Reviews

The lymphatics in kidney health and disease

Michael D. Donnan 1,2, Yael Kenig-Kozlovsky​ 3 and Susan E. Quaggin1,2 ✉ Abstract | The mammalian vascular system consists of two networks: the blood vascular system and the lymphatic vascular system. Throughout the body, the lymphatic system contributes to homeostatic mechanisms by draining extravasated interstitial fluid and facilitating the trafficking and activation of immune cells. In the kidney, lymphatic vessels exist mainly in the kidney cortex. In the medulla, the ascending vasa recta represent a hybrid lymphatic-like​ vessel that performs lymphatic-​like roles in interstitial fluid reabsorption. Although the lymphatic network is mainly derived from the venous system, evidence supports the existence of lymphatic beds that are of non-venous​ origin. Following their development and maturation, lymphatic vessel density remains relatively stable; however, these vessels undergo dynamic functional changes to meet tissue demands. Additionally, new lymphatic growth, or lymphangiogenesis, can be induced by pathological conditions such as tissue injury, interstitial fluid overload, hyperglycaemia and inflammation. Lymphangiogenesis is also associated with conditions such as polycystic kidney disease, hypertension, ultrafiltration failure and transplant rejection. Although lymphangiogenesis has protective functions in clearing accumulated fluid and immune cells, the kidney lymphatics may also propagate an inflammatory feedback loop, exacerbating inflammation and fibrosis. Greater understanding of lymphatic biology, including the developmental origin and function of the lymphatics and their response to pathogenic stimuli, may aid the development of new therapeutic agents that target the lymphatic system.

In mammals, the circulatory system consists of the blood role of dermal lymphatics in fluid homeostasis4 and the and the lymphatic vascular systems, which perform contribution of cardiac lymphatic remodelling to cardio- complementary roles in maintaining body homeostasis. vascular disease and atherosclerosis5. These studies have In contrast to the continuous arterial to venous circuit of also led to the identification and characterization of lym- the blood vascular system, the lymphatic system consists phatic vessels in tissues previously thought to be devoid of a blind-ended​ network of vessels, which provides uni- of lymphatics. For example, the discovery of meningeal directional, low-​flow, transport from peripheral tissues lymphatics in the central nervous system has redefined towards the central venous system. This network trans- our understanding of how the brain responds to inflam- ports interstitial fluid, immune cells, antigens, lipids mation and degeneration6. Hybrid lymphatic-like​ vessels and associated macromolecules — collectively known have also been discovered in multiple tissues through- 1Feinberg Cardiovascular & as lymph — which is integral to the maintenance of body out the body, including the Schlemm’s canal in the eye7, Renal Research Institute, fluid homeostasis, immune cell trafficking, inflamma- the spiral arteries in the placenta8 and the ascending Northwestern University 1 9 Feinberg School of Medicine, tion and regulation of blood pressure . Despite intensive vasa recta (AVR) within the kidney . In this Review, we Chicago, IL, USA. efforts to extensively detail the blood vascular system discuss the origins and organization of the lymphatic 2Division of Nephrology & in development and disease, the lymphatic system has system and hybrid lymphatic-​like vessels, their role in Hypertension, Northwestern historically been overlooked, in part owing to difficulty kidney function and disease and the potential role of the University Feinberg School of in visualizing the typically small and sparse lymphatic lymphatic system as a therapeutic target. Medicine, Chicago, IL, USA. vessels. However, advances in imaging and genetic 3 Department of Nephrology technologies have accelerated our understanding of the Lymphatic structure and organization & Hypertension, Rambam Medical Center, Haifa, Israel. specialized functions of the lymphatic vascular system. The lymphatic system comprises lymphatic vessels, lym- ✉e-mail:​ quaggin@ These studies have provided insights into unique char- phoid tissues and lymphoid organs that exist through- northwestern.edu acteristics of many organ-​specific lymphatic systems, out the body (Fig. 1). A network of highly branched, https://doi.org/10.1038/ including structural characteristics of the intestinal lacte- blind-​ended lymphatic capillaries spans almost every s41581-021-00438-y​ als that underlie their ability to absorb dietary fats2,3, the vascularized tissue, and enables uptake of surrounding

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Key points lymphatic vasculature are not uniform throughout the body and may differ between species. For example, • Advances in imaging and genetics technologies have furthered our understanding of the collecting lymphatics of the mouse lung are devoid the role of lymphatic vascular systems in both homeostasis and disease. of smooth muscle cells and fluid flow relies on changes • Key advances include the discovery of hybrid lymphatic-like​ vessels in multiple tissues in respiratory pressures13, whereas lymphatic flow in the including in the kidney, where the ascending vasa recta express a combination of lungs of sheep seems to be regulated by intrinsic lym- both blood and lymphatic endothelial markers and perform a lymphatic-like​ role in phatic pumping in addition to passive factors14. These reabsorbing interstitial fluid in the medulla. differences underscore the organ and species-​specific • Kidney lymphangiogenesis is strongly associated with injury, inflammation and the heterogeneity of lymphatics. progression of fibrosis. Peripheral lymphatic vessels direct antigen and • Lymphangiogenesis can perform a protective role in clearing the accumulated fluid immune cell-​containing lymph towards local draining and immune cells associated with inflammation from the interstitial space; however, 15 the kidney lymphatics also function to propagate an inflammatory feedback loop in lymph nodes (DLNs) . Over 200 DLNs exist throughout coordination with the draining lymph nodes, which may exacerbate inflammation and the human body, the development of which is closely fibrosis. In addition, chronic inflammation can result in the disorganized growth of aligned with those of lymphatic vessels. Within the leaky, poorly functioning lymphatic vessels, further contributing to tissue injury. DLNs, cytokines, immune cells and antigens interact • Targeting the lymphatic system is a potential future direction for new therapeutics with native dendritic cells and recruited leukocytes for kidney disease, and several therapies are undergoing investigation in preclinical within highly specialized lymph node compartments, models. allowing selective filtration of these inflammatory cells • Better understanding of the context-​dependent consequences of kidney and molecules, and enabling modulation of the immune lymphangiogenesis, as well as the mechanisms of action and potential off-target​ response16,17. Lymph then continues through these DLNs consequences of targeting the proposed molecular pathways are needed prior to into larger lymphatic ducts, including the thoracic their clinical use. duct, which eventually returns the lymph to the central circulation18.

interstitial fluid and immune cells. Similar to other Lymphatic development and function capillary structures, a single layer of endothelial cells A variety of signalling molecules and pathways coordi- forms the innermost layer of the lymphatic vasculature. nate the development and remodelling of the lymphatic However, in contrast to blood endothelial cells (BECs), vasculature. Several of these pathways are also involved lymphatic endothelial cells (LECs) exhibit unique inter- in development of the blood vasculature; however, spe- cellular junctions that enable enhanced reabsorption cific factors contribute to the determination of the fate of fluid and molecules. In the blind-​ended initial lym- of LECs (Table 1). As described below, these unique fac- phatic capillaries, discontinuous, punctate, button-​like tors can help to distinguish lymphatic endothelium from junctions anchor the sides of overlapping flaps of oak blood vascular endothelium, and can be used to identify leaf-​shaped LECs. Together with the thin, discontinu- potential hybrid structures. ous basement membrane of the lymphatic capillaries and absence of supporting mural cells, these specialized Transcription factors. Prospero-​related homeobox junctions are integral to the high permeability of lym- transcription factor 1 (PROX-1) is considered to be phatic capillaries and allow the uptake of fluid and cells the master regulator of lymphatic fate specification. from the surrounding tissues. Loss of these button-like​ It is expressed by all LECs and is required for their ini- junctions results in impaired capillary permeability and tial differentiation. In mice, PROX-1 is expressed from disruption of fluid uptake2. embryonic day (E) 9.5 by endothelial cells in the ante- The lymphatic capillaries converge towards progres- rior side of the cardinal vein, which give rise to LEC sively larger and fewer collecting lymphatic vessels. The progenitors19. Downregulation of Prox-1 at any stage LECs in collector vessels become more elongated, and during development will induce LECs to reprogramme cell-​to-​cell adhesions transition from the button-​like back to BECs20. In the kidney, PROX-1 is highly junctions of the capillaries to conventional, continu- expressed in classic lymphatic vessels that run alongside ous, zipper-​like junctions that are more restrictive to the arcuate and interlobular arteries in the corticom- interstitial fluid permeability10. Despite the dramat- edullary junction21,22 and at lower levels in the AVR9. ically different morphology of these junction types, In non-​kidney tissues, PROX-1 is also expressed in a both types of LECs express similar adherens junc- variety of extra-​lymphatic cell types, including hepat- tion proteins, including VE-​cadherin, β-​catenin and ocytes, neuroendocrine cells and cardiac muscle cells, p120-​catenin, and tight junction proteins including indicating additional roles for this transcription factor zonula occludens-1 (ZO-1), occludin, claudin-5, junc- beyond LEC specification23,24. tional adhesion molecule-​A (JAM-​A) and endothelial In addition to PROX-1, the forkhead domain-​ cell-​selective adhesion molecule (ESAM)11. Collecting containing transcription factors FOXC1 and FOXC2 are lymphatic vessels are surrounded by a layer of circum- required for proper lymphatic formation. In compar- ferential smooth muscle cells and have a prominent ison with PROX-1, FOXC2 and FOXC1 are required basement membrane that limits their permeability. during later stages of lymphatic development. FOXC1 Peristaltic action of the surrounding smooth muscle regulates cytoskeletal organization during lymphatic cells, in addition to intraluminal lymphatic valves, pro- valve maturation25. FOXC2 regulates the recruitment motes the unidirectional flow of lymphatic drainage12. of mural cells that surround the collecting lymphatics, However, these traditional characteristics of the as well as the formation of lymphatic valves26. Genetic

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a b Button-like junctions Zipper-like junctions

Adherens Oak leaf- Elongated Lymph vessel Thymus junction shaped LECs proteins: LECs • VE-cadherin Lymph • β-catenin nodes • p120-catenin

Spleen Tight junction proteins: • ZO-1 • Occludin • Claudin-5 • JAM-A • ESAM

Appendix Peyer’s patches

c d VEGFC Collecting lymphatic vessel Capillaries • Zipper junctions • Button junctions VEGFR-3+ • Prominent basement • Thin basement membrane membrane • Surrounding pericytes • No surrounding PROX-1+ and SMCs mural cells Cardinal vein

