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Ascending Vasa Recta Are /Tie2- Dependent Lymphatic-Like Vessels

Yael Kenig-Kozlovsky,1,2 Rizaldy P. Scott ,1,2 Tuncer Onay ,1,2 Isabel Anna Carota,1,2 Benjamin R. Thomson,1,2 Hyea Jin Gil,1,2 Veronica Ramirez,1,2 Shinji Yamaguchi,1,2 Christine E. Tanna,1,2 Stefan Heinen,3 Christine Wu,1,2 Radu V. Stan,4,5 Janet D. Klein,6 Jeff M. Sands ,6 Guillermo Oliver,1,2 and Susan E. Quaggin1,2

1Division of Nephrology and Hypertension and 2Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois; 3Sunnybrook Research Institute, Sunnybrook Hospital, Toronto, Ontario, Canada; 4Departments of Biochemistry and Cell Biology and 5Pathology, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire; and 6Division of Renal Medicine, Emory University School of Medicine, Atlanta, Georgia

ABSTRACT Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected BASIC RESEARCH from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the endothelial 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with nor- mal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, express- ing lymphatic markers (prospero homeobox 1 and vascular endothelial receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle–associated protein). Taken together, our data redefine the AVRs as Tie2 signaling– dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the counter- current exchange mechanism and the structural integrity of the renal medulla.

J Am Soc Nephrol 29: 1097–1107, 2018. doi: https://doi.org/10.1681/ASN.2017090962

