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

  • Valerio Brizi
    Affiliations
    Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy
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  • Christodoulos Xinaris
    Correspondence
    Address correspondence to Christodoulos Xinaris, Ph.D., Department of Molecular Medicine, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Via Stezzano, 87 24126 Bergamo, Italy.
    Affiliations
    Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy

    University of Nicosia Medical School, Nicosia, Cyprus
    Search for articles by this author
Open ArchivePublished:October 16, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.10.002

      Growing Embryonic Kidneys in Living Animals

      Although approximately 18,000 patients with end-stage kidney disease receive a donor organ each year in the USA,
      Centers for Disease Control and Prevention
      National Chronic Kidney Disease Fact Sheet: General Information and National Estimates on Chronic Kidney Disease in the United States. 2010.
      there are more than 5 times as many patients on waiting lists. The shortage of donor organs has caused the waiting period to increase to over three years and the mortality rate by 5% to 10%. Growing metanephroi (fetal kidney primordia) in living animals that could be used for replacement therapies may help bridge this gap.
      • Hammerman M.R.
      Organogenesis of kidneys following transplantation of renal progenitor cells.
      ,
      • Xinaris C.
      • Yokoo T.
      Reforming the kidney starting from a single-cell suspension.
      Over the last two decades, studies have shown that it is possible to grow new kidneys in adult living hosts or to integrate new filtering nephrons into neonatal kidneys. Embryonic kidney rudiments implanted beneath the renal capsule,
      • Dekel B.
      • Burakova T.
      • Arditti F.D.
      • Reich-Zeliger S.
      • Milstein O.
      • Aviel-Ronen S.
      • Rechavi G.
      • Friedman N.
      • Kaminski N.
      • Passwell J.H.
      • Reisner Y.
      Human and porcine early kidney precursors as a new source for transplantation.
      ,
      • Rogers S.A.
      • Lowell J.A.
      • Hammerman N.A.
      • Hammerman M.R.
      Transplantation of developing metanephroi into adult rats.
      within the omentum
      • Rogers S.A.
      • Lowell J.A.
      • Hammerman N.A.
      • Hammerman M.R.
      Transplantation of developing metanephroi into adult rats.
      ,
      • Matsumoto K.
      • Yokoo T.
      • Matsunari H.
      • Iwai S.
      • Yokote S.
      • Teratani T.
      • Gheisari Y.
      • Tsuji O.
      • Okano H.
      • Utsunomiya Y.
      • Hosoya T.
      • Okano H.J.
      • Nagashima H.
      • Kobayashi E.
      Xenotransplanted embryonic kidney provides a niche for endogenous mesenchymal stem cell differentiation into erythropoietin-producing tissue.
      or into tunnels in the cortices of host kidneys
      • Woolf A.S.
      • Hornbruch A.
      • Fine L.G.
      Integration of new embryonic nephrons into the kidney.
      become integrated into living animals and exhibit key renal functions, such glomerular filtration and macromolecule reabsorption. Cultured fetal kidneys
      • Robert B.
      • St John P.L.
      • Hyink D.P.
      • Abrahamson D.R.
      Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts.
      or organoids made from embryonic kidney cells
      • Xinaris C.
      • Benedetti V.
      • Rizzo P.
      • Abbate M.
      • Corna D.
      • Azzollini N.
      • Conti S.
      • Unbekandt M.
      • Davies J.A.
      • Morigi M.
      • Benigni A.
      • Remuzzi G.
      In vivo maturation of functional renal organoids formed from embryonic cell suspensions.
      also become vascularized and form mature and functional glomeruli following in vivo transplantation. More recent studies have shown that mouse
      • Francipane M.G.
      • Lagasse E.
      The lymph node as a new site for kidney organogenesis.
      and human
      • Francipane M.G.
      • Han B.
      • Oxburgh L.
      • Sims-Lucas S.
      • Li Z.
      • Lagasse E.
      Kidney-in-a-lymph node: a novel organogenesis assay to model human renal development and test nephron progenitor cell fates.
      kidney primordia transplanted into the lymph node (LN) can also grow and mature into functional miniorgans.
      Growing embryonic anlagens in situ has important technical and clinical benefits/advantages. The developing kidney is less immunogenic than the adult kidney because it contains fewer antigen-presenting cells and expresses fewer MHC class I and class II antigens.
      • Hammerman M.R.
      Organogenesis of kidneys following transplantation of renal progenitor cells.
      Compared to embryonic stem cells or induced pluripotent stem cells (PSCs), metanephric cells are already committed to a genetic program of renal development, obviating the need to preprogram cell fate and engineer a complete organ de novo. Nevertheless, there are also important challenges that must be addressed before these approaches achieve clinical relevance. First, unnatural ectopic environments do not allow fetal kidneys to develop into fully structurally and functionally mature organs (embryonic grafts reach approximately 4% of natural kidney size). Second, the ability of transplanted fetal kidneys to grow differentially according to the environment, results in a great variation in the outcomes of transplantation (in terms of efficient vascularization, and structural and functional maturation). Surprisingly, fetal organs grow differentially, even in the same animal, when transplanted into different sites.
      • Matsumoto K.
      • Yokoo T.
      • Yokote S.
      • Utsunomiya Y.
      • Ohashi T.
      • Hosoya T.
      Functional development of a transplanted embryonic kidney: effect of transplantation site.
      Identifying the cellular and molecular mechanisms that coordinate ectopic organ development would significantly improve the efficacy of the existing methods and hopefully produce kidney tissues that can be used for replacement therapies.
      One important step toward understanding the mechanisms that rule ectopic organogenesis has been taken by the studies conducted by Francipane et al,
      • Francipane M.G.
      • Han B.
      • Lagasse E.
      Host lymphotoxin-beta receptor signaling is crucial for angiogenesis of metanephric tissue transplanted into lymphoid sites.
      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 (LTβR) antagonist treatment or the omenta of Ltβr−/− mice, Francipane et al demonstrate that host LTβR signals are crucial for the development of a well-vascularized kidney graft, as well as its survival and growth. The absence of LTβR signals also impairs compensatory glomerular growth in response to renal mass reduction, without affecting normal kidney development.
      This commentary discusses the significance of these findings as well as points out the current challenges in utilizing these findings in regenerative nephrology. It further highlights the avenues that should be explored next.