Lymph sac

Unidirectional flow SMC Valve Fig. 1 | Lymphatic structure and development. a | The lymphatic system contains a network of vessels that spans almost every vascularized tissue in the body, often running in parallel to the blood vasculature, and performs a complementary role in maintaining fluid homeostasis. Several lymphoid tissues and organs, including widely distributed lymph nodes, function in a highly coordinated manner to regulate immune cell function. Peyer’s patches are lymphoid follicles in the small intestine, which act similar to lymph nodes in performing immune surveillance and coordinating the immune response within the intestinal mucosa. b | Capillary lymphatics contain overlapping oak leaf-shaped​ endothelial cells connected by discontinuous button-like​ junctions that facilitate the uptake of surrounding interstitial fluid. By contrast, collecting lymphatic endothelial cells (LECs) have continuous zipper-like​ junctions that limit their permeability. Similar adherens junction and tight junction proteins border the LECs of both capillary and collecting vessels. c | The lymphatic capillaries have a sparse, discontinuous basement membrane and lack mural cells, aiding their ability to take up interstitial fluid, molecules and immune cells. They drain into a hierarchal system of collecting lymphatics, which have surrounding smooth muscle cells (SMCs) and contain luminal valves that enable the unidirectional flow of lymph. d | During development, prospero-​related homeobox transcription factor 1 (PROX-1+) cells bud off the cardinal vein; vascular endothelial growth factor C (VEGF-​C)–vascular endothelial growth factor receptor 3 (VEGFR-3) signalling drives their expansion, resulting in formation of the initial lymphatic sac. Many lymphatic beds originate from venous or other lymphatic vessels.

mutations in the genes that encode these proteins also known as FLT4)33 and stimulate the expression of result in syndromic diseases as well as lymphoedema neuropilin 2 (ref.34). in humans27,28. The expression of PROX-1 is activated by two Vascular signalling pathways. In addition to regulat- key transcription factors: SOX18 and COUP-​TFII. ing lymphatic fate, the abovementioned transcription SOX18 mutants fail to induce PROX-1 expression, factors also regulate components of vascular signalling leading to arrested lymphatic development in mice pathways that contribute to lymphatic development. and hypotrichosis–lymphoedema–telangiectasia (HLT) The most potent stimulus for lymphangiogenesis in in humans29. Expression of SOX18 is needed only development and disease is VEGFR-3 signalling35.

Hypotrichosis–lymphoedema for the initiation of LEC fate, in contrast to PROX-1, This tyrosine kinase receptor is a downstream target of 30 36 –telangiectasia which is necessary for maintaining lymphatic fate . PROX-1 (ref. ), but also regulates PROX-1 expression (HLT). A rare genetic syndrome COUP-​TFII belongs to the nuclear receptor superfam- during development via a feedback loop37. VEGFR-3 characterized by lymphoedema ily. It is expressed on venous endothelium at E8.5 in mice expression is first detected around E8.5 in mice, local- in the lower limbs and eyelids, and is important for endothelial cells to acquire a venous ized to early vascular progenitors, venous endothelium cutaneous telangiectasia 31 and dilatations of superficial fate . In LECs, COUP-​TFII interacts with PROX-1 to and early LECs. At later stages, its expression becomes 32 38 vasculature, and defects in hair maintain LEC fate , regulate the expression of vascu- more restricted to LECs . However, VEGFR-3 remains follicle development. lar endothelial growth factor receptor 3 (VEGFR-3; expressed in several specialized BEC types, including

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Table 1 | Lymphatic markers and signalling pathways Protein Localization Function Extra-lymphatic​ Animal KO phenotype Human Refs expression (germline null) phenotype PROX-1 Nucleus TF; specifying and maintaining Endothelial cells of the Fail to develop Mutations 9,23,24,98,204 LEC identity AVR, tubule epithelial lymphatic vasculature found in several cells of the loop of malignancies Henle, hepatocytes, neuroendocrine cells, cardiac muscle cells LYVE-1 Membrane Receptor for hyaluronan; Subset of glomerular LYVE-1 is non-​essential None reported 21,55 and cytosol allows DC entry into lymphatic endothelial cells, blood for lymphatic vessels vessels within lung, liver vasculature sinusoids, HEVs of lymph development nodes, some macrophages and dendritic cells VEGFR-3 Membrane Receptor for VEGF-C​ and Fenestrated blood Disrupted lymphatic Hereditary 9,37,41 and cytosol VEGF-D;​ regulates PROX-1 endothelium, including and blood vascular lymphoedema (endocytosed expression and is required the peritubular capillaries, development type 1 A (OMIM upon ligand for angiogenesis and the glomerular capillary 153100; AD) binding) lymphangiogenesis loops and AVR PDPN Membrane O-​linked sialoglycoprotein, Podocytes, lung epithelial Disrupted separation None reported 72,99 and cytosol LEC specification and cells, fibroblastic reticular of blood and lymphatic differentiation, initiation of cells in lymph nodes vasculature systems platelet aggregation with blood-​filled lymphatics ANGPT2 Secreted Required for sprouting Expressed in blood and Abnormal lymphatic Primary 51,205 ligand, development of lymphatic LECs with increased patterning and limited lymphoedema stored in vessels. Acts as a context- ​ expression during postnatal angiogenesis. Weibel– dependent agonist or endothelial cell activation Can be rescued by Palade antagonist of Angpt-1–Tie2 ANGPT1 bodies in blood vessels TIE1 Membrane Required for lymphatic Expressed throughout Abnormal lymphatic None reported 54 development. Regulates blood and lymphatic patterning and blood context-​dependent TIE2–TEK endothelium vascular development. signalling Impaired glomerular capillary integrity TIE2/TEK Membrane Receptor for ANGPT2 and BECs, haematopoietic Severe defects Venous 53 ANGPT1 cells in cardiovascular malformations development (OMIM 600195; AD), primary congenital glaucoma (OMIM 617272, AD) NRP2 Membrane Receptor for VEGF-C.​ BECs in some venules Reduction in small None reported 63,64 Augments VEGF-C​ binding to lymphatic vessels and VEGF receptors; important for lymphatic capillaries lymphatic vessel sprouting FOXC1 Nucleus TF required for maturation of Podocytes Aberrant LEC Axenfeld–Rieger 27,100 collective lymphatics proliferation with syndrome, abnormally enlarged type 3 (OMIM lymphatic vessels 602482; AD) FOXC2 Nucleus TF required for maturation of Podocytes Failure of lymphatic Lymphoedema– 26,100 collective lymphatics, mural valve formation distichiasis cell recruitment and lymphatic syndrome (OMIM valve formation 153400) CCL21/SLC Cytosol, Chemokine involved in HEVs of lymph nodes, Delayed T cell None reported 206 secreted recruiting CCR7+ T and DCs Peyer’s patches, thymus, localization and from Golgi spleen and mucosal tissue extravasation into apparatus peripheral tissues and lymphoid organs D6 Membrane Decoy chemokine receptor, Haematopoietic cells and Increased adhesion None reported 207 chemokine scavenging certain leukocytes of immature DCs to receptor, controls inflammatory LECs, which displaces leukocyte interactions with mature DCs LECs, regulates the ability of LECs to discriminate between mature and immature DCs

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Table 1 (cont.) | Lymphatic markers and signalling pathways Protein Localization Function Extra-lymphatic​ Animal KO phenotype Human Refs expression (germline null) phenotype SOX18 Nucleus TF; induces PROX-1 in the Cardiac ventricles, some Blockade of LEC Hypotrichosis– 29,30 cardinal vein BECs, and vascular differentiation from the lymphoedema– smooth muscle cells cardinal vein, resulting telangiectasia in fetal lethality by E14.5 (OMIM 697823; AR) COUP-​TFII Nucleus TF; co-regulator​ of PROX-1, Developing blood vessels Fetal demise prior to None reported 31–34 regulates VEGFR-3 expression E10.5, with conditional and stimulates NRP2 KO; inactivation results in failure to form lymphatic vessels SVEP1 Secreted; Cell signalling through Primary non-​endothelial Abnormal lymphatic Primary 66,208 extracellular interaction with integrins expression by adjacent remodelling congenital matrix (α9 integrin) mesenchymal cells or glaucoma protein pericytes ITGA9 Membrane An integrin that participates Airway epithelium, Deficient lymphatic Case report of 209 in cell adhesion with various keratinocytes, muscle development resulting severe congenital ligands, including VEGF cells, hepatocytes, in mortality at 12 days of chylothorax; neutrophils, osteoclasts, age due to chylothorax candidate gene oocytes for primary lymphoedema owing to valve formation failure AD, autosomal dominant; ANGPT, angiopoietin; AVR, ascending vasa recta; BECs, blood endothelial cells; DC, dendritic cell; HEVs, high endothelial venules; KO, knockout; LEC, lymphatic endothelial cell; LYVE-1, lymphatic vessel endothelial hyaluronic acid receptor 1; PROX-1, prospero-related​ homeobox transcription factor 1; TF, transcription factor; VEGF, vascular endothelial growth factor; VEGFR-3, vascular endothelial growth factor receptor 3.

the fenestrated glomerular, cortical peritubular and Membrane-​associated molecules. The lymphatic vessel AVR endothelial cells of the kidney9,39,40. VEGFR-3 endothelial hyaluronic acid receptor 1 (LYVE-1) is an signalling is primarily activated by its cognate secreted integral membrane glycoprotein that is expressed on ligand vascular endothelial growth factor C (VEGF-C)​ 41 the walls of lymphatic vessels55. During mouse embry- and to a lesser extent VEGF-​D42. However, VEGFR-3 ogenesis it is detected by E9.5 in the cardinal vein. also demonstrates ligand-​independent and passive It is also expressed during development and adulthood signalling activity43,44. Expression of VEGF-​C, which is in several non-​lymphatic cells, including the mouse produced in the kidney by circulating macrophages45, yolk sac, intersomitic blood vessels, liver sinusoids podocytes46 and tubule epithelial cells45, varies depend- and macrophages56,57. Unexpectedly, deletion of Lyve-1 ing on developmental stage and environmental context. does not cause any overt defects in lymphatic struc- VEGF-​C signalling is not exclusive to the lymphatic ture, suggesting that it is not essential for lymphatic endothelium: proteolytically processed VEGF-​C also development58. However, LYVE-1 might function to interacts with VEGFR-2 and VEGFR-2/3 heterodimers facilitate adhesion and transmigration of dendritic cells expressed by BECs, including glomerular endothelial across LECs59. cells40,47. In addition, VEGF-A​ — which is traditionally Podoplanin (encoded by PDPN) is a membrane gly- associated with angiogenesis and BECs — signals via coprotein that is highly expressed by LECs but is absent VEGFR-2 localized to the surface of LECs, and has an from other endothelial cell populations, making it a increasingly recognized role in LEC proliferation and highly useful lymphatic marker. Its expression in mice migration48,49. begins at E10.5 when the LECs bud off from the car- A second tyrosine kinase vascular signalling path- dinal vein60,61. Podoplanin activates C-​type lectin-​like way, involving angiopoietin–TIE signalling, is also receptor 2 (CLEC-2) expressed by platelets, resulting in required for lymphatic development. Angiopoietin-2 platelet aggregation — a process that is important for (ANGPT2) is the most potent lymphatic-​associated the separation of blood and lymphatic vessels62. Deletion growth factor in this pathway. Mice lacking Angpt2 of Pdpn causes abnormal lymphatic patterning, which survive embryonic development but exhibit lymphatic results in respiratory failure secondary to impaired vascular deficits, including chylous ascites, a lack of pulmonary development, ultimately leading to early lymphatic valves, and reduced lymphangiogenesis50,51. postnatal demise60. The cognate angiopoietin receptor TIE2 (also known Neuropilin 2 (encoded by NRP2) is a transmem- as TEK) is required for the appropriate development of brane protein that is expressed by lymphatic and venous some lymphatic hybrid vessels (for example, Schlemm’s endothelial cells that can participate in inward as well as canal in the eye and AVR in the kidney) as well as outward signalling events63. Mice with homozygous Nrp2 Chylous ascites for cardiovascular development9,52,53. Importantly, a mutations demonstrate normal development of collect- The accumulation of lipid-rich​ lymph in the peritoneal cavity related orphan receptor, TIE1, appears to be the dom- ing lymphatic vessels but a significantly reduced growth 64 as a result of lymphatic vessel inant TIE receptor required for lymphatic vascular of small lymphatic vessels and capillaries . Neuropilin dysfunction. development54. 2 acts as a co-​receptor for VEGF-​C and VEGF-​D,