Renalbloodflowaccounts for approximately 20% of renal medullary vasculature consists of fenestrated total cardiac output in humans, but the kidneys ascending vasa recta (AVR) and capillary plexus constitute ,1% of body mass. Not surprisingly, along with the nonfenestrated descending vasa the renal vasculature is one of the most complex among highly vascularized organs, and it supports the many activities of the kidneys that include Received September 5, 2017. Accepted November 7, 2017. plasma filtration, maintenance of fluid, electrolyte Published online ahead of print. Publication date available at and acid-base balance, and endocrine functions.1,2 www.jasn.org. The various vascular beds of the kidney differ in Correspondence: Dr. Susan E. Quaggin, Feinberg Cardiovascular expression of endothelial markers, presence or ab- Research Institute, Northwestern University, 303 East Superior sence of fenestrations, variable mural cell coverage, Street, Chicago, IL 60611. Email: [email protected] and architectural organization.3 In particular, the Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 1097–1107, 2018 ISSN : 1046-6673/2904-1097 1097 BASIC RESEARCH www.jasn.org recta (DVR).4 This vasculature plays a major role in maintain- Significance Statement ing hyperosmolarity of the medullary interstitium to facilitate fluid resorption and promote fluid recycling and homeostasis. The countercurrent multiplication mechanism in the kidney medulla The proximity of the AVR and DVR is believed to be central to underlies the exquisite regulation of urine concentration and facil- fl the countercurrent exchange mechanism, because it enables itates water and electrolyte excretion to maintain uid homeostasis. The medullary interstitium is, therefore, an important site where transport of solute and water while preventing washout of the interstitial fluid accumulates and is returned to the systemic circu- osmotic gradient in the inner medullary interstitium, which is lation. We discovered that the ascending vasa recta (AVRs) are needed for maximal urine concentration ability.2,5,6 To date, specialized hybrid vessels, having properties of both blood and studies with different transgenic mouse lines with knockout of lymphatic vessels. Attenuation of angiopoietin/Tie2 signaling spe- fi various transporters and channels have confirmed the impor- ci cally leads to loss of the AVR, causing dilution and increased output of urine, and the formation of interstitial cysts in the medulla. tance of the nephrons and DVR for urinary concentrating Our findings provide formal genetic proof that the AVR with its novel capacity.7 However, no such results have been reported for lymphatic-like features is key for fluid drainage in the medulla, a the AVR. region devoid of classic lymphatic vasculature. Despite the critical roles of renal vasculature, very little is known about how most renal vascular beds develop and dif- RESULTS ferentiate. This major knowledge gap must be filled to permit development of targeted vascular therapies and accelerate de- Lineage Tracing of Tie2-Expressing Cells velopment of vascularized organoids and bioengineered kid- To better understand the importance of angiopoietin-Tie2 sig- neys. To date, genetic knockout studies in mice have led to the naling in the kidney, we first assessed the renal pattern of Tie2 identification of cytokines and growth factors, which are rel- promoter activity. Without a suitable Tie2 antibody for tissue evant to renal vascular development, including the .8 immunolabeling,wegeneticallylabeledcellsderivedfromtheTie2- The angiopoietin ligands Angpt1 and Angpt2 and their cog- expressing lineage by intercrossing Tie2-Cre and reporter trans- nate endothelial mTmG lacZ genicmice(Rosa26 and Rosa26 ,whichresultsinenhanced (Tie2)/Tek are essential for the development and homeostasis green fluorescent protein (EGFP) and b-galactosidase expression 9,10 of blood and lymphatic vasculature. Angpt1-mediated ac- Tg/+ mTmG/+ in the presence of Cre activity). At P1, Tie2-Cre :R26 tivation of Tie2 supports endothelial cell proliferation, sur- hemizygotes show EGFP expression pattern consistent with the vival, cell-cell junction formation, and regulation of mural distribution of glomerular and peritubular capillaries (Figure 1, cell recruitment, thereby promoting blood vessel quies- A–C). EGFP colocalizes with most, if not all, Emcn-expressing cence.11–13 The specific role of Angpt2 is cell context depen- vessels in both the cortex and the medulla (Supplemental Figure dent. In the blood vasculature, Angpt2 can antagonize Tie2 Tg/+: lacZ/+ 1). In adult Tie2-Cre R26 mice, b-galactosidase enzy- activation, promoting permeability, sprouting, destabiliza- matic activity reveals a pattern of Tie2-expressing cell derivatives tion, and regression of blood vessels.14–19 In contrast, during that closely recapitulates the microvascular organization of the lymphatic development, Angpt2 serves as the major endoge- kidney, including prominent staining in glomeruli and medullary nous Tie2 agonist.20,21 In the renal vasculature, deletion of vascular bundles (Figure 1D). These findings confirm that Tie2- Angpt1 at embryonic day E10.5 leads to partial defects in expressing cells give rise to these specialized renal vascular beds. the specialized capillary bed of the glomerulus, with dilated capillary loops and disorganization of the glomerular base- ment membrane observed at E17.5.22 Angpt2 deletion leads Late Gestational Deletion of ANGPT-TIE2 Signaling to disorganization of the cortical peritubular capillaries.23 In Leads to Renal Cysts the anterior chamber of the eye, both angiopoietin ligands Using a Tet-inducible Cre recombination strategy, we inacti- have been shown to cooperate in activation of Tie2 signaling, vated Angpt1, Angpt2, and Tie2 at late gestation (E16.5) in which is required for formation of Schlemm canal (SC), an mice. This overcomes the early lethality of constitutive loss of unusual specialized hybrid vessel with both blood vessel and these and allows us to study their relevance in kidney lymphatic-like characteristics.24 development and function. Homozygous conditional knock- Using a variety of newly generated transgenic mouse lines, out mutants are bornwithin expected Mendelian ratios and live we show that loss of both Angpt1 and Angpt2 or the receptor upuntiladulthood. They are referredtofromhereon as follows: D D Tie2 at embryonic day E16.5 leads to a cystic renal phenotype Angpt1 knockout (A1 E16.5), Angpt2 knockout (A2 E16.5), D and urinary concentrating defect, the result of a dramatic loss Angpt1/Angpt2 double knockout (A1/A2 E16.5), and Tek/Tie2 D of AVR. Furthermore, we show that most, if not all, AVRs have a knockout (Tie2 E16.5). Other genotypes were classified as con- D unique molecular phenotype characterized by lymphatic and trols and did not manifest overt renal phenotypes. A1/A2 E16.5 blood venous molecular markers. These findings show that the mutant mice have 92% reduction in Angpt1 and 84.5% re- AVR represent hybrid vessels with lymphatic-like features that duction in Angpt2 mRNA transcript levels in the kidneys com- are needed to perform the crucial role of interstitial fluid pared with control littermates as determined by quantitative drainage in the medulla, a region devoid of classic lymphatic RT-PCR analysis (Supplemental Figure 2, A and B). Tie2 protein D vasculature. levels in the kidneys were diminished 66.8% in Tie2 E16.5 mutant

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the cystic lesions obscure. The cysts are typically lined with flattened cells, which in adult mutants, show increased matrix deposition (Figure 2, K and L). Cellular infiltration, likely from inflammatory macrophages, and erythrocyte-filled cysts are occasionally seen in severely cystic mutant kidneys (Figure 2M). In contrast, singular D D A1 E16.5 or A2 E16.5 mutants do not develop renal cysts, indicating that a compound loss of Angpt1 and Angpt2 is necessary to mitigate cyst formation.

