Implications of Lymphatic Biology for Kidney Diseases

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Beyond a Passive Conduit: Implications of Lymphatic Biology for Kidney Diseases

Daniyal J. Jafree 1,2 and David A. Long 1

1Developmental Biology and Cancer Programme, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom 2MB/PhD Programme, Faculty of Medical Sciences, University College London, London, United Kingdom

ABSTRACT The kidney contains a network of lymphatic vessels that clear fluid, small molecules, three-dimensional (3D) imaging cou- and cells from the renal interstitium. Through modulating immune responses and via pled with quantitative analysis have crosstalk with surrounding renal cells, lymphatic vessels have been implicated in the characterized lymphatic development progression and maintenance of kidney disease. In this Review, we provide an over- in mouse and human embryonic kid- view of the development, structure, and function of lymphatic vessels in the healthy neys (Figure 1).16 In mouse, the meta- adult kidney. We then highlight the contributions of lymphatic vessels to multiple nephros, the precursor to the adult kid- forms of renal pathology, emphasizing CKD, transplant rejection, and polycystic ney, begins to form around embryonic kidney disease and discuss strategies to target renal lymphatics using genetic and day (E) 10.5.17 At early stages of its de- pharmacologic approaches. Overall, we argue the case for lymphatics playing a velopment,18 the embryonic kidney is fundamental role in renal physiology and pathology and treatments modulating devoid of lymphatics. It is not until these vessels having therapeutic potential across the spectrum of kidney disease. E14.5, after considerable development of the blood vasculature,19,20 that lym- JASN 31: 1178–1190, 2020. doi: https://doi.org/10.1681/ASN.2019121320 phatic endothelial cells (LECs) are de- tectable as a cellular plexus using immunohistochemistry for lymphatic markers Lymphatic vessels serve as a conduit for the disease.6–8 However, in spite of an in- (Table 1) such as Prospero homeobox clearance of tissue fluid, cells, and small creasing understanding of lymphatic bi- protein 1 (PROX1) or vascular endothe- molecules, within a protein-rich fluid ology in other organs (reviewed in9–11), lial growth factor (VEGF) receptor 3 termed lymph, from the interstitial com- the kidney’slymphaticshavereceived (VEGFR-3). The developing kidney partment of vertebrate organs. This fluid relatively little attention. In this Review, lymphatics wrap around the base of the enters the lymphatic system via lymphatic we first outline how lymphatics form nascent kidney pelvis and are suggested capillaries within tissues, traveling down a during kidney development and de- to be continuous with an extrarenal net- hierarchic network of collecting vessels be- scribe their structure and function in work supplying the ureter, adrenal 12,14 fore reaching lymph nodes which drain to adult kidneys. We then combine our cur- gland, and gonad. Between E15.5 large ducts, eventually returning lymph to rent understanding of kidney lymphatics and E18.5, the hilar lymphatics rapidly the venous circulation. Through the iden- with parallels drawn from lymphatic bi- remodel and expand, forming lume- tification of molecular markers of lym- ology in other organs to discuss their po- nized vessels that extend alongside arte- tential contribution to and therapeutic rioles into the renal cortex. During this phatics, advances in imaging technologies, implications for several renal diseases. time, a complex collecting duct network and novel genetic tools to visualize and manipulate their function, the roles of lymphatic vessels have expanded to include Published online ahead of print. Publication date cholesterol transport,1 clearance of cere- DEVELOPMENT, ARCHITECTURE, available at www.jasn.org. brospinal fluid,2 electrolyte homeostasis,3 AND FUNCTION OF KIDNEY Correspondence: Dr.DavidA.Long,De- and regulation of peripheral tolerance LYMPHATICS velopmental Biology and Cancer Programme, Great Ormond Street Institute of Child Health, 4,5 and innate and adaptive immunity. University College London, London WC1N 1EH, The kidney possesses a lymphatic sys- Development United Kingdom. Email: [email protected] tem that has been implicated in the pro- A combination of immunohistochemi- Copyright © 2020 by the American Society of – gression and maintenance of kidney cal studies12 15 and, more recently, Nephrology

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Figure 1. Development, structure, and function of kidney lymphatics. During early nephrogenesis at E12.5, the kidney is devoid of lymphatics. Thereafter, kidney lymphatic development proceeds in three distinct phases: first, the appearance of a plexus of LECs in the kidney at E14.5; then, the remodeling of LECs into a patent vascular network by E16.5; and finally, the extension of these vessels toward the renal cortex at E18.5. All stages are presented in the context of important morphologic and differentiation events during renal development. The specification of the blood vasculature,19,20 ureteric bud branching and nephron generation,21 stages of nephron differentiation,18,22 the appearance of stromal derivatives such as pericytes and mesangial cells,23 and initiation of urinary function17,24 are taken from the indicated references. In the adult kidney, lymphatics reside in the cortical interstitium and drain to large lymphatic vessels in the hilum. The renal medulla is devoid of lymphatics. Drainage begins in the cortical interstitium. Increased pressure in this compartment causes ECM-bound anchoring filaments to force LECs apart, thus allowing tissue fluid, immune cells (such as dendritic cells and neutrophils), and small molecules (such as soluble antigen and antibodies) to enter lymphatic capillaries. is established;21 fully differentiated cell with blind-ended lymphatic capillaries single, continuous layer of LECs. Unlike types within the kidney emerge such as draining into arcades running with most blood vessels, lymphatic capillaries the mature glomerulus, segments of the arcuate arteries at the corticomedullary have a sparse, discontinuous basement nephron,22 perivascular, and mesangial junction (Figure 1).35,36 The cortical membrane and lack supporting