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augmenting its interaction with VEGFR-3 and promo­ the possibility that considerable heterogeneity may exist ting receptor phosphorlyation65. In this role, neuropilin between and within organs, including the kidney. 2 selectively modulates lymphatic endothelial tip cell extension and promotes LEC sprouting at the leading Kidney lymphatics ends of lymphatic vessel sprouts63. Organization and development. The anatomy of the kid- ney lymphatic system was first described several decades Extracellular matrix proteins. SVEP1 (Sushi domain, von ago by analysing the drainage of dye injected directly Willebrand factor, EGF containing, Pentraxin domain into the kidney interstitum80–84 and by measuring the containing-1) is a huge extracellular matrix (ECM) pro- lymphatic uptake of labelled albumin injected into tein that is expressed in a variety of tissues surrounding the kidney parenchyma85. These imaging techniques lymphatic and lymphatic-​like vessels, including the tra- provided an initial, albeit low-resolution,​ snapshot of the becular meshwork of the Schlemm’s canal. It binds both lymphatic organization of the kidney. We now know that ANGPT1 and ANGPT2 (ref.66) and is also the high affin- the kidney cortex is drained by two lymphatic systems ity ECM ligand for α9 integrin (encoded by ITGA9)67, (Fig. 2). The first system begins as blind-​ended cortical which is expressed by a subset of LECs and hybrid vessels. intralobular lymphatic capillaries, which run adjacent Mice with deletion of Svep1 or Itga9 have lymphatic vas- to tubules, arterioles, and in close proximity to glomer- cular defects, including valve defects and chylous ascites uli. These capillaries drain back through a hierarchical similar to ANGPT2-knockout​ mice51,68,69, suggesting that network of interlobular lymphatic capillaries, interlobar the interactions of SVEP1 with angiopoietins and α9 and arcuate collector lymphatics, before draining into integrin are physiologically important. the hilar lymphatics. From there, lymph can drain into the local DLNs and continue its transit towards the Developmental origin of systemic lymphatics. In thoracic duct before its eventual return to the systemic humans, the lymphatic vascular system emerges in the circulation21,22,86. Within the kidney, the lymphatic ves- sixth to seventh week of embryonic development70. sels run in close proximity to the arteries of the same The notion that the lymphatic system originates from the name80,86. In addition, a second and more superficial venous system was first proposed in 1902 (ref.71). Over lymphatic system exists on the surface of the kidney 100 years later, it is well-established​ that a cluster of cells capsule, which has been shown to drain portions of the that emerge from the cardinal vein develop into the cortex87. Apart from the apparent absence of intralobu- initial lymphatic sac72 (Fig. 1). In mice, LEC progenitors lar vessels in the rabbit88,89, and the capsular lymphatic within the cardinal vein begin the process of budding network in sheep90 and mice91, the distribution of the and fate specification — processes that are dependent on cortical lymphatic system seems to be generally uni- PROX-1 — by E9.5 (refs4,73,74). In addition to the cardi- form among species21,22,80,88,92. This general structure is nal vein, LECs also emerge from intersomitic veins and consistent with the network of lymphatic vessels in the superficial venous plexus75. The sprouting, proliferation human kidney93,94. However, it should be emphasized and migration of LECs from these structures results that despite general acceptance of uniformity, exten- in the formation of new lymphatic vessels in a process sive characterization of kidney lymphatics has not been called developmental lymphangiogenesis. Similar to performed in any species. Moreover, although advances angiogenesis, migrating tip cells on lymphatic sprouts have been made in methods to interrogate and visualize sample the surrounding pericellular environment for the lymphatic vasculature, they remain limited in detect- both growth factor (primarily VEGF-​C) and mechani- ing the small capillary vessels, although they do continue cal stimuli76,77. In response to these stimuli, LECs within to serve a purpose in the clinical setting (Box 1). the stalk, residing behind the tip cell, proliferate, elon- In the past decade, use of immunohistochemical gating the lymphatic branch78. Subsequent maturation staining with a combination of LEC markers has pro- and stabilization of these new vessels is then dependent vided insights into lymphatic vessel development and on ANGPT2 signalling51. Iterations of this process result structure in the kidney (Fig. 2). LEC staining of rat in the development of an expansive lymphatic vascular and mouse kidneys over progressive stages of kidney network, which primarily traces its origin back to the development revealed the formation of a rich lymphatic venous endothelium. plexus in the kidney hilum around E13 in mice21 and Of note, however, use of sophisticated lineage-​tracing around E17–20 in rats22, well after the development of techniques also provides support for the existence of other lymphatic plexuses outside of the kidney. The kid- some lymphatic beds that are of non-​venous, or dual, ney hilar plexus is continuous with an existing lymphatic origin. These studies have shown that isolated clusters network in the renal pelvis, which also supplies the adre- of precursor LECs contribute to the development of nal gland, ureter and gonad95. In the past few years, tis- tissue lymphatic networks in a process termed lymph- sue clearing, combined with high-resolution​ microscopy vasculogenesis. These precursor LECs may derive from and three-dimensional​ imaging of whole-mount​ kidneys several non-​venous lineages. For example, in the mes- stained with a combination of LEC molecular markers, entery and heart, some LECs originate from cKit+ hemo- has provided more detailed insights into the develop- genic endothelium and Pax3+ intermediate mesoderm, ment of the kidney lymphatic system91. Specifically, this respectively73,79. Non-​venous progenitors also contrib- work showed that the early mouse embryonic kidney is ute to the development of dermal lymphatic vessels4. devoid of lymphatic structures. In the mouse, a cellular These new findings have fuelled debate on the origin of plexus of LECs emerges near the kidney hilum around organ- and tissue-specific​ lymphatic cells, and highlight E14.5, subsequent to the initial expansion of the kidney

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blood vasculature. From E15.5 through to E18.5, the development, the mature lymphatic network in the kid- hilar lymphatics rapidly expand into lumenized vessels ney may involve dual contributions from venous and alongside the arterioles. By E18.5, an expanded network non-​venous origins. Clusters of PROX-1+, LYVE-1+, of lymphatic vessels is present in the cortex and hilum of VEGFR-3+, podoplanin+ cells that are structurally dis- the kidney, but is absent from the medulla and kid- tinct from LECs of the maturing hilar lymphatic plexus ney capsule. As in other tissues, including the heart96, have also been identified in the E14.5–E16.5 mouse mesentery73,74 and skin4, where lymphvasculogenesis kidney cortex91. These clusters demonstrated minimal from a non-​venous origin contributes to lymphatic expression of traditional BEC markers such as PECAM1

a Capsule Capsular lymphatics Arteries and Capillaries Cortical lymphatic capillaries arterioles Glomerular capillaries Arcuate Cortex Peritubular capillaries artery Ascending vasa recta Interlobar artery Descending vasa recta Large artery

Lymphatics Veins and Arcuate Capsular lymphatics venules artery Subcapsular lymphatics Large vein Cortical lymphatic Interlobar vein capillaries (interlobular Arcuate lymphatics) Arcuate vein vein Arcuate lymphatics Interlobar lymphatics Hilar lymphatics Interlobar lymphatic vessel

Ascending vasa recta Capsule Cortex Medulla Glomerulus Ureter Medulla

b Adrenal gland Kidney lymphatic development

E 9.5–10.5 Isolated Initial LEC progenitors bud from the LEC clusters caridnal vein. The early developing kidney is devoid of lymphatics.

E 13.5–14.5 An initial lymphatic plexus extends from the renal pelvis into the kidney hilum. Initial distinct clusters of isolated LECs are found in the developing kidney. Kidney hilar plexus E 15.5–18.5 Rapid expansion of the kidney hilar plexus into lumenized lymphatic vessels. Dissapearance of isolated clusters of LECs.

Fig. 2 | Structure and development of kidney lymphatics. a | The vasa recta (AVR), which represent hybrid, lymphatic-​like structures mature kidney contains a sparse network of blind-​ended lymphatic characterized by the expression of a combination of blood and lymphatic capillaries in the kidney cortex, often localized adjacent to cortical molecular components, contribute to the reabsorption of the surrounding tubules, arterioles and glomeruli. These capillaries drain out of the kidney interstitial fluid. b | During development of the mouse kidney, lymphatic through a hierarchical network of collecting lymphatics, which run in structures are first seen around embryonic day (E) 14. These structures parallel with the larger interlobar and arcuate arteries and veins, and originate from an existing plexus in the renal pelvis, which spans between eventually drain from the hilar lymphatics. A more superficial lymphatic the adrenal gland and the ureter. Small, isolated clusters of lymphatic system exists in the kidney capsule. However, no lymphatic capillaries endothelial cells (LECs) are also observed that are potentially of have been observed in the kidney medulla. Instead, the ascending non-venous​ origin.