Renal Cysts Express Myofibroblast but Not Epithelial or Endothelial Markers We performed multiple marker analyses through antibody and lectin staining of kidney sections to characterize the renal cysts D D found in A1/A2 E16.5 and Tie2 E16.5 mutants (Figure 3A). The cysts lack expression of multiple epithelial tubule markers: Na+/K+-ATPase, Lotus tetraglobulus lectin for proximal tubules, uromodulin for the thick ascending limb of the loop of Henle, Ksp-cadherin for distal tubules, and aquaporin-2 and pancy- tokeratin for collecting ducts. The cysts also do not express the Figure 1. Lineage-tracing analysis reveals Tie2 expression pat- common blood endothelial cell markers Emcn, Cd34, and tern in the renal microvasculature. (A) Cells with prior or current Pecam1. Podxl, normally expressed by peritubular capillaries Tie2 promoter activity observed as EGFP fluorescence in a P1 and large caliber renal blood vessels, and Plvap/PV1, which is ex- animal doubly hemizygous for the transgenes Tie2-Cre and pressed in diaphragm-containing capillaries, were also negative Rosa26mTmG (R26mTmG). (B) Higher magnification pattern of Tie2 in the renal cysts. The lymphatic endothelial marker Lyve1 is promoter activity in a P1 kidney showing EGFP expression in glo- also absent in the cysts. In contrast, several myofibroblast meruli (white arrowheads) and peritubular capillaries within the markers strongly and uniformly stain the cysts, including cortex and medulla. (C) LacZ histochemical staining of a vibratome- a-smooth muscle actin (Acta2), calponin-1 (Cnn1), SM22a/ sectioned adult kidney from a mouse hemizygous for both Tie2-Cre transgelin (Tagln), vimentin (Vim), desmin, and PDGFRb lacZ R26lacZ D and Rosa26 ( ) transgenes showing Tie2 promoter activity receptor. Microcysts in younger (P2–P5) A1/A2 E16.5 and consistent with the renal vascular network. (D) Cortical and outer D Tie2 E16.5 mutant animals also show prominent expression medullary lacZ staining pattern in an adult kidney showing prior fi or current Tie2-Cre activity in glomeruli, cortical peritubular of the same myo broblast markers but do not show prominent capillaries, medullary capillary plexus, and medullary vascular expression of epithelial or endothelial markers. DE16.5 bundles (black arrowheads). Scale bars, 100 mm. Ultrastructural imaging of the renal cysts in Tie2 mu- tant animals reveals that the cysts are lined by nonciliated mice compared with kidneys from their control cohorts (Supple- squamous cells (Figure 3, B–D). These flattened cells are rela- D mental Figure 2, C and D). Adult (8–10 weeks old) A1/A2 E16.5 tively smooth and lack luminal villous brush border–like projec- D and Tie2 E16.5 mutant mice compared with control litter- tions. The presence of fibrillar matrix directly underneath these mates have 49.5% and 26.9% reduction, respectively, in GFR, an spindle-shaped cyst wall cells is evident in transmission electron D index of diminished renal function (Figure 2, A and B). However, micrographs of Tie2 E16.5 mutant kidneys (Figure 3D). D D neither A1/A2 E16.5 nor Tie2 E16.5 mutants show overt proteinuria and have insignificant urinary albumin-to-creatinine ratios relative Angpt1/2-Tie2 Signaling Affects Patterning of Renal to controls, despite having markedly reduced GFR (Supplemental Vasculature D D Figure 3, A and B). Consistently, A1/A2 E16.5 and Tie2 E16.5 Serial sectioning of postnatal P5 kidneys followed by staining mutants have normal glomerular histology and ultrastructure for Emcn and L. tetraglobulus lectin (to demarcate the cortex (Supplemental Figure 3, C and D). from the medulla) reveals that Emcn+ve vessels are notably D D D Unexpectedly, kidneys dissected from adult A1/A2 E16.5 sparse in A1/A2 E16.5 and Tie2 E16.5 but are not notably sparse D D D and Tie2 E16.5 mutant animals were grossly cystic (Figure 2, in singular mutants A1 E16.5 or A2 E16.5, particularly within C–F). Histologic analysis reveals the presence of multiple mac- the outer medulla (Figure 4A). A striking paucity of Emcn+ve D D rocysts in 8- to 10-week-old A1/A2 E16.5 and Tie2 E16.5 mu- outer medullary vascular bundles is observed in P14 kidneys D D tants (Figure 2, G–I). As early as postnatal age P2, microcysts from both A1/A2 E16.5 and Tie2 E16.5 kidneys (Figure 4B). starttoemergewithvariablepenetrancewithintherenalouter Quantification of Emcn-stained areas relative to total tissue medulla, although these medullary microcysts become clearly area indicates that overall vascular density is reduced by 38.3% D D D D prominentatageP10inkidneysfromA1/A2 E16.5 and Tie2 E16.5 and 26.9% in A1/A2 E16.5 and Tie2 E16.5 relative to control mutants (Figure 2J). However, in adult mutant mice, huge cysts littermates, respectively (Figure 4, C and D). More pro- have distorted the renal parenchyma, making the exact location of nounced microvascular rarefactionisobservedwithinthe