cells cells23 and renal excretory function is ini- lymphatics then follow the interlobar such as vascular smooth muscle cells, 17,24 tiated. By the end of mouse gestation, blood vessels, descending toward the re- pericytes or fibroblasts.13 Instead, LECs lymphatics are present in both the hilum nal pelvis. Finally, the lymphatics drain lining lymphatic capillaries overlap, are and the cortex. A similar pattern of lym- out of the kidney through hilar lymphatic held together by specialized button-like phatic vessels is established by the end of vessels, located adjacent to the major re- junctions37 and are physically connected the first trimester in humans.16 nal arteries and veins as they enter and to surrounding extracellular matrix exit the kidney. (ECM) by fibrillin-rich anchoring pro- Macroarchitecture Cellular Architecture teins.38,39 As fluid leaks across blood In the mature adult kidney, lymph drain- Initially within organs, lymph enters capillaries, the ECM in the interstitium age begins in the cortical interstitium, lymphatic capillaries, which consist of a expands, causing anchoring proteins to

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Table 1. Molecular markers of lymphatic endothelium in the adult kidney LEC Marker Molecular Function Non-LEC Expression in the Kidneya PROX1 Transcription factor involved in the Thick ascending limbs of the loop of maintenance of lymphatic identity25 Henle restricted to the inner medulla26 and reported in ascending vasa recta27 LYVE-1 Membrane glycoprotein and receptor for Some endothelial cells in the hyaluronan facilitating DC entry into glomerulus12 lymphatic vessels28 VEGFR-3 Receptor tyrosine kinase required for LEC Ascending vasa recta27 and cortical tip function and vessel sprouting29 peritubular capillaries30 PDPN Membrane glycoprotein maintaining Podocytes32 separation between blood and lymphatic endothelium31 Neuropilin 2 (NRP2) Transmembrane protein interacting with Blood endothelial cells34 b VEGFR-3 promoting VEGF- C–mediated lymphatic sprouting33 aDetermined from studies of mouse or rat adult kidney. bDerived from single-cell RNA sequencing, because no published immunostaining is available for NRP2. pull on LECs. Consequently, button-like endothelial markers have been identi- In the cortex, a mismatch between tubular junctions between LECs open, allowing fied.27,30 Peritubular capillaries—which reabsorption and the capacity for uptake the constituents of lymph to enter lym- facilitate the reabsorption of fluids and by peritubular capillaries may raise corti- phatics paracellularly (Figure 1).40 Sol- molecules from adjacent cortical tubular cal interstitial pressure49 and facilitate utes may alternatively enter lymphatics epithelium—express CD31 and VEGFR- lymphatic clearance. Early functional transcellularly via vesicle formation and 3, but not the other lymphatic markers studies indicate a functional interplay be- transcytosis across LECs.41 Lymphatic PROX1, lymphatic vessel endothelial hy- tween kidney lymphatic flow, venous capillaries drain lymph into functional aluronan receptor 1 (LYVE-1), or podo- pressure50–54 and solute load55,56 that war- units of precollecting and collecting ves- planin (PDPN).30 Conversely, in a recent rants further investigation.57–60 sels13 known as lymphangions. Within study, ascending vasa recta, which main- Rich in Igs and albumin,48 lymph, these larger caliber vessels, LECs are tain the medullary osmotic gradient crit- like plasma, also contains complement lined by continuous zipper-like junc- ical for urinary concentrating ability, cascade components, coagulation fac- tions, are supported by smooth muscle were found to express PROX1 and tors, ECM proteases and their inhibitors and mural cells and contain valves to fa- VEGFR-3 but not LYVE-1 or PDPN.27 and enzymes involved in cellular metab- cilitate unidirectional lymph flow.37,42 Unlike LECs, specialized functions of olism. Lymph is also enriched in nuclear peritubular capillaries and ascending histones, cytosolic enzymes, transcrip- vasa recta are facilitated by endothelial tion factors, and ribosomal compo- Heterogeneity of Lymphatics and fenestrations, which are identifiable in nents61 likely derived from cellular apo- “Lymphatic-like” Vessels electron micrographs as transcellular ptosis.62 Few studies have examined the Lymphatic vessels have a unique molec- channels of approximately 70 nm in di- composition of renal lymph. Sodium, ular signature distinguishable from that ameter.46 Whether these vessels, which chloride,potassium,andcalciumcon- of blood endothelia.43 Some markers are have been termed lymphatic-like, share tent of lymph draining from the kidneys expressed in all lymphatic vessels, such other molecular or structural features may have physiologic relevance.51,56 as PROX1 and VEGFR-3. However, with LECs is yet to be determined, but Analysis of rodent models of renal there is heterogeneity in the molecular clearly multiple markers in parallel are ischemia-perfusion injury have identi- profile between lymphatic capillaries required to reliably distinguish kidney fied that renal lymph contains cytokines and precollecting and collecting vessels.9 lymphatics from other cell types in the such as IL-1b and IL-6, TNF-a and Heterogeneity of the adult kidney’s kidney. monocyte chemoattractant protein 1,63 blood vascular system is well recognized, albeit at low quantities compared with with blood endothelial cells molecularly Function and Composition of Renal blood draining from the kidney. Renal distinguishable between cortex, me- Lymph draining lymph nodes receive dendritic dulla, and glomerulus.44,45 Whether Based on their anatomical location47 and cells(DCs)andTandBlymphocytes similar molecular diversity exists within uptake of radiolabeled albumin,48 kid- from afferent renal lymphatics.64 These kidney lymphatics remains unexplored. ney lymphatics are proposed to drain studies advocate roles for kidney lym- Populations of hybrid kidney blood ves- the interstitium of the renal cortex and phatics in the maintenance of peripheral sels expressing both blood and lymphatic hilum interstitium, but not the medulla. tolerance and clearance of cellular debris

