd1DTA mutants compared to controls. At E13.5 Wnt11 expression is confined to the ureteric tips in controls (I and M) however in the mutants it can be seen extending down into the ureteric trunk (J and N). At E16.5 Wnt11 expression can similarly be seen extending beyond the tips in mutants (L and P) while controls are confined to the tips (K and O). A–D and I–L Scale bar = 200 µm, E–H and M–P, Scale bar = 50 µm.

Figure 7. Differentiated nephron structures migrate into the medulla in Foxd1DTA mutants.

A–B. E16.5 NCAM staining. In mutant kidneys (B), we observed a lack of organization in differentiated nephron structures in comparison to controls (A). Furthermore, the differentiated structures, normally in the cortex, abnormally expand into the medulla of mutant kidneys (arrows). C–D: E16.5 Jagged 1 staining. Differentiated nephron structures could again be seen deep in the medulla of the mutant kidneys (arrow). E–F: E16.5 Lhx1 staining. Differentiated nephron structures could again be seen deep in the medulla of the mutant kidneys (arrow). Scale bar = A–F:100 µm.

Figure 8. Foxd1DTA mutants have thickened vessels and abnormal patterning.

A–B: E18.5 PECAM and Six2 immunofluorescence staining. Mutant kidneys (B) showed irregular vessel formation on the outside of nephron progenitor units (arrow heads) and thickened vessels between nephron progenitor caps (arrows), both of which are areas typically occupied by the renal stroma. C–D: E16.5 wholemount stains of Six2 and PECAM. Again in the mutant kidney (D), the vessels are much thicker (white arrow) than the control (C). Also, the extent of the vasculature overgrowth on the outside of the nephron progenitor cap in mutants is more apparent in the whole mount staining showing the vessels piling up thickly over top of the progenitor caps (D). A–B:50 µm; C–D:100 µm.

Figure 9. Glomeruli that form have Mesangial cells and renin-producing cells that are normally distributed in Foxd1DTA mutants.

A–F: Renin and PDGFRB immunofluorescence staining within glomeruli. Renin is clearly localized in the juxaglomerular apparatus of the control (concave arrow, A and C) and mutant (concave arrow, D and F) glomeruli. While PDGFRB staining is seen localized within the mesangial cells of the glomeruli (arrows). Scale bar = 150 µm.