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D Figure 2. Renal cysts develop in the absence of Angpt1/2-Tie2 signaling. (A) Reduced GFR in A1/A2 E16.5 mutants. (B) Reduced GFR in D Tie2 E16.5 mutants. Asterisks in A and B denote statistical significance at P,0.05. Gross kidney appearances in adult mice showing renal cysts in D D (DandF)compoundAngpt1/2(A1/A2 E16.5)andTie2(Tie2 E16.5) knockout mutants in comparison with (C and E) their control littermates, D respectively. (G–I) Stitched images of hematoxylin and eosin histology of adult kidneys showing multiple cysts (asterisks) in A1/A2 E16.5 and D D Tie2 E16.5 mutants. (J) Medullary microcyst (asterisks) development in a P10 Tie2 E16.5 kidney. (K) Higher magnification image showing squa- D mous cells lining renal cysts. (L) Van Gieson staining reveals collagen matrix accumulation around cysts (shown from a representative A1/A2 E16.5 D kidney). (M) Occasional hemorrhage in renal cysts (yellow asterisk) shown from a representative of hematoxylin and eosin–stained A1/A2 E16.5 kidney. Specimen ages: 10 weeks in A, B, and E–I;20weeksinC,D,andK–M; and P10 in J. Scale bars, 100 mminJ–M. medulla, where Emcn+ve-stained areas are reduced by 59.6% vascular bundles is due to specific loss of the AVR. Angpt1/2- D D and 45.4% in A1/A2 E16.5 and Tie2 E16.5 mutants, respec- Tie2 signaling is, therefore, essential for the establishment of tively, compared with controls (Figure 4, C and E). the AVR and the interbundle capillary plexus. We then stained kidney sections with Plvap, an integral protein of fenestral diaphragms found in AVR and interbundle Urine Concentrating Ability Is Reduced in the Absence capillary plexus.25 Consistently, as seen after Emcn staining, of Angpt1/2-Tie2 Signaling Plvap+ve vessels are scant in the outer medulla of kidneys from The AVR, DVR, and medullary capillary plexus play important D D both A1/A2 E16.5 and Tie2 E16.5 in P14 pups (Figure 4, F and roles in fluid recycling and concentration of urine. The pro- G). Plvap staining reveals that vessel rarefaction occurs within nounced simplification of the medullary capillary bed due to the medullary vascular bundles and interbundle capillary inactivation of Angpt1/2-Tie2 signaling, therefore, led us to plexus of mutant kidneys. In contrast, the abundance of UTB+ve suspect that fluid resorption through medullary vessels could D D DVR clusters is comparable between mutants and control be compromised. Indeed, A1/A2 E16.5 and Tie2 E16.5 mutant kidneys (Figure 4, F and G). Thus, the disappearance of mice kept in metabolic cages have increased urinary outputs

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Figure 3. Renal cysts arising from loss of Angpt1/2-Tie signaling uniformly express myofibroblast markers. (A) Representative im- D munofluorescence analysis of renal cysts (yellow asterisks) found adult A1/A2 E16.5mutants showing lack of expression of epithelial tubule–specific(L. tetraglobulus lectin [LTL], Na+/K+-ATPase [Atp1b1], uromodulin [Umod], Ksp-cadherin [Cdh16], aquaporin-2 [Aqp2], and pancytokeratin [PCK]) and endothelial (Emcn, Cd34, Pecam1, Plvap, Podxl, and Lyve1) markers but strong expression of myofi- broblast markers (Acta2, Cnn1, Tagln, Vim, desmin [Des], and PDGFRb receptor [Pdgfrb]). (B and C) Scanning electron and (D) D transmission electron micrographs of renal cysts (yellow asterisks) in an adult Tie2 E16.5 kidney showing the squamous mesenchymal morphology of cells lining cysts (nuclei labeled m). Fibrillar deposits are also visible underneath the cyst linings (arrows). Scale bars, 100 mminA;500mminB;200mminC;10mminD.