1180 JASN JASN 31: 1178–1190, 2020 www.jasn.org REVIEW in adult renal physiology. Renal lymph clearance function, modulating the potent effect of VEGF-D on renal canalsodrainreninandangiotensin inflammatory environment, and pre- lymphangiogenesis.90 II,63,65,66 but the physiologic relevance venting fibrotic remodeling in CKD Connective tissue growth factor of this route to the systematic circulation (Figure 2). Whether enhancing lym- (CTGF), an ECM-associated heparin- is unclear. phangiogenesis may also exert beneficial binding protein, has been identified as effects through clearance of interstitial a contributor to renal lymphangiogene- edema or inflammatory macromole- sis. CTGF is highly expressed by dam- cules within the kidney is not known. aged tubular epithelium and interstitial ROLES OF LYMPHATICS IN cells (likely macrophages91 or fibro- KIDNEY DISEASES Cellular and Molecular Mechanisms blasts92) in human kidneys with urinary of Lymphangiogenesis in the obstruction or DKD.93 Following total Lymphangiogenesis and Its Role in Diseased Kidney knockout of CTGF in adult mice, UUO Chronic Renal Injury and Fibrosis Studies of lymphangiogenesis in devel- resulted in reduced lymphangiogenesis Structural changes to the vasculature are opment79 or pathology80 have identi- and VEGF-C mRNA levels compared a prominent feature of CKD. Whereas fied a plethora of growth factors that with wild-type obstructed kidneys. In peritubular capillaries undergo rarefac- promote or inhibit lymphatic vessel culture, CTGF induces VEGF-C production, potentially triggering interstitial growth. Of those studied in the kidney, tion in immortalized mouse and human hypoxia and generating a profibrogenic VEGF-C and VEGF-D are central for proximal tubular epithelial cell lines and environment within diseased kidney,67 lymphangiogenesis in renal disease. binds directly to VEGF-C in a dose- renal lymphatics proliferate and sprout, These growth factors predominantly dependent manner.93 Whether and giving rise to new vessels in a process trigger lymphangiogenesis by activa- how CTGF exhibits activity directly termed lymphangiogenesis.68 In biop- tion of VEGFR-3, but VEGF-C can upon LECs has not yet been examined. sies of IgA nephropathy, focal glomeru- also act via VEGFR-2 to stimulate Inflammatory mediators, secreted by a losclerosis, lupus, ANCA-related GN, LEC and blood vessel proliferation variety of cell types upon tissue injury, diabetic kidney disease (DKD) and and migration.81 VEGF-C is highly also have roles in lymphangiogenesis. chronic interstitial nephritis acquired expressed by macrophages in the rat Upon stimulation of LECs by the inflam- from patients with disease ranging remnant kidney,71 murine unilateral matory milieu, PROX1 is activated and from moderate CKD to ESKD, the ureteral obstruction (UUO),82 human VEGFR-3 is upregulated downstream of cross-sectional area of lymphatics is sig- biopsies of IgA nephropathy, DKD36 NF-kΒ, increasing the responsiveness of nificantly greater than in nondiseased and chronic allograft rejection.83 These lymphatics to VEGF-C and VEGF-D.94 kidneys;36,69 a finding replicated in mul- macrophages, which are likely derived One inflammatory mediator which stim- tiple murine models of CKD.64,70–74 from bone marrow and infiltrate the ulates renal lymphangiogenesis is Lymphangiogenesis facilitates the diseased kidney from the vasculature,84 lymphotoxin-a, with overexpression in clearance of inflammatory cells from may be modulated by TGF-b1and mice proximal tubules leading to expansion the damaged tissue environment, which TNF-a release70,85 from damaged renal of cortical lymphatics accompanied by T is a vital step in the resolution of inflam- cells86,87 or from activation of hypoxia- and B lymphocyte–rich infiltrates.95 How- mation and prevention of fibrotic re- inducible factor 188 due to local renal ever, it was not determined whether modeling.Thisfunctionisillustrated hypoxia.67,89 Proximal tubular and col- lymphotoxin-a stimulates LECs directly or by ligating lymphatics exiting the kidney lecting duct epithelium are also poten- acts indirectly through other renal cell types. in rats, leading to loss of renal function tial sources of VEGF-C upon renal with tubulointerstitial fibrosis and me- injury,77,85 although the relative con- Lymphvasculogenesis in Renal Injury sangial expansion.75,76 However, the tributions of individual cell types to In addition to lymphangiogenesis, some accumulation of DCs, T and B lympho- lymphangiogenesis are unknown. Ex- evidence suggests that a small propor- cytes, and fibroblasts along with accom- pression of VEGF-D is increased in kid- tion of LECs arise from differentiation panying tubulointerstitial fibrosis in ney lysates from a mouse model of of tissue-resident or circulating progen- rodent or human CKD still occurs de- UUO70 and immunostaining demon- itors in a process termed lymphvasculo- spite a lymphangiogenic response.36,64,77 strates injured tubular epithelium as a genesis.9–11 In sex-mismatched renal An explanation for this may come from potential cellular source in cisplatin- transplants, in which male recipients studies from other organs which indicate induced nephrotoxicity and ischemia- received female donor kidneys, immu- that lymphangiogenesis, when occur- reperfusion injury (IRI) in mice.74 nohistochemical analysis demon- 1 1 ring as a response to chronic inflamma- Induction of VEGF-D in the tubular strated that 4.5% of PROX1 PDPN tion, results in leaky vessels with a epithelium of otherwise healthy adult lymphatics contained a single Y chro- reduced capacity for clearance.78 Thus, mice resulted in a four-fold expansion mosome, indicating a host-derived con- therapies to target lymphangiogenic of the mean cross-sectional area of tribution to graft lymphatics.96 From pathways hold potential for restoring kidney lymphatics, demonstrating the these experiments, it was proposed that

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Figure 2. Lymphatic expansion in chronic renal injury and its targeting using prolymphangiogenic therapies. In mouse models of chronic renal injury and in human CKD, lymphangiogenesis (the expansion of lymphatics via proliferation and sprouting of existing lymphatic endothelium) occurs and is considered the predominant mechanism of lymphatic expansion in disease. A number of cell types in the inflammatory milieu including injured tubular epithelium, activated T and B lymphocytes, neutrophils, DCs and activated fibroblasts secrete growth factors (VEGF-C, VEGF-D, CTGF) and inflammatory mediators (lymphotoxin-a [LT-a], TNF-a, TGF-b) which act directly or