      Vascularization of Glomeruli Engrafted into Mouse LN Might Be Driven by the Host LTβR/NIK Axis

      Earlier studies by Francipane showed that embryonic kidneys transplanted into mouse LNs develop mature nephrons with a vascular network that originates from both host and graft-derived vascular cells.
      • Francipane M.G.
      • Lagasse E.
      The lymph node as a new site for kidney organogenesis.
      ,
      • Francipane M.G.
      • Han B.
      • Oxburgh L.
      • Sims-Lucas S.
      • Li Z.
      • Lagasse E.
      Kidney-in-a-lymph node: a novel organogenesis assay to model human renal development and test nephron progenitor cell fates.
      ,
      • Francipane M.G.
      • Lagasse E.
      Maturation of embryonic tissues in a lymph node: a new approach for bioengineering complex organs.
      Although these studies demonstrated that the LN provides an appropriate niche for transplanting and growing embryonic kidneys, the cellular and molecular mechanisms that govern organogenesis within the LN remain unknown.
      In this new study, Francipane et al investigated the molecular signals that support kidney vascularization in host lymphoid sites and omenta. Based on the observation that most glomerular endothelial cells (CD34+) express nuclear factor kB-inducing kinase (NIK)—a downstream target of LTβR that is a key regulator of angiogenesis
      • Noort A.R.
      • van Zoest K.P.M.
      • Weijers E.M.
      • Koolwijk P.
      • Maracle C.X.
      • Novack D.V.
      • Siemerink M.J.
      • Schlingemann R.O.
      • Tak P.P.
      • Tas S.W.
      NF-ΚB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis.
      and LN development
      • Dejardin E.
      • Droin N.M.
      • Delhase M.
      • Haas E.
      • Cao Y.
      • Makris C.
      • Li Z.W.
      • Karin M.
      • Ware C.F.
      • Green D.R.
      The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-KappaB pathways.
      —the authors speculated that the host LTβR/NIK axis could play a role in ectopic kidney organogenesis. Francipane et al tested this hypothesis using two independent strategies: Two days before metanephros transplantation into the LNs, recipient mice were treated with a recombinant LTβR-Fc fusion protein that antagonizes LTβR-mediated effects by engaging soluble and cell surface forms of LTβR ligands, and transplanted kidney rudiments into the omenta of LTβR knockout (Ltβr−/−) mice. Both strategies showed that defective host LTβR signaling is associated with decreased expression of endothelial and angiogenic markers in kidney grafts, indicating that host LTβR signals are important for the vascularization of ectopically grown embryonic kidneys. Surprisingly, the defective host LTβR signaling impaired glomerular vascularization in engrafted kidneys, despite the fact that the main cell source of glomerular endothelium was of graft origin.
      LTβR was not only decisive for vascularization but also for the growth of renal structures. Compared with their control counterparts, metanephroi engrafted into the Ltβr−/− omentum exhibited structural alterations overtime, with complete loss of glomeruli and tubules after six weeks. Interestingly, similar results were obtained when newborn kidneys were transplanted (instead of embryonic ones), indicating that host LTβR signals are important for the survival and growth of the developing tissue, even when the graft is extensively vascularized before transplantation.