(2.7- and 3.9-fold higher compared with controls, respec- that the AVRs were genetically labeled with Prox1. Prox1 re- tively) within a 24-hour period (Figure 5, A and B). Addition- porter activity was found coexpressed with Pecam1, CD34, and D D ally, urine obtained from A1/A2 E16.5 and Tie2 E16.5 mutant Emcn in vascular bundles in the outer medulla of control an- mice is significantly more dilute relative to that of controls on imals (Figure 6, A–C). More importantly, these bundled vessels the basis of osmolality measurements (approximately 52% and expressing both PV1 and Prox1 promoter activity are distinct from 2 2 66% of controls, respectively) (Figure 5, C and D). These find- nearby Prox ve/PV1 ve/UTB+ve DVR (Figure 6D). These Prox+ve ings, therefore, strongly implicate Angpt1/2-Tie2 signaling in vessels, therefore, are identical to the AVRs. Unlike the capil- indirect regulation of urine concentration and fluid homeostasis. lary plexus, VEGFR2 expression is absent in these Prox1+ve AVR vessels (Figure 6E). Instead, expression of promoter ac- AVRs Atypically Express Lymphatic Markers Prox1 and tivities for Prox1 and VEGFR3 (markers strongly expressed in VEGFR3 and Depend on Angpt1/2-Tie Signaling for lymphatic vessels28) colocalizes in the AVR (Figure 6F). How- Development ever, the AVRs lack expression of other canonical lymphatic On the basis of previous studies from our laboratory, where we markers, such Lyve1 and podoplanin (Pdpn)30,31 (Supplemen- identifiedakeyroleofangiopoietin-Tie2signalingintheformation tal Figure 4). Interestingly, these Prox+ve medullary vascular D of lymphatic-like SC in the eye,24 we wondered if any of the vessels bundles are dramatically lost in kidneys from Tie2 E16.5 mu- lost in the kidney also exhibited lymphatic or hybrid endothelial tants (Figure 6G). In contrast, Lyve1+ve/Pdpn+ve arcuate lym- D characteristics. We incorporated Prox1 fluorescent (EGFP or phatic vessels remain present in kidneys from A1/A2 E16.5 and D tdTomato) transgenes into our Tie2 conditional genetic crosses Tie2 E16.5 mutants. Altogether, these findings indicate that the to assess potential restructuring of Prox1+ve lymphatic or hybrid AVRs are Tie2-dependent hybrid vessels with intermediate vessels. Similar to lymphatic vessels and the SC,26–29 we found properties of blood and lymphatic endothelial cells.

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Figure 4. AVRs are lost and renal vascular density is reduced upon attenuation of Angpt1/2-Tie2 signaling. (A) Sagittal sections of P5 kidneys doubly stained for Emcn and L. tetraglobulus lectin (LTL) showing attenuated vascular density in the absence of both Angpt1 and Angpt2 together and Tie2 but not in singular loss of Angpt1 or Angpt2. (B) Immunohistochemical staining for Emcn in adult kidneys showing persistence of renal D D vascular density reduction in A1/A2 E16.5 and Tie2 E16.5 mutants and notable loss of medullary vascular bundles (yellow arrowheads). cKO, ho- mozygous conditional knockout mutant. (C) Representative sagittal section of a P5 kidney indicating the demarcation (white dotted line) of cortical and medullary regions on the basis of LTL-stained proximal tubules for quantification of Emcn staining density. (D) Emcn staining area normalized D D to total tissue area in fully sectioned kidneys showing reduced total renal vascular densities in A1/A2 E16.5 and Tie E16.5 mutant compared with D D control littermates. (E) Emcn staining density indicating significant medullary vascular reduction in A1/A2 E16.5 and Tie E16.5 mutants relative to controls. (F) Plvap and UTB coimmunostaining of transverse kidney sections at the level of the outer medulla showing diminished Plvap-stained D D microvasculature in a representative kidney from an A1/A2 E16.5 mutant. AVR clusters but not the DVRs are lost in A1/A2 E16.5 mutants. (G) Di- D D minished Plvap-stained microvasculature in the outer medulla in representative kidney from a Tie E16.5 mutant. Similar to A1/A2 E16.5 mutants, D Tie E16.5 mutants have distinctive loss of AVRs but not the DVRs. (F and G) Vascular bundles formed by Plvap+ve AVR and UTB+ve DVR are en- circled. Scale bars: 200 mminA–C, F, upper panel, and G, upper panel; 50 mm in F, lower panel, and G, lower panel. Statistically significant means (P,0.05) are denoted by asterisks in D and E.