1182 JASN JASN 31: 1178–1190, 2020 www.jasn.org REVIEW bone marrow–derived macrophages can while reducing renal macrophage and T these results suggest that augmentation, transdifferentiate into lymphatic endo- lymphocyte or DC accumulation. This rather than inhibition, of lymphangio- thelium in inflammatory contexts.97 led to an decrease in systolic BP in both genesis is beneficial in CKD. models,100 but the effect of VEGF-D In another study,64 the kidneys of In contrast, a study performing para- 1 overexpression on fibrotic remodeling mice with LYVE-1 cells ablated and biosis between green fluorescent protein in the hypertensive kidney was not ex- then subjected to either UUO or IRI har- (GFP) transgenic and wild-type mice or fi adoptive transfer of GFP-expressing plored. Moreover, a servo-control tech- bored lower numbers of in ltrating DCs, nique to maintain renal perfusion pres- T and B lymphocytes, macrophages, bone marrow in murine UUO64 showed sure was not applied, so it is not clear neutrophils, natural killer cells and genes low levels of co-expression of GFP and 1 whether the mechanism of injury arises encoding for inflammatory cytokines LYVE-1 lymphatics occurred, although as a direct consequence of nitro-L-argi- and those associated with renal fibrosis this was not quantified. It is not clear nine methyl ester on the kidney or indi- 7 days after injury. Reduction of the in- whether the efficiency of GFP or time rectly from hypertension. Another study flammatory milieu was also observed in point of the experiment contributed to used mice overexpressing VEGF-C from UUO and IRI mice administered soluble the difference between the above studies. More extensive lineage tracing is re- podocytes in streptozocin-induced LYVE-1 or VEGFR-3 fusion proteins. DKD. Podocyte VEGF-C overexpres- The anti-inflammatory and antifibrotic quired to validate these findings but, un- 1 sion significantly reduced the effects of LYVE-1 cell ablation and fu- til then, a myeloid origin of LECs during hallmarks of early DKD, including al- sion protein delivery were attributed to renal injury cannot be ruled out. buminuria, mesangial expansion, and inhibition of lymphangiogenesis, be- Targeting Lymphangiogenesis in decreased glomerular collagen deposi- cause both strategies reduced the density 1 Chronic Renal Injury tion.101 This effect was attributed to and decreased proliferation of LYVE-1 Several strategies have been implemen- restoration of glomerular endothelial lymphatics in diseased kidneys. To what ted to augment renal lymphangiogenesis barrier function, but the authors did extent these strategies target non-LECs in preclinical studies (Table 2). Daily in- not examine enhanced renal lymphan- or cells outside the kidney to exert their traperitoneal administration of a re- giogenesis as a potential cause. beneficial effects is unclear. combinant isoform of VEGF-C protein Two other rodent studies explored (VEGF-C156S), which binds preferen- the hypothesis that inhibition of lym- Contribution of Lymphatics to tially to VEGFR-3 over VEGFR-298,led phangiogenesis might be beneficial in Adaptive Immunity in Renal to an expansion of the periarterial renal CKD. Rats, which had adriamycin deliv- Transplant Rejection lymphatic network but not blood micro- ered intravenously to trigger protein- A lymphangiogenic response has been vasculature in murine UUO. VEGF- uria, were treated with a monoclonal documented in rejected human renal al- C156S also attenuated collagen I/III anti-VEGFR3 antibody (IMC-3C5)102 lografts83,96,103–105 and rodent models deposition, reduced proinflammatory from 6 weeks after induction of ne- of transplant nephropathy.106–108 In macrophage number, and lowered total phropathy. At 12 weeks of follow-up, this context, lymphangiogenesis is asso- TGF-b1 in UUO kidneys compared with treatment with IMC-3C5 significantly ciated with lymphocyte-rich infiltrates untreated controls.99 To what extent reduced the mean cortical lymphatic and correlates with allograft fibrosis VEGF-C156S may exert these beneficial vessel number in both healthy and and impaired graft function.83 A body 1 effects through VEGFR-3 blood endo- adriamycin-treated kidneys, without al- of evidence points toward a fundamen- thelial cells in the kidney27,30 is unclear. tering leukocyte count, collagen deposi- tal role for lymphatics in the generation Another strategy to augment lym- tion, or interstitial fibrosis in the injured and maintenance of adaptive immune phangiogenesis in CKD has been the ec- kidneys. Although the authors con- responses in renal transplants. Across topic expression of pro-lymphangiogenic cluded tubulointerstitial inflammation organs, LECs maintain peripheral toler- growth factors in transgenic mice. In and fibrosis to be independent of lymphan- ance by actively modulating immune adult mice with either salt-sensitive or giogenesis in adriamycin nephropathy, the cell function, including T and B lym- nitro-L-arginine methyl ester–induced hy- late onset of treatment may influence the phocytes and DCs, by activating or in- pertension, tubular VEGF-D overexpression efficacy of IMC-3C5. Nevertheless, when hibiting their maturation, driving the increased cortical lymphatic density interpreted in light of above studies, proliferation or apoptosis of these cells, indirectly on lymphatic endothelium to support lymphangiogenesis. The boxes indicates other possible factors which have been implicated in lymphangiogenesis in other organs but have not been explored in the context of renal injury. Some studies suggest that lymphvasculogenesis; the transdifferentiation of other cell types such as macrophages, into LECs and their integration into lymphatic vessels, as an alternative mechanism of lymphatic expansion in chronic renal injury. The premise of prolymphangiogenic therapies, such as growth factors or genetic approaches in mice, is to augment the expansion of lymphatics to increase the clearance of the activated immune cells. A number of studies show that this alleviates renal inflammatory and reduces fibrotic remodeling in the kidney. ANGPT1/2, angiopoietin 1/2; FGF2, fibroblast growth factor 2; MMP, matrix metalloproteinase.