      These observations may lead to the assumption that LTβR plays an important role in kidney organogenesis, and consequently transplanted kidneys cannot grow normally in the presence of defective LTβR signaling. However, this was not the case: LTβR was not essential for kidney development during embryogenesis. Ltβr−/− mice exhibited normal kidney structure and vasculature, suggesting that LTβR signaling is essential only for ectopic but not for normal organogenesis. To explain this paradox, Francipane et al assumed that LTβR signaling is important for angiogenesis only and not for vasculogenesis, which is the main mode of vascularization during normal kidney development. However, this assumption can only partially explain the observed phenomena, because the glomerular endothelium derived almost exclusively from the donor and was indeed affected by the defective host LTβR signaling. Still, the lymphoid microenvironment may affect the graft's vasculogenesis indirectly: it can provide the signaling cues to induce host angiogenic responses that are necessary for the survival and growth of the graft early after transplantation, and then the graft's intrinsic vascularization machinery (through secretion vasculogenic/angiogenic molecules and differentiation of endogenous endothelial progenitors) may take over to self-sustain its vascularization. In the absence of functional LTβR signaling, both angiogenesis and vasculogenesis are affected.
      Another interesting aspect of the study that requires further investigation is the contribution of LN stroma to ectopic organogenesis. Francipane et al showed that engrafted glomeruli are embedded in a host LTβR-reactive extracellular matrix (ECM) secreted by LN stromal cells. Although LN stromal cells constitute only 1% to 5% of the LN tissue,
      • Malhotra D.
      • Fletcher A.L.
      • Astarita J.
      • Lukacs-Kornek V.
      • Tayalia P.
      • Gonzalez S.F.
      • Elpek K.G.
      • Chang S.K.
      • Knoblich K.
      • Hemler M.E.
      • Brenner M.B.
      • Carroll M.C.
      • Mooney D.J.
      • Turley S.J.
      Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.
      they have a number of crucial functions, including the secretion of ECM components and chemokines that support hematopoietic cell maturation,
      • Malhotra D.
      • Fletcher A.L.
      • Turley S.J.
      stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity.
      and the regulation of vascular endothelial growth factor levels for endothelial cell proliferation.
      • Chyou S.
      • Ekland E.H.
      • Carpenter A.C.
      • Tzeng T.C.J.
      • Tian S.
      • Michaud M.
      • Madri J.A.
      • Lu T.T.
      Fibroblast-type reticular stromal cells regulate the lymph node vasculature.
      Therefore, identifying which type(s) of LN stromal cells support kidney vasculogenesis/angiogenesis, as well as the stromal cell–derived ECM proteins and chemokines that are involved in these processes, would be very useful for kidney tissue engineering. This information may help improve existing methods for the differentiation of PSCs into glomerular endothelial cells, and design matrices that may enhance kidney organoid vascularization and maturation.
      After observing that the number of glomerular endothelial cells that express NIK (a key regulator of neovascularization)
      • Noort A.R.
      • van Zoest K.P.M.
      • Weijers E.M.
      • Koolwijk P.
      • Maracle C.X.
      • Novack D.V.
      • Siemerink M.J.
      • Schlingemann R.O.
      • Tak P.P.
      • Tas S.W.
      NF-ΚB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis.
      decreased in the absence of functional LTβR, the authors prudently speculated that the LTβR/NIK axis mediated the angiogenetic signals that are required for ectopic kidney organogenesis. However, the transplantation of kidney rudiments into the omenta of Nik−/− mice revealed that NIK is dispensable for ectopic kidney vascular integration and maturation. This finding suggests either the existence of different targets downstream of LTβR that lead to angiogenesis or the activation of compensatory angiogenic pathways in the absence of NIK. Another crucial issue that remains to be addressed is how the defective LTβR signaling is associated with glomerular and tubular loss in late-stage grafts.