Angpt1/2-Tie Signaling Regulates the Patterning of ullary interstitium concomitant with the thinning of vascular D D the Renal Interstitium arrays in A1/A2 E16.5 and Tie2 E16.5 mutants (Figure 7, A and Histologic examination of late embryonic kidneys (E18.5) and B). We, therefore, inferred that loss of vascular bundles could postnatal P2 kidneys reveals a notable expansion of the med- have resulted in altered patterning of perivascular support

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conventional lymphatic vessels are absent in the renal medulla,32–34 although it has a great need for clearance of interstitial fluid. Instead of these vessels, the renal medulla is thought to rely on fenestrated AVRs and their connection to the arcuate veins for efficient removal of medullary intersti- tial fluid and preservation of the crucial medullary osmotic gradient.3,6,35–37 The specialized features of the AVR, such as abundant fenestrations, lack of mural cell coverage, and strategic organization into vascular bundles in the outer medulla, are believed to contribute to their high hy- draulic conductance.4,38–41 However, the molecular basis of development and differ- entiation of the AVR have not been report- ed previously. Furthermore, formal genetic proof has not been provided to show their DE16.5 DE16.5 Figure 5. A1/A2 and Tie2 mutants have urine concentration defects. Total role in renal medullary fluid removal. urine collected within a 24-hour period showing increased urine output from (A) A1/ D D This study shows that angiopoietin-Tie2 A2 E16.5 and (B) Tie2 E16.5 mutants. Osmolality measurements on urine samples D D signaling is vital for the normal develop- showing dilute urine production from (C) A1/A2 E16.5 and (D) Tie2 E16.5 mutants. ment of medullary microcirculation and Statistically significant means (P,0.05) are denoted by asterisks. in particular, the AVR. When genetically cells and the surrounding interstitium. We first analyzed the attenuated, angiopoietin-Tie2 signaling results in the absence renal expression of the mesenchymal marker Cnn1 during late of vascular bundles of the outer medulla and the rarefaction of gestation (E18.5) and perinatally (P1, P2, and P5). We found surrounding capillary plexus. Although lineage tracing studies that Cnn1 has markedly broader expression in all four time revealed that Tie2-expressing cells give rise to the majority, if D D points examined in both A1/A2 E16.5 and Tie2 E16.5 mutants not all, of the vessels within the medullary vascular bundles, the consistent with the histologic findings (Figure 7, C and D). AVRs but not the DVRs seem to be absent when Tie2 signaling Similar elevated and widened expression patterns are seen is lost at E16.5. This finding suggests a time-sensitive require- with other myofibroblast markers that we found expressed ment for Angpt-Tie2 signaling in AVR formation. in renal cysts, such as Acta2, Pdfgfrb, Tagln, and Vim (Figure Using different transgenic fluorescent reporter mice, we 7E). In kidneys of P1 pups, transcripts for Pdgfb were elevated identified expression in the AVR of the transcription factor 85% relative to controls (Figure 7F). Thus, loss of Angpt1/2- Prox1, a master regulator of lymphatic cell fate.26,27 Similarly, Tie2 signaling upregulates the expression of PDGFB, a growth characterization of reporter mice indicated that the AVRs also factor that likely promotes the hyperproliferation of coexpress VEGFR3, a tyrosine kinase receptor required for PDGFRb-expressing medullary interstitial or perivascular lymphangiogenesis.26,42,43 In addition to the expression of cells. Intriguingly, the same myofibroblast markers are also both Prox1 and VEGFR3, markers normally ascribed to lym- the ones that we found expressed by the squamous cells lining phatic vessels,28 the AVRs also express blood endothelial– D D the cysts of kidneys from A1/A2 E16.5 and Tie2 E16.5 mutants. specificmarkers(Emcn,CD34,Pecam1,andPlvap).Conversely, In adult control animals, the expressions of the myofibroblast the AVRs do not express other canonical lymphatic markers, markers Acta2, Cnn1, and Tagln are restricted to smooth mus- such as Lyve1 and Pdpn.30,31 Notably, VEGFR2, the expression cles covering large caliber blood vessels, whereas Vim expres- of which is typical of many blood vessels, including peritubu- sion in addition is also found strongly expressed in podocytes lar renal capillaries,44 is undetectable in the AVR. Thus, the (Supplemental Figure 5). AVRs seem to represent unusual hybrid vessels that carry mo- lecular features of both lymphatic and blood vascular endo- thelial cells. The expression of Prox1 and VEGFR3 in the AVR DISCUSSION together with their abundant fenestration help explain the relatively high hydraulic conductivity of the AVR and Excess interstitial fluid is generally removed by the lymphatic their role in the reuptake of large volumes of interstitial system and recirculated back into the systemic blood vascula- fluid4,38,39,41—features typically found in lymphatics. Prox1 ture. In the kidney, definitive lymphatic vessels surround hilar is known to regulate Vegfr3 expression positively,45 whereas renal arteries and cortical interlobular and arcuate arteries, and VEGFR3 receptor activity has been implicated in vascular re- they are found within the renal capsule.32,33 Paradoxically, modeling and widening of vessel diameters to accommodate