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Table 2. Preclinical strategies targeting lymphangiogenesis in chronic renal injury Effect of Strategy on Model of Strategy Mechanism of Action and Model Effect on Renal Lymphatics CKD VEGFC-156S Mutated form of VEGF-C and Increase in cross-sectional area of Reduction in fibrotic remodeling 1 selective agonist of VEGFR-3 to LYVE-1 total and perivascular (Sirius red stain) promote lymphangiogenesis.98 renal lymphatics compared with Reduction in collagen I deposition 10 mg/d delivered IP over 14 d in control (Western blot) murine UUO99 Reduction in infiltrating macrophages (IHC) Reduction in TGF-b1 expression (Western blot) Ksp-rtTA;TRE-Vegfd mice Transgenic mice with doxycycline- Increase in cross-sectional area of Reduction in infiltrating 1 dependent overexpression of LYVE-1 cortical renal macrophage and T lymphocytes VEGF-D from tubular epithelium lymphatics and branches per with high-salt diet (flow to promote lymphangiogenesis. artery compared with control cytometry) Tested in L-NAME–dependent Reduction in infiltrating hypertensive nephropathy for 2 wk macrophage and DCs without with or without 3 wk high-salt high-salt diet (flow cytometry) diet100 Pod-rtTA;TRE-Vegfc mice Transgenic mice with doxycycline- Effects attributed to glomerular Reduction in urinary albumin- dependent overexpression of VEGF activity, so renal creatinine ratio (ELISA) VEGF-C from podocytes. Diabetic lymphatics were not Reduction in mesangial matrix nephropathy induced using investigated expansion (histology) 50 mg/kg STZ per day for 5 d.101 Reduction in collagen I (Sirius red Doxycycline either given before or stain) 4 wk after STZ injection IMC-3C5 Anti–VEGFR-3 antibody delivered to Reduction in cross-sectional area No significant reduction in 1 inhibit lymphangiogenesis in rats of PDPN cortical lymphatics in infiltrating macrophage or T with adriamycin nephropathy.102 both adriamycin-treated and lymphocytes (IHC) At 6 wk after adriamycin treatment, non-adriamycin-treated kidneys No significant reduction in 40 mg/kg body wt of IMC-C35 compared with controls tubulointerstitial fibrosis delivered three times per wk IP (histology) No significant reduction in collagen I (IHC and qPCR) Lyve1-Cre;R26R-DTR mice Transgenic mice expression DTR in Significant reduction in cross- Reduction in DCs, macrophages, T 1 1 LYVE-1 cells and their progeny. sectional area of LYVE-1 and B lymphocytes, neutrophils, Tested in murine UUO and IRI with lymphatic vessels assessed at and NK cells in UUO (flow 7 d follow-up after a single IP dose 3 d after DT administration cytometry) of 1.25 ng/kg body wt DT64 Reduction of inflammatory cytokines in UUO (qPCR) Reduction in fibrosis (qPCR, IHC, and Sirius red) LYVE-1 or VEGFR-3 soluble fusion Soluble LYVE-1 or VEGFR-3 proteins Significant reduction in cross- Reduction in DCs and T 1 proteins hypothesized to inhibit sectional area of LYVE-1 lymphocytes in UUO kidneys lymphangiogenesis through lymphatic vessels assessed at (flow cytometry) sequestering lymphangiogenic 7 d after UUO surgery Reduction of inflammatory growth factors. Injected via tail cytokines in UUO (qPCR) vein before or after UUO or IRI Reduction in fibrosis (qPCR, IHC, induction in mice64 and Sirius red)

IP, intraperitoneal; IHC, immunohistochemistry; L-NAME, nitro- L -arginine methyl ester; STZ, streptozocin; qPCR, quantitative PCR; DTR, diphtheria toxin receptor; DT, diphtheria toxin; NK, natural killer. generating chemokine gradients for their Injured cells within the tubulointersti- in acute allograft rejection in humans.103 1 chemotaxis, or controlling their trafﬁck- tium in renal allograft rejection may guide Abundance of CXCR7 lymphatics corre- ing and efﬂux through lymph.4 In differ- the sprouting of lymphatic endothelium. late with the serum creatinine level in ent contexts, it has emerged that LECs One potential candidate is C-X-C motif these patients. At the same time, the may themselves archive soluble antigen chemokine receptor 7 (CXCR7), which mRNA level of the CXCR7 ligands, C-X- for presentation to DCs.109,110 is upregulated on kidney lymphatic vessels C motif chemokine ligand type 11

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(CXCL11) and CXCL12, was significantly modulate immune tolerance in renal crosstalk.123 However, polycystin 1 and 2 higher in the tubulointerstitium of hu- transplantation by mobilizing antigen- —encoded by PKD1 and PKD2, respec- man renal allografts with borderline le- presenting cells through lymphatics tively—are also expressed within lym- sions or undergoing acute rejection. The and toward lymph nodes (Figure 3). phatics.124 Zebrafish with a loss-of- cellular sources of CXCL12 and CXCL11 Therapies to inhibit lymphangiogen- function mutation in the pkd1a gene were not identified in this study but, in esis may represent a strategy to improve (lyc1), a duplicate gene encoding poly- renal ischemic injury, injured tubular ep- the outcome and survival of renal allo- cystin 1, have lymphatic defects where ithelium was shown to secrete grafts. To our knowledge, the targeting the main