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Box 1 | Imaging of the kidney lymphatics fenestrated and are more numerous in comparison. These characteristics allow the AVR to efficiently trans- Accurate imaging of the lymphatic system is important for understanding the structure port high volumes of interstitial fluid while maintaining and function of the vessels in development and disease, and for aiding the diagnosis and a low rate of flow, thereby promoting solute exchange treatment of conditions such as lymphoedema, malignant metastasis, lymph leakage and preventing vascular washout of the medullary and lymphocele formation. Unlike blood vessels, which are easy to visualize and 97 cannulate for angiography techniques, the lymphatic vessels carry mostly clear fluid, osmotic gradient through countercurrent exchange . and cannulation is very challenging. Hence, most imaging techniques rely on the ability Evidence for this role is supported by findings in mice, of the lymphatics to take up labelled tracers injected into the tissue. Injection of India in which loss or simplification of the AVR leads to a mas- ink and labelled albumin into the kidney medulla has been used to characterize the sive accumulation of fluid in the medullary interstitial anatomy and function of kidney lymphatics in animal models80,90. In the clinic, lymphatic space and disrupts urinary concentrating ability9. imaging of the kidney is used mainly for diagnosis and treatment of chyluria — a rare The highly fenestrated, large-​diameter AVR share condition characterized by anomalous communication between lymphatic vessels and many features with traditional lymphatic capillaries, the urinary system. Diagnostic lymphangiography with sequential CT or unenhanced including a discontinuous basement membrane and an MR lymphography can be used to diagnose lympho-urinary​ fistulas, characterize the absence of surrounding mural cells. The origin of AVR is distribution of collateral lymphatic vessels, and identify hydrodynamic pressure yet to be experimentally defined but seem distinct from abnormalities to guide treatment in these patients211,212. Lymphatic leakage and lymphocele formation are frequent complications after kidney transplantation. that of the DVR, which are derived from the efferent Lymphangiography can be used to diagnose the leakage site, followed by an arterioles. At a molecular level, the AVR express both embolization procedure to seal the leak213. Interestingly, lymphangiography alone is lymphatic and blood endothelial proteins and might effective as a therapeutic intervention in this setting as the injected material induces therefore be best described as hybrid vessels (Fig. 3). a local inflammatory reaction that can seal the leak214. Finally, because lymphatic In addition to common BEC markers, such as endomu- drainage of renal cell carcinoma is unpredictable, intra-tumoural​ injection of cin (encoded by Emcn), CD31, CD34 and TIE2, the AVR 99mTc-​nanocolloid with planar sequential lymphoscintigraphy and single photon also express low levels of PROX-1 and VEGFR-3 (ref.9), emission CT–CT imaging can be used to identify the sentinel nodes and plan the which are expressed in all LECs. However, in contrast to 215 surgery . High-resolution,​ live imaging techniques to visualize intra-​kidney lymphatics classic LECs, the AVR do not express LYVE-1 or podo- are not yet available. However, tissue clearing and 3D reconstructive methods are planin. The expression of PROX-1, which is known to providing new insights and enabling the construction of visual maps of developing (ref.37) lymphatics in rodent and human kidneys91. positively regulate the expression of VEGFR-3 , is consistent with lymphatic-​like functions of the AVR and may contribute to the larger size of these vessels and endomucin, suggesting a non-venous​ origin. Similar compared with the DVR43. However, whether the AVR PROX-1+ and podoplanin+ endothelial cell clusters were are dependent on VEGF-​C–VEGFR-3 signalling for also found in human embryonic kidneys at a similar development is not yet known. The absence of podo- developmental stage. The precise lineage of these LEC planin, which, as described earlier, activates CLEC-2 clusters remains undetermined. to induce platelet aggregation, also seems appropriate given the direct connectivity of the AVR to the blood The ascending vasa recta. Although cortical lymphatic circulation62. Of note, the AVR share several of these spe- vessels have been observed in all species studied, kid- cialized hybrid blood vessel and lymphatic-like​ features ney medullary lymphatics have not been identified in with other hybrid vascular structures (Fig. 3; Box 2). dogs80,84, sheep90, mice21,91 or rats22,85. Similarly, most published studies of healthy human kidneys have not Extra-​lymphatic expression of lymphatic markers. identified classic lymphatic systems in the medulla94. Although the above-described​ markers are useful for the By contrast, radiographic studies have demonstrated identification of LECs, many of them are also expressed in the existence of medullary lymphatics in pigs82. Humans non-​lymphatic cells, including multiple cell types within filter ~180 l of plasma a day but only produce ~2 l of the kidney. For example, a subset of glomerular endothe- urine, with the majority of fluid requiring drainage lial cells, macrophages and dendritic cells express LYVE-1 back into the systemic circulation. Given this contin- (ref.21). As mentioned earlier, VEGFR-3 is expressed uous accumulation of interstitial fluid in the medulla, within the fenestrated endothelial cells of the glomeru- it is surprising that no lymphatic vessels exist in this lus, the cortical peritubular capillaries and the medullary space to manage excess fluid similar to their functions AVR 9. In addition to its expression by LECs and AVR, in other tissues. Instead, the medulla contains a complex PROX-1 is also expressed by NKCC2-positive​ tubule epi- and unique bundling of capillaries and tubules. Inside thelial cells within the thick ascending limb of the loop of these bundles, the AVR, together with the descending Henle98. Its tubular expression is upregulated by increas- vasa recta (DVR), exist in close proximity to the loop of ing osmolality and is critical for the differentiation and Henle. The vasa recta function not only to bring oxygen maturation of the thick and thin ascending limbs of the and nutrients to the neighbouring tubules but also to loop of Henle98. Podoplanin and FOXC2 are expressed remove accumulating fluid from the medullary inter- by podocytes; deletion of the encoding genes — Pdpn or stitium. In particular, the AVR likely act as a substitute Foxc2 — results in severe podocyte defects99,100, although for traditional lymphatic vessels and mitigate the contin- the connection, if any, between their function in LECs and uous accumulation of interstitial fluid. Indeed, labelled podocytes is unknown. Currently, the functional role of albumin injected into the medullary interstitium is these genes in non-​lymphatic cells is not well studied — taken up by the AVR85. In comparison with the DVR, and as many molecular markers of LECs are non-specific,​ which are smaller diameter vessels lined by a continuous a combination of markers is often needed to identify and endothelium, the AVR have a large diameter, are highly distinguish LECs correctly within the kidney.

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Lymphatics and fluid homeostasis between LECs, thereby enhancing fluid uptake104. As dis- BECs of the capillaries are semipermeable under physi- cussed below, lymphangiogenesis can also be stimulated ological conditions, enabling continuous extravasation postnatally to enhance fluid reabsorption. of plasma into the extracellular interstitial compartment. Interstitial fluid within the kidney can exit through Although a small amount of this fluid is reabsorbed by the venous or lymphatic systems. In the kidney cortex, surrounding venules, the lymphatic system functions lymphatic vessels can reabsorb the interstitial fluid, cells to reabsorb the majority of extravasated fluid and and macromolecules that comprise the renal lymph macromolecules101. In humans, the lymphatic system (Box 3). Indeed, the majority of renal lymph seems to transports approximately 12 l of fluid per day back to the originate from the kidney cortex; lymphatic drainage systemic circulation102. At any one time, the lymphatic from the kidney medulla has never been convincingly system contains approximately 1–2 l of fluid. Fittingly, demonstrated101. Rather, and as discussed above, in most the most striking manifestation of lymphatic dysfunc- species the medulla does not contain classic lymphatic tion in humans is lymphoedema — a condition charac- vessels but instead contains the AVR — hybrid-​like terized by an overwhelming accumulation of interstitial structures that function to mitigate the accumulation of fluid. interstitial fluid in this region105. Extra-​renal lymphatic Anchoring filaments that connect lymphatic cap- collecting vessels are sensitive to both biomechanical illaries to the surrounding tissue extracellular matrix changes in transmural pressure, as well as vasoactive fac-

allow the lymphatic system to adapt and increase fluid tors such as PGE2 and nitrous oxide, which enable them uptake in the presence of increased volumes of intersti- to contract and pump in response to changing demands tial fluid103. As interstitial fluid builds, these filaments for kidney lymphatic drainage106. The consequence of are stretched and pull on capillary LECs to increase the disrupting kidney lymphatic drainage can be observed capillary diameter and further open the button junctions in the setting of kidney transplantation, in which the

Schlemm's canal Conjunctiva Cornea Anterior chamber Iris Aqueous vein (containing aqueous humour) Hybrid ECs Characteristics Schlemm’s + – canal • VEGFR-3 • LYVE-1 • Large diameter • PROX-1 • PDPN • Giant vacuoles Lens Sclera • CD34 • Discontinuous • PECAM1 basement membrane • TIE2 • No supporting • EMCN mural cells • PLVAP • Venous origin

Ascending vasa recta Remodelled placental spiral artery

Hybrid ECs Maternal artery Spiral artery + – Maternal • VEGFR-3 • VEGFR-2 vein • PROX-1 • LYVE-1 Trophoblast • CD34 • PDPN • PECAM1 • TIE2 • EMCN • PLVAP Placenta Characteristics Placenta Fetal circulation • Large diameter • Fenestrated Characteristics Ascending • Discontinuous Hybrid ECs • Large diameter vasa recta basement + – • Non-fenestrated membrane • VEGFR-3 • EMCN • Loss of surrounding • No supporting • PROX-1 • PDPN smooth muscle cells mural cells • LYVE-1 during remodelling • Unknown origin • PECAM1 • Arterial origin

Fig. 3 | Hybrid lymphatic structures. Several hybrid, lymphatic-​like vessels osmotic gradient. The spiral arteries of the placenta undergo active have been found in tissues otherwise devoid of lymphatic vessels. remodelling and take on lymphatic-​like characteristics during A combination of lymphatic and blood endothelial cell characteristics development. Although many differences exist in the morphology and enables these vessels to perform specialized tissue-​specific functions. For function of these vessels, they share some similarities, including a large example, the Schlemm’s canal in the eye mediates drainage of aqueous diameter that enables low resistance, high capacity flow. LYVE-1, lymphatic humour directly into a venous system to regulate intraocular pressure. vessel endothelial hyaluronic acid receptor 1; PROX-1, prospero-​related The ascending vasa recta (AVR) within the kidney medulla regulate the homeobox transcription factor 1; VEGFR, vascular endothelial growth reabsorption of medullary interstitial fluid and preserve the medullary factor receptor.