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Intriguingly, the development of the AVR and the SC depends on cooperative agonist functions of Angpt1 and Angpt2 on Tie2 activation.11,14,21,24 In contrast, with blood vascular development, Angpt1-dependent Tie2 activation is antagonized by Angpt2.11,14,21,24 The context-dependent requirement for Angpt1 and Angpt2 in AVR development, therefore, is consistent with its hybrid phenotype. As predicted, mice that lack AVRs ex- hibited reduced urinary concentrating ca- pacity. Unexpectedly, however, the mice also developed a dramatic cystic phenotype. Analysis of multiple markers indicated that the cysts are not derived from vascular or epithelial cells. Instead, the cysts are lined by squamous mesenchymal PDGFRb+ve cells that uniformly express multiple myo- fibroblast markers (Cnn1, Tagln, Acta2, and Vim). Interestingly, inactivation of angiopoietin-Tie2 signaling results in expansion of PDGFRb+ve and myofibroblast-like mesenchyme in the renal medulla. One plausible explanation for the increase in PDGFRb+ve medullary mesenchyme is the upregulation of the Pdgfb encod- ing the PDGFRb ligand PDGFB, which we D observed in Tie2 E16.5 mutant animals. Ex- cessive PDGFB levels in conjunction with increased interstitial fluid flow could also facilitate the differentiation of the intersti- tial mesenchyme into myofibroblasts.50,51 In venous malformations linked to hyper- activation of Tie2, perivascular support is diminished, whereas endothelial expression Figure 6. The AVR bundles lost on inactivation of Angpt1/2 signaling express Prox1 of Pdgfb is suppressed, a finding that indi- and Vegfr3. In Prox1-GFP reporter mice, vessels within medullary vascular bundles cates that Tie2 negatively regulates the ex- coexpress GFP with (A) Pecam1, (B) CD34, (C) Emcn, and (D) Plvap but not UTB. (E) pression of Pdgfb.52 The cysts progressively TdTomato and GFP expressions in Prox1-tdTomato (Prox1-tdT)/Vegfr2-GFP double- enlarge with age, suggesting fluid accumu- reporter mice do not overlap. Prox1-tdT+ve medullary vessels do not express GFP. lation. We posit that these cysts represent (F) In Prox1-tdT/Vegfr3-YFP double-reporter mice, YFP expression overlaps with fl tdTomato in medullary vascular bundles. (G) Prox1+ve AVRvesselsarelostinkidneysfrom collections of interstitial uid entrapped D fi Tie2 E16.5 mutants. Scale bars: 100mminA,D,F,andG;50mminB,C,andE. by myo broblast-like cell derivatives of the medullary interstitium or perivascular cells. The broadening of the medullary increased fluid flow.46,47 The activation of VEGFR3 by fluid interstitium with concomitant impaired circulatory drainage shear stress has been postulated to contribute to the regulation could contribute to altered fluid dynamics in the medulla and of vessel caliber.48 Indeed, the AVRs are approximately 60% subsequent fluid retention. The renal cysts are also reminiscent wider in diameter than the DVRs.36,40,41 of the dramatic buphthalmos (bulging of the eyes) that results The hybrid phenotypic and molecular characteristics of the from development failure of the SC and impaired aqueous humor AVR closely resemble those found in SC, a vessel that drains drainage due to loss-of-function mutations in angiopoietin-Tie2 back into the venous system the aqueous humor produced in signaling,24,53 providing additional parallels between function of the anterior chamber of the eye. Recently, the SC has been AVR and SC. described as a hybrid vessel expressing Prox1, VEGFR3, Overall, our data redefine the AVRs as hybrid vessels highly CD34, Emcn, and Plvap but not Lyve1 or Pdpn.29,49 analogous to SC in the eye with molecular features of both