axial lymphatic vessel fails to CXCL12.111 CXCL12 results in dose- of VEGFR-3/VEGF-C or other canonical form during development. In mice, dependent increases in migration of mu- lymphangiogenic pathways have not knockout of Pkd1 or Pkd2 results in rine and human LECs in vitro and pro- been tested in animal models of renal blood-filled lymph sacs, severe edema motes tube formation in the latter.112 transplant, although it has been shown in the absence of structural heart defects, Thus, chemokines such as CXCL12, gen- to be beneficial in cardiac graft survival hemorrhaging, cutaneous lymphatic ves- erated by inflamed regions, may guide in rat.120 It is emerging that existing clin- sel defects, and early lethality.124 The lymphangiogenesis toward sites of injury ical therapies that promote renal trans- lymphatic defects in the skin were repli- in rejecting allografts (Figure 3). plant survival, such as sirolimus (an cated in mice with conditional knockout inhibitor of mammalian target of rapa- of Pkd1 using an endothelial Sox18- fl T2 Increasing the ef ux of immune cells mycin), may exert their effects directly CreER mouse line.125 Small interfering from renal allografts is associated with on LECs by inhibiting lymphangiogene- RNA–mediated knockdown of PKD1 or poorgraftfunctioninratrenaltrans- sis.121,122 Another therapeutic strategy PKD2 in human LECs results in loss of plant.113 One factor responsible for im- 1 1 tested in rat renal allografts is inhibition cell number, filopodial abnormalities, mune cell mobilization is CD9 CD63 of Rho-associated protein kinase disorganized adherens junctional com- exosome-rich endothelial vesicles, (ROCK). By treating transplant- plexes, and impaired capillary network fi which are identi ed surrounding lym- recipient rats with lysozymes conjugated formation and cell migration in wound- phatic vessels in chronic allograft ne- to a ROCK inhibitor (Y27632), glomer- healing scratch assays. In this model and phropathy. Exposure of cultured LECs ular and tubulointerstitial macrophage also in Pkd1-null mice, the orientation of a to TNF- , which is released from dam- influx was decreased at 1 and 4 days the Golgi apparatus in individual LECs 86,87 aged renal cells, increases the che- post-transplantation, respectively, ac- was found to be randomized. Together, mokine and growth factor content of companied by a significant decrease in these findings suggest that the polycys- exosome-rich endothelial vesicles lymphatic vessel abundance. However, tins cell-autonomously regulate LEC ori- (EEVs). These EEVs promote the trans- BP and proteins associated with fibrosis, entation and migration and are required migration of human DCs across LECs. A including vimentin and procollagen- for normal lymphatic development. a key chemokine supporting this process, 1 1, did not significantly change upon Using whole-mount immunofluores- a released by LECs stimulated by TNF- , Y27632 treatment.106 Whether inhibi- cence, optical clearing, and high-resolution is chemokine (C-C motif) ligand 21 tion of mammalian target of rapamycin 3D imaging,16,126 we examined the kid- (CCL21).114,115 CCL21 encourages che- or ROCK directly exert antilymphangio- ney lymphatics in mice homozygous for RC motaxis of DCs toward lymphatic ves- genic effects, or whether lymphatic a p.R3277C allele (Pkd1 ), a slow pro- sels,116 their transmigration across abundance decreases as a secondary con- gressing model of autosomal dominant LECs114 and the egress of DCs through sequence of immunosuppression, is PKD. We found that lymphatic vessels lymphatics and toward lymph nodes.117 not clear. and corticomedullary cysts in this In human chronic renal allograft rejec- model sat in close proximity, suggestive tion, CCL21-secreting LECs are found in Polycystic Kidney Disease of fluid transport41 or molecular cross- close association with nodular infil- The hallmark of polycystic kidney dis- talk between LECs and cyst epithelium. trates83 containing multiple leukocyte ease (PKD), the most common form of We found complex lymphatic defects in RC/RC subtypes expressing the CCL21 receptor, which is autosomal dominant PKD and homozygous Pkd1 mice, including chemokine (C-C motif) receptor 7,118 is caused by mutations in PKD1 or a stunting of the lymphatic network rel- including T and B lymphocytes and PKD2, is the formation and growth of ative to the volume of the kidney and a DCs. Increased expression of CCL21 is multiple epithelial fluid-filled cysts significant decrease in the diameter of associated with recurrent nephropathy within the kidney which drive inflam- large hilar lymphatics. The presence of in patients who have received renal mation and fibrosis, resulting in a pro- these defects at E18.5; an early time transplants independent of their age, gressive decline in renal function. Our pointofcystprogressioninthismouse sex, or expression levels of chemokine understanding of the progression of model of PKD, suggests that these de- (C-C motif) receptor 7 within the trans- PKD has largely focused on renal epithe- fects could arise directly due to loss of planted kidney.119 Thus, LEC-generated lial cell metabolism, fluid transport, Pkd1 in LECs, rather than secondarily EEVs and chemokine gradients likely survival and differentiation or molecular from structural changes to the kidney