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Box 2 | Hybrid vasculatures outside the kidney lymphatic vessels then transport the activated lympho- cytes back to the blood circulation. This system can In addition to the ascending vasa recta (AVR), several other hybrid endothelial structures function to promote cross-​tolerance against innocuous have been identified that demonstrate functional and molecular characteristics of both circulating antigens. K14-​Vegfr-3-​Ig transgenic mice blood and lymphatic endothelial cells216 (Fig. 3). Together, these structures highlight the that lack dermal lymphatic vessels demonstrate greatly heterogeneity and plasticity of endothelial cells and represent important structures for further investigation to determine their functional roles. impaired immune responses to exogenous antigen and often develop signs of autoimmunity115. Schlemm’s canal Of note, this role in cross-​tolerance is also observed Schlemm’s canal (SC) is a lymphatic-like​ vessel that is found in the anterior chamber of in the kidney lymphatic system. Filtered antigens that the eye. SC functions to drain aqueous humour fluid9,217 and is necessary to maintain intraocular pressure. The endothelial cells of SC exhibit giant vacuoles, have are smaller than albumin and are reabsorbed into the discontinuous basement membranes and lack surrounding mural cells. They also tubular compartment are then collected by the renal similarly express both blood endothelial cell (BEC) markers including EMCN, CD34 and lymph. Resident dendritic cells within DLNs then cap- TIE2 and the lymphatic endothelial cell (LEC) markers vascular endothelial growth ture these kidney-derived​ antigens and present them to factor receptor 3 (VEGFR-3) and prospero-related​ homeobox transcription factor 1 CD8+ T cells, promoting cross-​tolerance116. The role of (PROX-1). SC is dependent upon both Angiopoietin–Tie2 and vascular endothelial the lymphatics in the development of adaptive immunity growth factor C (VEGF-​C)–VEGFR-3 signalling for development7,52. implicates this system in inflammatory kidney diseases Placental spiral artery and transplant rejection, as discussed later. The placental spiral arteries (SAs) are responsible for the transport of maternal blood to the fetal side of the placenta. Prior to fetal development, these vessels exhibit The lymphatic system in disease traditional arterial features, including a continuous basement membrane and coverage In line with the essential role of the lymphatic system in by smooth muscle cells. However, during fetal growth, the SA undergoes active maintaining immune surveillance and interstitial remodelling to support the increasing blood flow requirements of the fetus. In response fluid drainage, loss or dysregulation of these vessels can to VEGF-​C–VEGFR-3 signalling the SA remodels into a more lymphatic-like​ vessel, contribute to multiple pathological conditions. For exam- widening in diameter and loss of its surrounding smooth muscle cells8. These lymphatic- ​like characteristics permit low-resistance,​ high-capacity​ blood flow to the placenta. ple, lymphatics are required for the resolution of inflam- The endothelial cells of remodelled SAs have increased expression of the traditional mation after myocardial ischaemia and infarct, and lymphatic markers PROX-1, lymphatic vessel endothelial hyaluronic acid receptor 1 absence of a functioning lymphatic system in mice results (LYVE-1) and VEGFR-3. However, in contrast to the SC and AVR, the SA is not fenestrated in exacerbation of inflammation and deterioration of and does not function to resorb or drain fluid. cardiac function117. In healthy individuals, the network of lymphatic vessels throughout the body remains rela­ tively static after initial development and maturation. kidney lymphatics are ligated and often clipped dur- However, the lymphatic system can be stimulated to grow ing the surgical process. During transplantation, kid- and remodel postnatally. This new lymphatic growth, ney allograft size can increase as a result of interstitial or neo-​lymphangiogenesis, is frequently observed edema, and can be resolved by mechanically draining the at sites of tissue injury, interstitial fluid overload118, renal lymph107. Following transplantation, kidney lymph hyperglycaemia119 and inflammation, and is found in can leak from the allograft, resulting in lymphocele, in many kidney diseases, correlating with the severity of 0.6–33.9% of transplants108. However, these compli- tubulointerstitial fibrosis120. The extent to which this pro- cations are typically temporary and lymph flow is cess is protective rather than maladaptive in these settings restored through new lymphatic connections after remains unclear and may be context dependent121–124. transplantation109. Newly formed lymphatic vessels maintain tissue health by clearing injury-​related tissue oedema and inflam- Lymphatics and immune regulation matory infiltrates. However, disorganized expansion of In addition to draining interstitial fluid, the lymphatic the lymphatic system can hinder immune cell clearance, system is also responsible for trafficking immune cells, contributing to the development of chronic inflammation soluble antigens and cytokines from peripheral tissues to and eventual fibrosis125 (Table 2). DLNs, before returning them to the blood circulation110 (Fig. 4). LECs express several cytokines and adhesion Acute injury and inflammation. Acute kidney injury molecules, including the chemokine CCL21, which is associated with the development of both intrarenal promotes entry of antigen-​presenting cells (APCs) and and systemic inflammation126. In the aftermath of tissue leukocytes into lymphatic vessels111. In addition, LECs injury, capillary vascular permeability increases, result- can adapt to their microenvironment to directly suppress ing in the migration of fluid and cellular infiltrates into the local immune response. For example, in response the kidney interstitium. Inflammatory cytokines, includ- to IFNγ, LECs can express the checkpoint protein ing IFNγ, TNF and TGFβ stimulate macrophages and PDL1, which limits further local accumulation of CD8+ proximal tubule epithelial cells to secrete nitric oxide, T cells112. Within DLNs, LECs can capture and ‘archive’ pro-​inflammatory cytokines (including IL-1, IL-6, local antigens, storing them for later presentation to IL-12, IL-23 and TNF), connective tissue growth factor recruited circulating APCs113. Fibroblastic reticular (CTGF), VEGF-C​ and VEGF-D​ 124,127–129 (Fig. 4). VEGF-C,​ cells within DLNs also secrete chemokines (for exam- and to a lesser extent VEGF-​D, subsequently promote ple, CCL19, CCL21 and CXCL12), which recruit naive expansion and remodelling of the lymphatic capillary net- Lymphocele A post-surgical​ complication in T cells via specialized vessels known as high endothelial work through activation of VEGFR-3 on existing LECs, 114 which lymphatic fluid collects venules (HEVs) . Inside the DLNs, APCs prime the resulting in post-​developmental lymphangiogenesis (also in the body. recruited T cells to induce adaptive immunity. Efferent referred to as neo-lymphangiogenesis)​ 120,124.

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In the kidney, lymphangiogenesis can occur irrespec- CLEC-2 on extravasated platelets results in the release of tive of the nature of kidney injury and functions to facil- sphingosine-1-​phosphate (S1P), which promotes itate the clearance of interstitial fluid as well as cellular VE-​cadherin expression by neighbouring HEVs, main- infiltrates and debris resulting from tissue injury124,130,131. taining their barrier integrity136. Within DLNs, recruited Interestingly, these newly formed lymphatic vessels seem immune cells interact with kidney-​derived APCs, after to emerge via the expansion of existing donor kidney which the activated immune cells are returned to the lymphatic vessels (lymphangiogenesis) as well as via blood circulation and can subsequently return and con- the recruitment and differentiation of host circulating tribute to the inflammatory infiltration of the kidney precursor cells (lymphvasculogenesis). A 2021 study after injury. Thus, the kidney lymphatic system and reported that classically activated (M1) macrophages can DLNs have an integral role in the pro-inflammatory​ feed- also transdifferentiate into LECs, form tube-like​ struc- back loop needed to activate leukocytes in the immune tures and colocalize with lymphatic vessels in response response to kidney injury. Removal of kidney DLNs or to unilateral ureteral obstruction (UUO)-​induced kid- disruption of this feedback loop ameliorates the devel- ney injury132. This finding suggests that macrophages opment of kidney injury in animal models129,137. In a can promote lymphangiogenesis through growth factor model of rapidly progressive glomerulonephritis, mice signalling, and contribute directly to the formation of that underwent bilateral resection of the DLN demon- new lymphatic vessels after injury — a proposal that strated significantly reduced macrophage infiltration into is supported by an analysis of newly developed lym- the kidney and lower numbers of crescentic glomeruli phatic vessels within female-​to-​male sex-​mismatched than mice with intact DLNs137. Direct inhibition of kid- kidney allografts, in which 4.5% of LECs contained ney lymphangiogenesis, blockade of DLN recruitment a recipient-​derived Y chromosome, suggesting a of CCR7+ APCs, or injection of anti-​PDPN antibody host-​derived, likely circulating cell origin133. After res- to disrupt the functional microarchitecture of the DLN olution of the stimuli that prompted lymphangiogene- also attenuated intrarenal inflammation in multiple sis, some regression of these newly formed vessels can injury models, including ischaemia–reperfusion injury, occur, although its extent seems to be tissue and context UUO and nephrotoxic serum nephritis129,137. Together, dependent134. The mechanisms by which regression of these studies suggest that disrupting this injury-​related newly formed lymphatic vessels occurs also remains pro-inflammation​ feedback loop between the kidney and unknown. DLNs might represent a future approach to mitigating The induction of lymphangiogenesis in response to the inflammatory response to kidney injury. inflammation enables transport of antigen and immune Of note, inflammation is a complex biological process cells from the site of injury to surrounding DLNs to ini- that also serves a protective role, and care must there- tiate an immune response. During inflammatory kidney fore be taken when considering anti-​lymphangiogenic injury, APCs interact with and are activated by antigens approaches. Lymphangiogenesis can contribute in the interstitial space (Fig. 4). CCL21 expressed by LECs to the removal of debris and noxious stimuli to promote enables the kidney lymphatic system to attract and reab- the resolution of inflammation138. In addition, LECs sorb these CCR7+ APCs and lymphocytes129, which are are adaptive and can directly suppress local immune then transported to DLNs129,135. Podoplanin expressed by cell infiltration112. Finally, without proper clearance of fibroblastic reticular cells surrounding the HEVs helps to interstitial fluid by the lymphatic system, tissue oedema maintain the integrity of these specialized vessels, facili- and fluid overload can further exacerbate tissue injury, tating the organized entry of naive T cells to DLNs from particularly in encapsulated organs such as the liver and the blood circulation. Podoplanin-mediated​ activation of the kidney that have limited capacity to accommodate increases in interstitial volume139. Increased kidney subcapsular pressure can reduce kidney blood flow and Box 3 | Composition of kidney lymph glomerular filtration rate140. Lymphangiogenesis acts The cortical kidney lymphatic system acts to drain interstitial fluid to maintain fluid to clear interstitial oedema from the kidney in these homeostasis as well as reabsorb interstitial plasma proteins, electrolytes, cytokines, settings and mitigates the development of injury107,141. growth factors and immune cells. Thus, kidney lymph contains both common and Thus, complete inhibition of lymphangiogenesis may organ-specific​ factors that represent the microenvironment in which it was collected. not be desirable and a major challenge in develop- The composition of kidney lymph has been characterized by measuring the ratios of ing therapeutics that target the lymphatic system will 218 219 220 218 lymphatic to plasma inulin , creatinine , labelled glucose and electrolytes be the identification of approaches to separately influ- exiting the kidney. These studies have revealed that kidney lymph comprises fluid, ence the protective versus the detrimental functions of electrolytes and small proteins that arise from both the capillary and tubular filtrate with a solute concentration close to that of plasma221. In addition to common these vessels. components of interstitial fluid, renin and angiotensin II are also detected in the kidney lymph, potentially at higher concentrations than in kidney venous plasma, suggesting Kidney fibrosis. Pathological kidney fibrosis, includ- secretion of these components into the kidney interstitum90,222. However the relevance ing glomerulosclerosis and tubulointerstitial fibrosis, of lymphatic transport of these molecules remains unknown. Kidney lymph also collects is a prognostic marker of poor outcomes, including filtered antigens that are smaller than albumin, as well as resident dendritic cells that tubular atrophy and rarefaction of the glomerular and are contained in the tubular compartment116. These protein and cellular components are peritubular capillaries142. Increased kidney lymphatic transported to local draining lymph nodes and help to maintain peripheral immune vessel proliferation and density strongly coincide with tolerance. In the setting of kidney injury, increased levels of pro-inflammatory​ cytokines the development of kidney fibrosis in both human and chemo-attractants​ are also observed in the lymph223, raising the possibility of and animal studies, and seem to occur irrespective of lymph-based​ diagnostics for kidney disease. the nature of the initial injury120,124,129,143. However, the

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a Afferent Kidney injury lymphatic Draining vessel lymph node

Antigen

Arterial return APC of effector cells Effector via the circulation T cell Trans- differentiation b c into LECs Platelet PDPN Cytokine ↑ Adhesion CLEC-2 molecules Macrophage ↑ IL-1 • IFNγ ↑ IL-6 S1P • TNF ↑ IL-12 • TGFβ ↑ IL-23

