Host Lymphotoxin-β Receptor Signaling Is Crucial for Angiogenesis of Metanephric Tissue Transplanted into Lymphoid Sites

Abstract

The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-b receptor (LTbR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTbR antagonist treatment or in the omenta of Ltbr knockout (Ltbr À/À ) mice, the host LTbR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTbR signaling correlated with decreased expression of endothelial and angiogenic markers in kidney grafts as well as structural alterations. Because the number of glomerular endothelial cells expressing the LTbR target nuclear factor kBeinducing kinase (NIK) decreased in the absence of a functional LTbR, it was speculated that an LTbR/NIK axis mediated the angiogenetic signals required for successful ectopic kidney organogenesis, given the established role of NIK in neovascularization. However, the transplantation of kidney rudiments in omenta of Nik À/À mice revealed that NIK is dispensable for ectopic kidney vascular integration and maturation. Finally, defective LTbR signaling impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTbR signals.

Blood Vessel Detection Algorithm for Tissue Engineering and Quantitative Histology

“…Metanephric (E14.5) kidneys were retrieved from timed-pregnant C57BL/6J mice as previously described 16 and transplanted in the omentum of recipient C57BL/6J mice. 15 After two weeks from the engraftment, the kidneys were retrieved from the mice and prepared for histological examination. Histological samples were fixed in 4% paraformaldehyde, infiltrated in 30% sucrose solution, embedded in OCT compound, and finally sectioned into 4 µm-thick sections for IF analyses.…”

“…The operator was blinded to the treatment groups and not involved in the projects. Finally, for the third dataset, 15 32 independent glomeruli, in five different images, were analyzed: N=5 from Image 1 , N=6 from Image 2 , N=7 from Image 3 , N=8 from Image 4 and N=6 from Image 5 . The field of view for each image acquisition was 0.46 mm 2 , which is equivalent to 1036x1024 pixels.…”

“… 19 Glomeruli from 5 sections/mouse were considered. The corrected total glomerular fluorescence (CTGF) was calculated using Eq.1 15 , 19 : …”

“…The method was tested and validated using data from three independent in vivo experiments that specifically focused on the characterization of angiogenesis. 7,15,35 Three different tissue types were adopted including: cardiac muscle, abdominal wall, and metanephric kidney engrafted in the omentum. IHC staining of vascular structures in the three in vivo studies was based on the labeling of the EC marker CD31, and/or aSMA.…”

Lymphotoxin-Beta Receptor Signaling Is Crucial for the Vascularization of Transplanted Metanephros

“…One important step toward understanding the mechanisms that rule ectopic organogenesis has been taken by the studies conducted by Francipane et al, 13 which are published in the current issue of The American Journal of Pathology. By transplanting kidney rudiments into either the LNs of mice undergoing lymphotoxin-beta receptor (LTbR) antagonist treatment or the omenta of Ltbr À/À mice, Francipane et al demonstrate that host LTbR signals are crucial for the development of a well-vascularized kidney graft, as well as its survival and growth.…”