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Figure 3. Context-specific lymphatic functions in transplant rejection and PKD. In chronically rejecting renal allografts from humans and rodent models, lymphatic vessels are found in close association with inflamed regions containing lymphocyte-rich infiltrates. Injured tubular epithelium and cells within infiltrates secrete guidance cues for lymphatic endothelium including CXCL11 and CXCL12 with cognate G protein–coupled receptors (CXCR4, CXCR7) expressed on lymphatic endothelium. Conversely, LECs secrete CCL21—apotent agent of chemotaxis for immune cell subtypes—and CD91 CD631 EEVs. EEVs contain immunomodulatory proteins (colony stimulating factor 1 [CSF-1]) and factors involves in leukocyte migration (CX3CL1, CCL2, CCL5). In the early stages of murine PKD, lymphatics are found in close association with cyst epithelium, potentially suggesting molecular crosstalk between lymphatic and cyst epithelium or the transport of cyst solute to lymph. Kidney lymphatics themselves are malformed, which may suggest that defects in the kidney lymphatic vasculature result in defective tissue fluid and immune cell clearance and contribute to cyst expansion and decline of renal function in PKD. due to cyst expansion and compression. FUTURE DIRECTIONS emerging approaches, such as antifi- In either case, defective lymphatic func- brotic drugs in CKD, immunosuppres- tion may contribute to cyst expansion There is a rapidly increasing body of ev- sive agents in renal transplant or through impaired clearance of cells or idence supporting a role for lymphatic epithelial-centric medication in PKD. tissue fluid (Figure 3). vessels across multiple forms of renal New advances are rapidly transform- disease; however, several questions re- ing our understanding of lymphatics in In a preclinical study, we targeted the main. Structural changes to renal lym- health and disease. Among these, the lymphatics in PKD by delivering re- phatics are not evident in murine or hu- concept of lymphatic heterogeneity— combinant VEGF-C intraperitoneally man AKI.36,74 However, the possibility that every organ possesses a unique lym- in two rapidly progressing mouse mod- that renal lymphatics are altered at the fi Pkd1nl/nl phatic vascular bed with organ-speci c els of PKD. In mice, which molecular or functional level in acute functions has not been approached in Pkd1 have hypomorphic alleles and re- nephropathy, and whether these can the kidney. We used 3D imaging to iden- 127 nal vascular malformation, VEGF-C be exploited to prevent the transition tify a population of highly dynamic LEC administration led to an expansion of from AKI to CKD, warrants further clusters present during mammalian re- 30 renal lymphatics. Additionally, study. The mechanisms of action of nal development.16 In other organs, VEGF-C restored the defective archi- fi 1 pro- or anti-lymphangiogenic factors these clusters represent tissue-speci c tecture of VEGFR-3 peritubular fi nl/nl to prevent brotic remodeling in pre- progenitors, which may impart molec- capillaries observed in Pkd1 mice. clinical models of CKD, whether ular and functional heterogeneity in the These changes were accompanied by through clearing inflammatory cells, adult lymphatic vasculature.9–11 An- reduced inflammation, decrease in modulating factors secreted by LECs, other emerging area, demonstrated by cyst size and normalization of the kid- effects on the blood vasculature recent studies in animal models and ney:body weight ratio. Our findings through VEGFR-2 activation, or conse- humans, is that lymphatics in skin 1 suggest targeting VEGFR-3 endothelium, quences on other cells in the fibrotic and muscle may be key players in the including lymphatics and lymphatic-like environment are yet to be elucidated. regulation of tissue fluid and sodium vessels, could reduce disease severity and Strategies to target lymphatics or mod- homeostasis,130,131 revealing an un- progressive decline in renal function ob- ulate lymph egress128,129 could be de- precedented relationship between ex- served in cystic renal disease. livered in synergy with pre-existing or trarenal lymphatics and kidney disease