HEV • TonEBP ↑CTGF ↑VE-CAD expression ↑TNF ↑ VEGF-C

T cell ↑VEGF-D VEGFR-3 recruitment FRC Proximal tubular epithelial cell Lymphangiogenesis

Fig. 4 | Lymphatic patterning and signalling in acute kidney injury. A feedback loop between the kidney and the draining lymph nodes (DLNs) is activated in response to acute kidney injury and injury-associated​ lymphangiogenesis. a | Antigen released in response to injury is picked up by CCR7+ antigen-presenting​ cells (APCs) within the interstitial space and trafficked via the lymphatics to the DLNs following a CCL21 gradient. b | High endothelial venules (HEVs) traffic naive T cells into the DLN where they interact with the activated APCs. Fibroblastic reticular cells (FRCs), which surround the HEVs, promote recruitment of naive T cells through the secretion of cytokines CCL19, CCL21 and CXCL12. They also maintain the integrity of HEV conduits through the cell-surface​ expression of podoplanin (PDPN), which interacts with C-type​ lectin-like​ receptor 2 (CLEC-2) on extravasated platelets, prompting the release of sphingosine-1-phosphate​ (S1P) and enhancing the expression of VE-cadherin​ on neighbouring HEV cells to maintain their barrier integrity. Activated effector T cells then return to the blood circulation, migrating back to the kidney where they release inflammatory cytokines. c | Inflammatory cytokines, prompt proximal tubule epithelial cells and macrophages within the kidney to release a variety of growth factors, including vascular endothelial growth factor C (VEGF-C)​ and VEGF-D,​ which further promote lymphangiogenesis, and increase expression of adhesion molecules such as ICAM, VCAM-1 and E-selectin​ on lymphatic endothelial cells, facilitating the drainage of fluid and removal of accumulated immune cells. LECs, lymphatic endothelial cells; VEGFR-3, vascular endothelial growth factor receptor 3.

pathophysiological relevance of this association remains from the kidney in rats exacerbated the expression a subject of debate. of TGFβ, tubule epithelial cell apoptosis and kidney Despite a number of studies showing that lymphang- fibrosis142,144,145. These data support a protective role iogenesis promotes an immune response that may for lymphangiogenesis in the chronic response to contribute to kidney injury, lymphangiogenesis might injury through the removal of inflammatory cells and also mitigate the transition from acute kidney injury pro-fibrotic​ factors. In addition, emerging research sug- to kidney fibrosis by draining pro-​fibrotic inflamma- gests that the lymphatics might directly promote tissue tory cytokines and immune cells, which are produced repair after injury through lymphangiocrine signalling. in response to kidney injury and can promote fibrotic In the heart, LECs secrete the extracellular protein reelin, remodelling. Augmentation of lymphangiogenesis by which promotes cardiomyocyte survival after myocar- administration of recombinant human VEGF-​C to dial infarction and attenuates scarring146. Whether sim- mice following UUO-​induced injury reduced levels of ilar lymphangiocrine signalling occurs in the kidney is pro-​fibrotic markers such as TGFβ within the kidney not yet known. and attenuated the development of kidney interstitial However, as discussed earlier, lymphangiogenesis fibrosis121. By contrast, ligation of lymphatic drainage in the setting of kidney injury is not always beneficial.

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The chronicity of injury and inflammatory responses Notably, this effect was attributed to the interaction of may influence the beneficial versus detrimental effects VEGF-​C with VEGFR-2 and VEGFR-3 heterodimers of lymphangiogenesis in kidney fibrosis. In mouse expressed by local glomerular endothelial cells rather models of chronic kidney disease and proteinuria, than effects on lymphangiogenesis. Further studies kidney lymphangiogenesis occurs prior to the devel- are needed to gain a greater understanding of the role opment of fibrosis147. Similar to other aetiologies of of lymphatics in fibrotic kidney disease and how they inflammation-​associated lymphangiogenesis, diabetic might be targeted therapeutically to harness their bene- mice show increased kidney expression of TGFβ, TNF ficial properties in clearing inflammatory immune cells and VEGF-​C concomitant with an increased num- and interstitial fluid, while preventing the formation ber of lymphatic vessels throughout the kidney148,149. of disorganized, leaky vessels that may hasten disease Of note, lymphangiogenesis and tubulointerstitial progression. fibrosis occur primarily in regions of inflammatory cell infiltration122,150. Despite a beneficial role of the Polycystic kidney disease. Polycystic kidney diseases lymphatic system in removing inflammatory and (PKDs) are a group of disorders characterized by growth pro-​fibrotic factors, lymphangiogenesis in the diabetic of fluid-​filled epithelial lined cysts within the kidney, kidney may eventually become pathological in the set- which subsequently drive kidney inflammation and ting of sustained inflammation122,148. Chronic inflam- fibrosis152. Over time, progressive cystic expansion mation in other tissues, such as the skin, is associated and associated fibrosis lead to a decline in kidney func- with a pathological enlargement of local lymphatic tion, which can progress to kidney failure. These cysts vessels that are hyperpermeable or ‘leaky’, resulting in most commonly result from genetic mutations in the impaired clearance of fluid from the interstitial space151. PKD1 and PKD2 genes, the protein products of which, Chronic inflammation and upregulation of VEGF-​C in polycystin 1 and 2 respectively, localize to the primary diabetic kidney disease might also lead to over-sprouting​ cilia of kidney tubule epithelial cells. The blood and of disorganized kidney lymphatics, which may pro- lymphatic microvasculature that surrounds the kidney mote the progression of fibrosis148. Directed suppres- tubules are thought to have a role in modulating kid- sion of lymphangiogenesis with a selective VEGFR-3 ney cyst growth. Kidney cysts appear in close proximity inhibitor slowed the progression of diabetic nephrop- to the cortical lymphatic vessels, suggesting that lym­ athy and fibrosis in mice122. However, the expression of phatic vessels could act to transport fluid that accumu­ VEGF-​C in the setting of diabetic kidney disease may lates within the growing cysts91. Progressive cystogenesis not always be detrimental. One study demonstrated that within the kidney is also associated with the develop- podocyte-specific​ overexpression of VEGF-C​ in mice led ment of a disorganized peri-​cystic CD31+, VEGFR-3+, to a reduction in glomerular injury and albuminuria47. PROX-1− vascular network123, which is thought to be

Table 2 | Consequences of lymphangiogenesis in kidney diseases and conditions Setting Beneficial effects Detrimental effects Refs Acute kidney Clearance of inflammation-associated​ interstitial Promotes further injury through a 129,137,131, injury fluid, noxious antigens and cellular debris to pro-inflammatory​ feedback loop with 138,139 mitigate injury local draining lymph nodes Kidney fibrosis Removal of inflammatory cytokines and immune Disorganized lymphatic expansion in 122,148,151, cells that persist after injury; reduces the chronic lymphangiogenesis results in 121,144,145 activation of tubule epithelial apoptosis and the poor lymphatic functioning, hindering development of fibrosis immune cell and fluid clearance and augmenting the progression of fibrosis Polycystic Increased clearance of inflammatory cells with No detrimental consequences directly 91,123 kidney disease reductions in cyst size and progression of cystic defined kidney disease Peritoneal No beneficial effects directly defined. However, Increased reabsorption of peritoneal 164,161,165 ultrafiltration peritoneal lymphangiogenesis occurs in response fluid, contributing to peritoneal failure to an accumulation of similar pro-fibrotic​ factors ultrafiltration failure and might have a role in mitigating chronic inflammation Hypertension Reduces dermal interstitial volume retention No detrimental consequences directly 118,177, and increases clearance of kidney immune defined 181,182 cell accumulation, resulting in attenuation of hypertension Kidney Immediately post-transplantation,​ restores Increases migration of antigens and 192,193, transplantation normal lymphatic efflux from the kidney, antigen-presenting​ cells from the 108,109 attenuating interstitial oedema and transplanted kidney, potentially preventing lymphocele. In one study using activating an alloimmune response and a rat kidney transplant model, induction of facilitating transplant rejection lymphangiogenesis attenuated the development of allograft rejection; however, the mechanism of this was not defined

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less efficient than normal vasculature in clearing fluid. lymphatics occurs in PKDs and that these changes In the hypomorphic Pkd1nl/nl mouse model of PKD, contribute to the progression of disease. exogenous treatment with VEGF-C​ was associated with remodelling of both the pericystic network of vessels Peritoneal ultrafiltration failure. Peritoneal dialy- and a widening of the larger lymphatic vessels, which sis is a form of kidney replacement therapy that relies increased clearance of inflammatory cells, and a markedly on a patient’s peritoneal membrane for fluid and sol- attenuated cystic disease123. Interestingly, polycystin 1 ute exchange. Waste products diffuse down a gradient and 2 are also expressed within LECs153. Polycystin 1 regu­ across a network of capillaries lining the peritoneum lates LEC migration and morphogenesis in zebrafish into dialysate that is instilled into the abdomen. Excess and mice153,154, and mice with disruption of Pkd1 develop fluid is also drawn from the peritoneal capillaries into severe oedema similar to other mouse models of dis- the abdominal cavity via an osmotic gradient produced rupted lymphatic development153,154. In the mouse kidney, by hypertonic glucose and other osmoles within the disruption of Pkd1 (in Pkd1RC/RC mice) leads to reduced dialysate. This fluid shift, or ultrafiltration, is balanced by lymphatic vessel branching and a reduction in the total continuous reabsorption through the system of peritoneal proportion of kidney volume occupied by lymphatic lymphatic vessels that drain fluid back into the central vessels91. Notably, these lymphatic defects are observed vascular system155. In effect, the total effective ultrafiltra- prior to the development of kidney cysts, suggesting tion during peritoneal dialysis amounts to transcapillary that mutations in Pkd1 could directly disrupt lymphatic fluid removal minus the lymphatic reabsorption156. development through its function in LECs. Together, Peritoneal ultrafiltration failure (UFF) (Fig. 5), defined these findings suggest that disruption of the kidney by high peritoneal solute transport and diminished

a Lymphatic Peritoneum b Peritoneal cavity Milky spot absorption Stoma channel Liver Mesothelial Tight Diaphragmatic cell junctions Stomach stomata (80%) Transverse colon Sub- Macrophage mesothelium Omentum Lacuna Small Omental and intestine mesenteric lymphatics Peritoneal Capillary Lymphatic capillaries (20%) cavity

Collecting lymphatics and blood vessels c Mesothelial-to-mesenchymal transition

Peritoneal fibrosis Fibroblast- like cell

• Peritonitis • Glucose TGF 1 VEGFR-3 • AGEs β CTGF • GDP VEGF-C • Lymphangiogenesis Ultrafiltration • ↑ Peritoneal fluid reabsorption failure

Fig. 5 | Peritoneal lymphatics and ultrafiltration failure. a | The parietal and visceral peritoneum form the peritoneal cavity within the abdominal cavity. The diaphragmatic stomata are responsible for 80% of lymphatic absorption from the peritoneal cavity whereas end lymphatic vessels in the omental and mesenteric visceral peritoneum make up a majority of the remaining lymphatic absorption. b | The apical surface of the peritoneum is lined by a layer of mesothelial cells that overlie a connective tissue layer containing blood and lymphatic capillaries. Adjacent mesothelial cells adhere by tight junctions except at regions of lymphatic stomata, which allow for the unidirectional flow of fluid into lymphatic cisterns known as lacunae. Lymphoid tissue, referred to as ‘milky spots’, provide an additional means for lymphatic vessels to connect with the peritoneal space210. c | Inflammatory events such as peritonitis result in increased expression of TGFβ1, which prompts macrophages to release vascular endothelial growth factor C (VEGF-C)​ and mesothelial cells and fibroblast like cells in the sub-mesothelial​ space to release connective tissue growth factor (CTGF). CTGF also prompts macrophages to release VEGF-C.​ Although CTGF can bind directly to VEGF-C​ and prevent its binding to vascular endothelial growth factor receptor 3 (VEGFR-3), the net effect of CTGF release is increased lymphangiogenesis. The resulting increase in peritoneal fluid reabsorption, in combination with mesothelial-to-​ ​mesenchymal transition and peritoneal fibrosis, results in ultrafiltration failure. AGE, advanced glycosylation end products; GDP, glucose degradation products. Part b adapted with permission from Sarfarazi et al. 210, Elsevier.