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D D Figure 7. A1/A2 E16.5 and Tie2 E16.5 mutants have abnormal expansion of the renal interstitium. (A and B) Periodic acid–Schiff his- D D tology of E18.5 and P2 kidney sections showing similar expansion of medullary interstitium (red asterisks) in A1/A2 E16.5 and Tie2 E16.5 mutants. (C and D) Immunofluorescence staining for the interstitial marker Cnn1 showing expansion of the interstitium at E18.5, P1, P2, D D and P5 in A1/A2 E16.5 and Tie2 E16.5 mutants. (E) Immunofluorescence staining for other myofibroblast markers (Acta2, PDGFRb re- ceptor [Pdgfrb], Tagln, and Vim) notably expressed in the medullary interstitium as seen in P1 kidneys showing increased expression of D these markers in the kidneys of A1/A2 E16.5 mutants. (F) Renal expression of Pdgfb is increased in the absence of Tie2 signaling on the basis of quantitative RT-PCR analysis. Pdgfb relative to Pecam1 as normalized to controls from P1 kidneys. Statistically significant means (P,0.05) are denoted by an asterisk. Scale bars: 200 mminAandB;400mminC–E. lymphatic and blood endothelia. The angiopoietin-Tie2 sig- Prox1-EGFP and Prox1-tdTomato, Veg fr3-YFP,andVeg fr2-GFP re- naling pathway is essential to the development of these hybrid porter mice were used to identify Prox1+ve, Vegfr3+ve, and Vegfr2+ve vessels. Given the susceptibility of the medullary microcircu- vessels. Mouse genotyping was done by PCR. Mouse rearing, hus- lation to injury in diseases, such as ischemia-reperfusion bandry, and phenotyping were carried out following ethical procedures AKI,54 and the fact that Tie2 signaling can be targeted clini- approved by the Institutional Animal Care and Use Committee of cally (e.g., with recombinant angiopoietins or mimetics),55,56 Northwestern University. Additional methods are included in Supplemental our findings pave the way for the development of novel ther- Material. apeutic renoprotective strategies and suggest that kidney structure and function should be investigated in patients with glaucoma caused by mutations in the TEK/TIE2 gene.53 ACKNOWLEDGMENTS

We are grateful to the staff of the Center for Comparative Medicine of CONCISE METHODS NorthwesternUniversityforassistanceinanimalcare.Weareindebtedto Douglas Holmyard (Mount Sinai Hospital, Toronto, Canada) for help Conditional floxed alleles of murine genes for Angpt1, Angpt2,and with electron microscopy. We are thankful to Dr. Peter S. Aronson (Yale Tek/Tie2 were described previously.24 These genes were bred ac- University) for his gift of Ksp-cadherin antibody. We would like to ac- cordingly with the transgenes R26-rtTAand tetO-Cre, allowing for knowledgeDouglasFambrough(JohnsHopkinsUniversity)andEugene doxycycline-induced whole-body inactivation of the floxed alleles. Butcher (Stanford University) for antisera against Na+/K+-ATPase and Doxycyline (0.5% [wt/vol]) was administered in drinking water of Plvap, respectively, which were obtained through the Developmental time-mated dams from the 16th day of gestation (E16.5) until pups Studies Hybridoma Bank (University of Iowa). Weexpress our gratitude were weaned (P21). Renal Tie2 expressionwas mapped by breeding to Thomas Pannebecker (University of Arizona) for helpful suggestions Tie2-Cre with R26-LacZ or R26-mTmG reporter mice. Transgenic on immunostainings of the vasa recta.

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Imaging work was performed at the Northwestern University 15. Ziegler T, Horstkotte J, Schwab C, Pfetsch V, Weinmann K, Dietzel S, Center for Advanced Microscopy, and it was generously supported by Rohwedder I, Hinkel R, Gross L, Lee S, Hu J, Soehnlein O, Franz WM, National Cancer Institute Cancer Center support grant P30 CA060553 Sperandio M, Pohl U, Thomas M, Weber C, Augustin HG, Fässler R, Deutsch U, Kupatt C: Angiopoietin 2 mediates microvascular and he- (to the Robert H. Lurie Comprehensive Cancer Center). This work was modynamic alterations in sepsis [published online ahead of print July 1, fundedwithresearchgrantsfromtheNationalInstitutesofHealth:grants 2013]. JClinInvestdoi:10.1172/JCI66549 1R01GM120592-01 (R.V.S.), R01-DK41707 (to J.D.K. and J.M.S.), 16. Roviezzo F, Tsigkos S, Kotanidou A, Bucci M, Brancaleone V, Cirino G, 1R01HL124120-01 (to S.E.Q.), R01EY025799 (to S.E.Q.), and Papapetropoulos A: Angiopoietin-2 causes inflammation in vivo by – 5T32DK108738-02 (to S.E.Q.). promoting vascular leakage. J Pharmacol Exp Ther 314: 738 744, 2005 17. Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, Bartol A, Kienast Y, Vettel C, Loos EK, Kutschera S, Bartels S, Appak S, Besemfelder E, Terhardt D, Chavakis E, Wieland T, Klein C, Thomas M, DISCLOSURES Uemura A, Goerdt S, Augustin HG: Angiopoietin-2 differentially reg- None. ulates through TIE2 and signaling. JClinInvest 122: 1991–2005, 2012 18. Hakanpaa L, Sipila T, Leppanen VM, Gautam P, Nurmi H, Jacquemet G, Eklund L, Ivaska J, Alitalo K, Saharinen P: Endothelial destabilization by REFERENCES angiopoietin-2 via integrin b1 activation. Nat Commun 6: 5962, 2015 19. Scharpfenecker M, Fiedler U, Reiss Y, Augustin HG: The Tie-2 ligand 1. 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