1186 JASN JASN 31: 1178–1190, 2020 www.jasn.org REVIEW or salt-sensitive hypertension. Ulti- 2. Louveau A, Plog BA, Antila S, Alitalo K, development and cystic kidney disease. mately, for the renal lymphatic field to Nedergaard M, Kipnis J: Understanding the eLife 8: e48183, 2019 functions and relationships of the glym- 17. McMahon AP: Development of the mam- move forward, novel technologies to phatic system and meningeal lymphatics. malian kidney. Curr Top Dev Biol 117: 132,133 134–136 visualize, ablate, and geneti- JClinInvest127: 3210–3219, 2017 31–64, 2016 cally manipulate137 lymphatic endothe- 3. Titze J, Luft FC: Speculations on salt and the 18. Brunskill EW, Park JS, Chung E, Chen F, lium need to be translated to renal research genesis of arterial hypertension. Kidney Int Magella B, Potter SS: Single cell dissection – to advance our understanding of this un- 91: 1324 1335, 2017 of early kidney development: Multilineage 4. Card CM, Yu SS, Swartz MA: Emerging roles priming. Development 141: 3093–3101, derstudied system in renal development, of lymphatic endothelium in regulating 2014 physiology, and disease. adaptive immunity. JClinInvest124: 19. Munro DAD, Hohenstein P, Davies JA: Cy- 943–952, 2014 cles of vascular plexus formation within the 5. Betterman KL, Harvey NL: The lymphatic nephrogenic zone of the developing mouse vasculature: Development and role in kidney. Sci Rep 7: 3273, 2017 ACKNOWLEDGMENTS shaping immunity. Immunol Rev 271: 20. Daniel E, Azizoglu DB, Ryan AR, Walji TA, 276–292, 2016 Chaney CP, Sutton GI, et al.: Spatiotem- 6. Seeger H, Bonani M, Segerer S: The role of poral heterogeneity and patterning of de- The authors would like to acknowledge lymphatics in renal inflammation. Nephrol veloping renal blood vessels. Angiogenesis Professors Peter Scambler and Christiana Dial Transplant 27: 2634–2641, 2012 21: 617–634, 2018 Ruhrberg (University College London [UCL]), 7. Yazdani S, Navis G, Hillebrands J-L, van 21. Short KM, Combes AN, Lefevre J, Ju AL, Paul Riley (University of Oxford), Adrian Goor H, van den Born J: Lymphangio- Georgas KM, Lamberton T, et al.: Global fi Woolf (University of Manchester), and Nor- genesis in renal diseases: Passive bystander quanti cation of tissue dynamics in the de- or active participant? Expert Rev Mol Med veloping mouse kidney. Dev Cell 29: man Rosenblum (The Hospital for Sick Chil- 16: e15, 2014 188–202, 2014 dren) for useful discussions about this work. 8. Russell PS, Hong J, Windsor JA, Itkin M, 22. Combes AN, Phipson B, Lawlor KT, Dorison Mr. Jafree wrote the original draft of the Phillips ARJ: Renal lymphatics: Anatomy, A, Patrick R, Zappia L,et al.: Single cell manuscript and both authors reviewed the physiology, and clinical implications. Front analysis of the developing mouse kidney manuscript after which revisions were made Physiol 10: 251, 2019 provides deeper insight into marker gene 9. Ulvmar MH, Mäkinen T: Heterogeneity in expression and ligand-receptor crosstalk and approved by both authors. the lymphatic vascular system and its origin. [published correction appears in Develop- Cardiovasc Res 111: 310–321, 2016 ment 146: dev182162, 2019]. Development 10. Petrova TV, Koh GY: Organ-specificlym- 146: dev178673, 2019 DISCLOSURES phatic vasculature: From development to 23. Sequeira-Lopez MLS, Lin EE, Li M, Hu Y, pathophysiology. J Exp Med 215: 35–49, Sigmund CD, Gomez RA: The earliest 2018 metanephric arteriolar progenitors and Dr. Long reports a patent JP6261617B2 (agents 11. Wong BW, Zecchin A, García-Caballero M, their role in kidney vascular development. that induce lymphangiogenesis used in the treat- Carmeliet P: Emerging concepts in organ- Am J Physiol Regul Integr Comp Physiol ment of cystic kidney disease) issued January 2018. specific lymphatic vessels and metabolic 308: R138–R149, 2015 Mr. Jafree has nothing to disclose. regulation of lymphatic development. Dev 24. Caubit X, Lye CM, Martin E, Coré N, Long Cell 45: 289–301, 2018 DA,VolaC,etal.:Teashirt3isnecessaryfor 12. Lee H-W, Qin YX, Kim YM, Park EY, Hwang ureteral smooth muscle differentiation FUNDING JS, Huo GH, et al.: Expression of lymphatic downstream of SHH and BMP4. Develop- endothelium-specific hyaluronan receptor ment 135: 3301–3310, 2008 LYVE-1 in the developing mouse kidney. 25. Srinivasan RS, Escobedo N, Yang Y, Mr. Jafree and Dr. Long are jointly funded by Cell Tissue Res 343: 429–444, 2011 Interiano A, Dillard ME, Finkelstein D, et al.: UCL Great Ormond Street Institute of Child 13. Tanabe M, Shimizu A, Masuda Y, Kataoka The Prox1-Vegfr3 feedback loop maintains Health and the National Institute for Health M, Ishikawa A, Wakamatsu K, et al.: Devel- the identity and the number of lymphatic Research Great Ormond Street Hospital Biomed- opment of lymphatic vasculature and mor- endothelial cell progenitors. Genes Dev 28: ical Research Centre Child Health Research PhD phological characterization in rat kidney. 2175–2187, 2014 Studentship and Doctoral Training Support Clin Exp Nephrol 16: 833–842, 2012 26. Kim Y-M, Kim WY, Nam SA, Choi AR, Kim H, Fund,theUCLMB/PhDprogram,Kidney 14. Munro DAD, Hohenstein P, Coate TM, Kim YK, et al.: Role of Prox1 in the trans- Research UK Project (Paed_RP_10_2018) Davies JA: Refuting the hypothesis that forming ascending thin limb of henle’sloop and Innovation (IN_012_2019) grants, and semaphorin-3f/neuropilin-2 exclude blood during mouse kidney development. PLoS Rosetrees Trust small project grant PGS19-2/ vessels from the cap mesenchyme in One 10: e0127429, 2015 10174. Dr. Long’s laboratory is further funded the developing kidney. Dev Dyn 246: 27. Kenig-Kozlovsky Y, Scott RP, Onay T, by Medical Research Council grant MR/ 1047–1056, 2017 Carota IA, Thomson BR, Gil HJ, et al.: As- P018629/1, Diabetes UK grants 13/0004763 and 15. 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