      LTβR Signaling Is Important for Glomerular Adaptive Responses to Uninephrectomy

      The above data also raise an interesting question of clinical relevance: does LTβR signaling play similar trophic and/or pro-angiogenetic roles in the adult kidney? To answer this question the authors uninephrectomized Ltβr−/− mice and studied the remnant kidney after 10 days. Although the kidneys from wild-type and Ltβr−/− mice had similar weights before surgery, the remnant kidneys of Ltβr−/− mice were of a lower weight. The contralateral kidneys in wild-type mice exhibited greater glomerular enlargement and contained more erythrocytes than Ltβr−/−-engrafted organs. Differences in adaptive responses appeared to be confined to the glomerulus, as tubular epithelial cells from both groups exhibited a similar signal pattern of the proliferation marker Ki-67. Future studies are needed to assess whether the role of LTβR in compensatory glomerular growth is specific to mass reduction or a general response to impaired renal function, and how LTβR blockers
      • Seleznik G.
      • Seeger H.
      • Bauer J.
      • Fu K.
      • Czerkowicz J.
      • Papandile A.
      • Poreci U.
      • Rabah D.
      • Ranger A.
      • Cohen C.D.
      • Lindenmeyer M.
      • Chen J.
      • Edenhofer I.
      • Anders H.J.
      • Lech M.
      • Wüthrich R.P.
      • Ruddle N.H.
      • Moeller M.J.
      • Kozakowski N.
      • Regele H.
      • Browning J.L.
      • Heikenwalder M.
      • Segerer S.
      The lymphotoxin β receptor is a potential therapeutic target in renal inflammation.
      (used against renal or non-renal inflammations) may affect this response.

      Conclusions

      As novel and challenging as these findings are, they also raise important new questions. Which LTβR-downstream pathway (or pathways) controls vascularization in ectopic kidney organogenesis? What is the precise mechanism of LTβR signaling-mediated glomerular and tubular loss in late-stage grafts?
      Although the remaining questions do not allow for straightforward conclusions regarding the cellular/molecular mechanisms that support kidney organogenesis in lymphoid sites, Francipane et al's studies certainly reveal the important role that the lymphoid stromal microenvironment plays in supporting angiogenesis in ectopically transplanted embryonic kidneys. A better understanding of the properties of the LN microenvironment might have implications for growing functional organs from fetal kidney rudiments or stem-cell–derived organoids. The lack of technologies that can replicate sufficiently the organogenesis niche, prevent the realistic recapitulation of in vivo organ sizes and structures, tissue patterning and vascularization, intercellular communication, and functions.
      • Xinaris C.
      Organoids for replacement therapy: expectations, limitations and reality.
      More in depth knowledge of the cellular/molecular mechanisms that drive ectopic kidney organogenesis will be crucial for developing protocols that will allow for better spatial and temporal control of vascularization, tissue differentiation, and maturation.

      Acknowledgments

      The authors thank Kerstin Mierke for the excellent editing work on the manuscript; and Rubina Novelli for constructive comments, critical reading, and corrections to the text.

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      Linked Article

      • Host Lymphotoxin-β Receptor Signaling Is Crucial for Angiogenesis of Metanephric Tissue Transplanted into Lymphoid Sites
        The American Journal of PathologyVol. 190Issue 1
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          The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-β receptor (LTβR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTβR antagonist treatment or in the omenta of Ltbr knockout (Ltbr−/−) mice, the host LTβR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTβR signaling correlated with decreased expression of endothelial and angiogenic markers in kidney grafts as well as structural alterations.
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