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

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Host Lymphotoxin-b Receptor Signaling Is Crucial for Angiogenesis of Metanephric Tissue Transplanted into Lymphoid Sites Maria Giovanna Francipane,*y Bing Han,* and Eric Lagasse* From the McGowan Institute for Regenerative Medicine and Department of Pathology,* University of Pittsburgh, Pittsburgh, Pennsylvania; and the Ri.MED Foundation,y Palermo, Italy Accepted for publication August 20, 2019. Address correspondence to Maria Giovanna Francipane, Ph.D., 450 Technology Dr., Ste. 333, Pittsburgh, PA 15219; or Eric Lagasse, Pharm.D., Ph.D., 450 Technology Dr., Ste. 300, Pittsburgh, PA 15219. E-mail: [email protected] or lagasse@ pitt.edu.

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The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-b receptor (LTbR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTbR antagonist treatment or in the omenta of LTBR knockout (LTbR/) mice, the host LTbR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTbR signaling correlated with decreased expression of endothelial and angiogenic markers in kidney grafts as well as structural alterations. Because the number of glomerular endothelial cells expressing the LTbR target nuclear factor kBeinducing kinase (NIK) decreased in the absence of a functional LTbR, it was speculated that an LTbR/NIK axis mediated the angiogenetic signals required for successful ectopic kidney organogenesis, given the established role of NIK in neovascularization. However, the transplantation of kidney rudiments in omenta of NIK/ mice revealed that NIK is dispensable for ectopic kidney vascular integration and maturation. Finally, defective LTbR signaling impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTbR signals. (Am J Pathol 2019, -: 1e18; https://doi.org/10.1016/j.ajpath.2019.08.018)

Chronic and end-stage kidney disease impose a large burden on human health worldwide. Projected prevalence trend lines are not comforting,1 suggesting an urgent need to explore new approaches to overcome limitations associated with traditional interventions. Dialysisdthe most common method used to treat advanced kidney failuredcan only replace 10% to 20% of kidney function and is associated with increased left ventricular mass, intramyocardial cell fibrosis, and capillary loss.2,3 Orthotopic kidney transplantation offers better quality of life and longer survivals,4 yet it is limited by organ shortage and the risks associated with immunosuppression, including increased vulnerability to infection and cancer, as well as organ toxicity.5 One novel solution to the shortage of human organs available for transplantation envisions growing new organs in situ via transplantation of developing anlagen from animal embryos.6 A few sites have been considered for renal anlage transplantation, and, paradoxically, the orthotopic

environment seems not to be the most appropriate. Indeed, transplantation of metanephric tissue into the adult mouse kidney does not lead to glomerular or tubular differentiation.7 Other sites have also been considered, including the anterior eye chamber,8 the omentum,9 the paraaortic area,10 and, recently, the lymph node (LN) has been identified as an attractive site for the maturation of mouse metanephroi into functional glomeruli and tubules.11,12 Here, we show that host lymphotoxin-b receptor (LTbR) pathway generates angiogenic cues to support kidney organogenesis in both LNs and omenta. Our hypothesis that

Supported by the Ri.MED Foundation (M.G.F.); NIH grant R01 DK085711 (E.L.); and The Pediatric Device InitiativeeMcGowan Institute for Regenerative Medicine, University of Pittsburgh e Commonwealth of Pennsylvania grant SAP4100073573 (E.L., M.G.F.). Disclosures: E.L. is the scientific founder and the Chief Scientific Officer of LyGenesis, Inc.

Copyright ª 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.ajpath.2019.08.018

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LTbR could affect kidney organogenesis in these sites sprang from the central role of LTbR in the development13,14 and homeostasis15 of lymphoid organs, as well as from our finding that glomeruli maturing in both LNs and omenta contained, during the early stages of engraftment, endothelial cells expressing the nuclear factor kB-inducing kinase (NIK), a downstream target, among others, of LTbR.16 Given an established role for NIK in neovascularization,17 and the presence of host endothelial cells in ectopic kidney grafts, it was speculated that a host LTbR/ NIK axis could allow successful kidney organogenesis. In accordance, defective LTbR signaling was found to be associated with decreased numbers of glomerular NIK-expressing endothelial cells, decreased absolute vascularization/angiogenesis, and structural alterations of the kidney grafts. However, kidney organogenesis was not affected in the absence of NIK. This result might suggest either the existence of different targets downstream of LTbR, leading to angiogenesis, or the activation of compensatory angiogenic pathways in the absence of NIK. Finally, the absence of LTbR signals impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTbR signals.