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ultrafiltration capacity, is an important and frequent Hypertension. Given the defined role of the lymphatic complication of peritoneal dialysis. It is a major fac- system in body fluid homeostasis, it has long been pro- tor in the discontinuation of peritoneal dialysis and is posed that derangements of the lymphatic system might associated with increased mortality157,158. The devel- contribute to the development of sodium-​dependent opment of UFF is associated with chronic peritoneal volume expansion and hypertension172. Moreover, stud- fibrosis characterized by sub-​mesothelial fibrosis and ies from the past few years have revealed the important neo-angiogenesis​ 159. A combination of increased vascular contribution of inflammation and activated immune surface area, higher vascular permeability, and impaired cell infiltration in the kidney to the development of channel-​mediated water transport is thought to be the hypertension173, providing another possible mechanism primary driver of ineffective ultrafiltration. However, by which the lymphatic system might influence the although UFF is associated with neo-​angiogenesis, development of this disease (Fig. 6). Indeed, lymphatic peritoneal biopsy samples from individuals with UFF vessel density is increased in both the skin and the kid- demonstrate a blood capillary density similar to that of ney in the setting of hypertension118,174, and a growing biopsy samples from individuals undergoing peritoneal body of literature suggests that enhancing lymphangi- dialysis without UFF159. This finding suggests that factors ogenesis improves blood pressure in hypertensive ani- other than blood capillary density may be responsible for mal models by modulating both fluid homeostatic and the increased peritoneal membrane fluid transport asso- inflammatory cell mechanisms. ciated with UFF. Rather, lymphangiogenesis resulting in High dietary sodium intake is associated with an increased lymphatic absorption from the peritoneal cav- increased risk of hypertension175. Dietary salt loading ity might be an important driver of UFF160. In contrast to prompts the skin interstitium to act as a buffer, by accu- blood capillary density, the number of lymphatic vessels mulating Na+ and Cl− and leading to hypertonic inter- and the rate of lymphatic absorption within the perito- stitial volume retention118,176–178. In hypertensive mice, neum is associated with both the duration of peritoneal the increase in osmolarity and extracellular ions within the dialysis and the development of UFF161,162. VEGF-C​ lev- dermis interstitium leads to direct activation of the tran- els within human dialysate also correlate with peritoneal scription factor tonicity-​responsive enhancer-​binding permeability163. Moreover, blocking of lymphangiogen- protein (TonEBP, also known as Nfat5) in macrophages esis with use of a soluble VEGFR-3 inhibitor improved and dendritic cells118,178. Activation of TonEBP induces impaired ultrafiltration in mice164. VEGF-C​ expression by these cells promoting lymphatic Lymphangiogenesis in the setting of peritoneal UFF expansion within the dermis. This lymphatic remodel- is closely linked to the development of peritoneal fibro- ling enables increased drainage of accumulated intersti- sis. Chronic inflammatory conditions such as peri- tial fluid and electrolytes, mobilizing them for external tonitis are associated with the development of UFF, renal clearance and resetting the interstitium as a buff- the development of peritoneal fibrosis, and increased ering reservoir accompanied by a reduction in blood lymphangiogenesis165. The molecular mechanisms pressure. In line with this process, the plasma concen- underlying peritoneal lymphangiogenesis are similar to tration of VEGF-C​ is higher in patients with refractory those underlying inflammatory-​associated lymphangi- hypertension than in normotensive controls118, and ogenesis in other tissues. For instance, TGFβ promotes lymphatic vessel density is higher in the skin of hyper- lymphangiogenesis and fibrosis within the peritoneum tensive than in normotensive mice179. Conversely, inhi- as well as other tissues143,161 in response to inflamma- bition of dermal lymphangiogenesis in mice, either by tion. In the peritoneum, TGFβ signals through receptors macrophage depletion, sequestration of VEGF-​C or expressed by mesothelial cells and macrophages to trig- blockade of VEGFR-3, results in increased interstitial ger the release of VEGF-​C, which acts on local LECs to fluid retention and elevated blood pressure when ani- promote lympangiogenesis161,165. The induction of peri- mals are challenged by a high-salt​ diet118,177. These stud- toneal mesothelial cells by TGFβ also promotes secretion ies demonstrate that the skin interstitium functions as of CTGF, which has been linked to peritoneal fibrosis and an extra-renal​ site of sodium and fluid homeostasis, and lymphangiogenesis165–167. A feedback loop enables CTGF that by acting on this reservoir the dermal lymphatics to conversely induce the release of TGFβ and VEGF-​C can partly influence, although not fully normalize, blood by peritoneal mesothelial cells168. However, CTGF also pressure. interacts directly with VEGF-C,​ limiting its ability to act Similarly to the skin, VEGF-​C-​mediated lymphang- on VEGFR-3 receptors expressed by LECs165,169. Despite iogenesis is also observed in the heart and kidney of this inhibitory effect, the available evidence suggests that rodents exposed to high salt intake180,181. In the kidneys overall, CTGF acts to promote peritoneal lymphangio- of hypertensive rodents, a correlation between hyperten- genesis. Inhibition of CTGF reduces UFF and the devel- sion, immune cell infiltration and increased lymphang- opment of peritoneal fibrosis, supporting its candidacy iogenesis is frequently observed182–184. In this setting, as a therapeutic target165,168,170. By contrast, blockade of lymphangiogenesis is thought to protect against kidney the VEGF-C–VEGFR-3​ pathway also improved impaired injury and the exacerbation of hypertension. Kidney infil- ultrafiltration but had no effect on the development of trating immune cells release pro-inflammatory​ and profi- peritoneal fibrosis164, suggesting that the development brotic cytokines, which interrupt blood flow and sodium of peritoneal fibrosis related to CTGF expression might excretion, contributing to both progressive hypertension rely on additional non-​VEGF-​C-​mediated mechanisms, and kidney injury174,184–186. Enhancing lymphangiogen- including the role of CTGF as a regulator of angiogenesis, esis, either through selective tubular overexpression of as well as its direct pro-fibrotic​ activity171. VEGF-​D or subcutaneous administration of VEGF-C,​ is

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a Hypertension b Epidermis

Dermis – Cl– Proteoglycan Na+ Cl + Na – + Cl Cl– Na Na+ Cl– Na+ + – Na Cl – Cl – Na+ Cl Na+ Na+ Na+ Macrophages • Salt-induced hypertonicity and DCs VEGF-C • Immune cell deposition

VEGFR-3

Lymphangiogenesis ↑TonEBP VEGF-C Lymphangiogenesis

Fig. 6 | Lymphatic regulation of hypertension. a | Hypertension is associated with the accumulation of interstitial fluid and the deposition of immune cells in several organs including the skin, heart and kidney. These processes trigger lymphangiogenesis, which acts to buffer fluid and immune cell accumulation. b | In salt-​induced hypertension, sodium binds to proteoglycans in the interstitial space of the skin, which acts as an additional compartment for regulating total body water content. In response to the local hypertonicity in these tissues, upregulation of tonicity-responsive​ enhancer-​ binding protein (TonEBP) in macrophages and dendritic cells promotes dermal interstitial sodium buffering via vascular endothelial growth factor C (VEGF-C)-​ induced​ lymphangiogenesis. DCs, dendritic cells; VEGFR-3, vascular endothelial growth factor receptor 3.

protective in mouse models of hypertension, associated circumstances is typically mediated by pre-​existing with a reduction in immune cell accumulation, attenuated antibodies in the setting of prior sensitization and fibrosis and a reduction in blood pressure181,182,187. In an can therefore occur independently of a cell-​mediated angiotensin II-​induced mouse model of hypertension, immune response. administration of a recombinant isoform of VEGF-​C, In rejecting human kidney allografts, diffuse mon- VEGF-​C156S, reversed the development of hyperten- onuclear infiltrates, as well as nodular infiltrates, are sion and mitigated kidney injury179. Surprisingly, the lym- found throughout the cortex. New lymphatic vessels phangiogenic response observed in kidneys in response extend up into areas of the parenchyma that contain to angiotensin II infusion was absent in the co-​treatment high densities of nodular cellular infiltrates189,190, and group. This effect is possibly a consequence of reduced the extent of lymphangiogenesis correlates positively lymphangiogenic drive in response to the reduction in with the severity of transplant rejection at the time blood pressure; however, this finding also suggests that of biopsy190,191. Similarly in rats, increased recruitment of the anti-hypertensive​ effect of VEGF-C156S​ is independ- APCs into kidney lymphatic vessels was associated with ent of kidney lymphangiogenesis. Although the exact worse allograft function192, inhibition of lymphangio- mechanisms of this antihypertensive effect remain unex- genesis with the mTOR inhibitor sirolimus attenuated plored, an increase in skin lymphatic vasculature density the development of chronic allograft injury to a greater was noted in the VEGF-C156S​ treatment group. Together, extent than ciclosporin193. These studies raise the con- these data suggest that enhancing lymphangiogenesis, in cern that lymphangiogenesis increases the delivery both the dermis and the kidney, might represent a thera­ of APCs to the recipient lymph node and initiates an peutic option for the treatment of hypertension and alloimmune response, facilitating transplant rejection. hypertensive kidney disease. However, transplant recipients with a higher density of kidney lymphatic vessels have a lower chance of pro- Transplant rejection. Transplant rejection is a major gression to interstitial fibrosis and/or tubular atrophy, risk factor for kidney allograft loss with the infiltra- which suggests a potential protective role194. This notion tion of inflammatory T-​lymphocytes acting as potent is further supported by the demonstration that induction mediators of rejection188. Inflammatory lymphocytes of lymphangiogenesis by overexpression of VEGF-​C in are trafficked to and from the kidney allograft via the mouse kidney allografts attenuates the development of blood circulation and via newly formed lymphatic transplant rejection and reduces mortality109. Similar to vessels. Although the lymphatic vessels of the donor their role in other aetiologies of injury and inflamma- kidney are not initially connected to the systemic circu- tion, new lymphatic vessels may drain interstitial fluid lation, studies in mice show that they grow quickly and and cellular infiltrates that accumulate with rejection; re-establish systemic lymphatic connections within days of however, the exact mechanisms by which lymphatics transplantation109. Kidney allografts can undergo acute provide kidney graft survival have not yet been defined. rejection within the first few days post-transplantation,​ Conflicting reports also exist with regard to the effects before kidney lymphatic vessels connect to the systemic of lymphangiogenesis on other organ allografts: lym- circulation. However, hyper-​acute rejection in these phangiogenesis has been reported to promote rejection

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of heart transplants5, but has conversely been reported lymphatics, normalization of the pericystic capillary to promote survival of lung transplants195. network and a reduction in pericystic macrophage infiltration, leading to a reduction in both cyst size and Therapeutic targeting of the lymphatics kidney inflammation123. Intracutaneous injections of The dynamic growth and regression of lymphatic ves- VEGF-​C156S also promoted kidney lymphangiogen- sels and their central role in regulating several key body esis and attenuated the development of kidney fibro- processes that are important for physiological func- sis after kidney injury in a mouse model of UUO121. tion, inflammation and fibrosis has stimulated much In keeping with a protective role from inflammation, interest in developing therapies that specifically target intracutaneous injections of VEGF-​C156S attenuated the lymphatic system. A number of lymphatic-​specific the development of dermal oedema and inflamma- treatments for lymphoedema and malignancy are now tion in a model of chronic cutaneous inflammation199. under study in clinical trials (reviewed elsewhere196). In addition, and as mentioned earlier, VEGF-C156S​ can Lymphatic-specific​ therapies for kidney diseases are less reverse angiotensin-II-​ mediated​ hypertension, although developed, but several are of potential interest based on whether this response is due to a lymphangiogenic effect insights from preclinical models. is unclear179. Exogenous VEGF-C​ therapy may also have potential therapeutic consequences in the kidney beyond VEGF-​C–VEGFR-3. As the primary regulator of lym- the promotion of lymphangiogenesis. VEGFR-3 is also phangiogenesis, the VEGF-C–VEGFR-3​ signalling path- expressed in the BECs of some fenestrated microvascu- way represents a prime target for lymphatic modulating lature beds and VEGF-​C can interact with VEGFR-2 therapies. Inhibition of VEGFR-3 has been proposed (refs9,40). In a mouse model of type 1 diabetes mellitus, as a therapeutic means of preventing pathogenic lym- podocyte-​specific overexpression of VEGF-C​ protected phangiogenesis. SAR131675 is a potent and selective glomerular endothelial cells through interaction with inhibitor of VEGFR-3, which blocks both its tyrosine VEGFR-2 and independent of effects on LECs, prevent- kinase activity and its ability to autophosphorylate. ing the development of early diabetic kidney changes47. In a mouse model of diabetic kidney disease, SAR131675 As discussed previously, conflicting studies exist with reduced kidney lymphangiogenesis, glomerulosclerosis regard to the effects of blocking VEGF-​C–VEGFR-3 and tubulointerstitial fibrosis122. A soluble VEGFR-3 activity in diabetic kidney disease122. Thus, further fusion protein (sVEGFR3-FC),​ which binds and seques- investigation is needed to better define the sites of action, ters VEGF-​C and VEGF-​D, also attenuated intrarenal mechanistic consequences and off-​target effects of lymphangiogenesis induced by UUO and ischaemia– modulating VEGF-C–VEGFR-3​ activity in the kidney. reperfusion, and ameliorated the development of kidney fibrosis129. In another study, however, adminis- Connective tissue growth factor. Insights into the role tration of an anti-​VEGFR3 antibody (IMC-3C5) to rats of CTGF in lymphangiogenesis, angiogenesis and the 6 weeks after induction of adriamycin-​induced kidney development of fibrosis has prompted investigations into injury successfully reduced lymphangiogenesis but did the possible therapeutic effects of blocking this growth not alter the development of fibrosis197. The reason for factor. For example, a monoclonal antibody, FG-3019, the differences in study outcomes remains unclear, which interferes with the action of CTGF, is in clinical but may be related to the nature of the initial kidney trials in patients with pulmonary fibrosis200. This trial injury, the timing of therapy relative to the progression raises the possibility that CTGF could also be targeted in of injury, or mechanistic differences in the approaches the context of kidney fibrosis. FG-3019 has been demon- used to target VEGF-​C–VEGFR-3 signalling. Outside strated to prevent UFF in animal models of peritoneal of the kidney, inhibition of VEGFR-3 activity using an fibrosis UFF170; however, the extent to which attenuation adenovirus-​expressing soluble VEGFR-3, which binds of UFF in this setting is due to the anti-lymphangiogenic​ and sequesters endogenous VEGF-C​ and VEGF-​D, has properties of FG-3019 versus its effects on angiogenesis been demonstrated to suppress lymphatic growth and and fibroblast activities remains to be determined. consequently improve net peritoneal ultrafiltration by reducing lymphatic-​mediated fluid reabsorption in Podoplanin. Anti-​podoplanin antibodies have been rodent models164. Although targeting of VEGFR-3 has investigated in preclinical studies for the treatment of been proposed as a means of preventing UFF in patients malignancies and thrombosis201,202. In the context on peritoneal dialysis, studies have so far been limited to of kidney disease, anti-​podoplanin antibody treatment preclinical models. protected against the development of kidney injury Conversely, activation of the VEGF-​C–VEGFR-3 in a mouse model of nephrotoxic serum nephritis137. In pathway to promote lymphangiogenesis has been shown that study, treatment disrupted the ability of fibroblas- to have therapeutic benefits in reducing the development tic reticular cells to recruit T cells, thereby preventing of kidney fibrosis and attenuating the development of their activation within the kidney DLNs, associated cystic kidney disease in mice and rats121,123. VEGF-C156S​ with markedly reduced expansion of the lymphatic vas- — which specifically targets VEGFR-3, in contrast culature. Mice treated with anti-​podoplanin antibody to endogenous VEGF-​C, which can activate both had improved kidney function, reduced infiltration VEGFR-2 and VEGFR-3 (ref.198) — has been sug- of inflammatory macrophages and a lower percentage of gested as a potential treatment for several forms of crescentic glomeruli compared with control mice that kidney disease. In animal models of PKD, injection received sham therapy. However, the mechanisms and of this compound led to enlargement of the kidney consequences of podoplanin inhibition within lymph

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node fibroblastic reticular cells, as well as the potential The potential to develop novel therapies to slow fibro- for off-​target effects on other podoplanin-​positive cells sis and cyst growth, and to improve success rates by such as podocytes, in which inhibition of podoplanin extending the life of the peritoneum for home dialysis is has been demonstrated to result in proteinuria203, need exciting. However, these potential therapies are limited to be further explored. by our understanding of how these signalling pathways may act in organ-specific​ and context-dependent​ ways. Conclusions As organ-​specific developmental signalling pathways Despite an enhanced focus on the role of lymphatic are often reactivated in the setting of disease, detailed vessels in a variety of disease processes, several gaps studies of lymphatic origin and growth in the kidney remain in our understanding of the function of the using state-​of-​the-​art technologies such as single-​cell lymphatic system in both health and disease. In the kid- RNA sequencing and lineage tracing hold promise in ney and organs of interest to nephrologists, improved bringing new therapies to clinical practice. understanding of the contribution of the lymphatics to protective versus pathogenic states is urgently needed. Published online 22 June 2021

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212. Arrive, L., Monnier-​Cholley, L. & El Mouhadi, S. 219. Cockett, A. T., Roberts, A. P. & Moore, R. S. Renal Author contributions Use of unenhanced MR lymphography to characterize lymphatic transport of fluid and solutes. Investig. Urol. All authors made substantial contributions to the drafting, idiopathic chyluria. AJR Am. J. Roentgenol, 211, 7, 10–14 (1969). researching and writing of this article. All authors reviewed W200 (2018). 220. Cook, V. L., Reese, A. H., Wilson, P. D. & Pinter, G. G. and/or edited this manuscript prior to submission. 213. Yildirim, I. O. et al. A novel technique in the treatment Access of reabsorbed glucose to renal lymph. of lymphoceles after renal transplantation: C-​arm cone Experientia 38, 108–109 (1982). Competing interests beam CT-guided​ percutaneous embolization of 221. Bell, R. D. Renal lymph flow and composition during S.E.Q. has applied for patents related to therapeutic target- lymphatic leakage after lymphangiography. acetazolamide and furosemide diuresis. Lymphology ing of the ANGPT–TEK pathway in ocular hypertension, glau- Transplantation 102, 1955–1960 (2018). 17, 10–14 (1984). coma and kidney disease, receives research support, owns 214. Iwai, T. et al. Experience of lymphangiography as a 222. Wilcox, C. S. & Peart, W. S. Release of renin and stocks in and is a director of Mannin Research, is an external therapeutic tool for lymphatic leakage after kidney angiotensin II into plasma and lymph during advisory board member of AstraZeneca and receives consult- transplantation. Transplant. Proc. 50, 2526–2530 hyperchloremia. Am. J. Physiol. 253, F734–F741 ing and advisory board fees from Roche, Janssen, Genentech (2018). (1987). and AstraZeneca. The other authors declare no competing 215. Kuusk, T. et al. Lymphatic drainage from renal tumors 223. Bivol, L. M. et al. Unilateral renal ischaemia in rats interests. in vivo: a prospective sentinel node study using induces a rapid secretion of inflammatory markers SPECT/CT imaging. J. Urol. 199, 1426–1432 to renal lymph and increased capillary permeability. Peer review information (2018). J. Physiol. 594, 1709–1726 (2016). Nature Reviews Nephrology thanks A. Phillips, who 216. Pawlak, J. B. & Caron, K. M. Lymphatic programing co-reviewed​ with P. Russell; J. Rutkowski; and J. Titze for their and specialization in hybrid vessels. Front. Physiol. 11, Acknowledgements contribution to the peer review of this work. 114 (2020). M.D.D. was supported by the National Institutes of Health 217. Thomson, B. R. et al. A lymphatic defect causes ocular (T32DK108738) and is currently supported by the American Publisher’s note hypertension and glaucoma in mice. J. Clin. Invest. Society of Nephrology Ben J. Lipps Research Fellowship. Springer Nature remains neutral with regard to jurisdictional 124, 4320–4324 (2014). S.E.Q is supported by the National Institute of Diabetes and claims in published maps and institutional affiliations. 218. Keyl, M. J. et al. Composition of canine renal hilar Digestive and Kidney Diseases (P30DK114857) and National lymph. Am. J. Physiol. 209, 1031–1033 (1965). Eye Institute (R01EY025799). © Springer Nature Limited 2021

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