mice (n Z 10 per group) or in the omenta of wild-type or NIK/ C57BL/6 mice (n Z 5 per group). Kidneys were split longitudinally in two pieces before transplantation. The kidney preparation was placed onto the exposed omentum, and the omentum was later folded onto itself. Growth Factor Reduced Matrigel (356231; Corning, Corning, NY) was used to glue the transplanted omenta. Intraomental kidney grafts from wild-type and LTbR/ mice were collected at different time points (2 weeks: n Z 1; 3 weeks: n Z 4; 4 weeks: n Z 1; 5 weeks: n Z 1; 6 weeks: n Z 3), whereas intraomental kidney grafts from wild-type and NIK/ mice were all collected 3 weeks after transplantation. Wild-type or LTbR/ C57BL/6 mice were also transplanted with GFPþ newborn (postnatal day 2) kidneys in their omenta (n Z 3 per group). Half of a kidney was transplanted in each mouse as described in the paragraph above. One mouse from each group was sacrificed every week, starting at 2 weeks after transplantation up to the fourth week. Images of kidney grafts were obtained with both the Olympus IX71 inverted fluorescence microscope (Tokyo, Japan) and the Leica EZ4D stereomicroscope (Wetzlar, Germany).

Materials and Methods

To assess LTbR pathway contribution to kidney organogenesis inside the LN, recipient mice were treated intraperitoneally with 100 mg of mouse LTbR-Fc fusion protein (71122; BPS Bioscience, San Diego, CA) or control immunoglobulin (PI31205; Pierce Biotechnology, Rockford, IL) 2 days before transplantation. Mice were further treated at day 8 and 15, and LNs were collected at day 22. LTbR-Fc concentration and exposure times were chosen based on previously published studies.19,20 LTbR blockage was confirmed by decreased glomerular NIK expression.

Ethics Statement

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Mice were purchased from Charles River Laboratories (Wilmington, MA) or The Jackson Laboratory (Bar Harbor, ME), and bred and housed in the Division of Laboratory Animal Resources facility at the University of Pittsburgh. Experimental protocols followed NIH’s Guide for the Care and Use of Laboratory Animals,18 and they were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Mixed sexes were used.

Tissues and Transplantations E14 kidneys were retrieved from timed-pregnant green fluorescent protein (GFP)þ or wild-type C57BL/6 mice under a dissecting microscope. GFPþ kidneys served for transplantation of wild-type C57BL/6 LNs to investigate host-derived vascularization (CD31 stain) and expression of LTbR, erasmus university rotterdamdthymic reticulum antibody 7 (ER-TR7), LT-b, and NIK (n Z 6) or to test the effect of LTbR-Fc treatment (n Z 6) (see below). Wild-type kidneys served for transplantation of wild-type C57BL/6 LNs (n Z 3) to analyze glomerular NIK expression. LN transplantation was performed as previously described,11 and LN kidney grafts were collected 3 or 6 weeks after transplantation, depending on the experiment. Alternatively, paired GFPþ E14 kidneys were transplanted in the omenta of wild-type or LTbR/ C57BL/6

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LTbR-Fc Treatments

Stains and Quantifications Engrafted LNs and omenta and mouse kidneys were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound or paraffin, and cut into 4e to 7em-thick sections for further analysis. Hematoxylin and eosin (H&E) staining was performed as described elsewhere.21 All other stains were performed as follows. Fluorescent Immunohistochemistry Staining of Frozen Tissue Sections Frozen sections were equilibrated at room temperature for 15 minutes, then rehydrated in phosphate-buffered saline (PBS) for 15 minutes, and permeabilized with cold 0.1% Triton X-100 for 10 minutes. After three washes in PBS supplemented with 0.05% Tween 20 (PBS-T), a solution of 3% bovine serum albumin was applied to the sections for 30 minutes to prevent nonspecific binding. Sections were then incubated with specific primary

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Growing Kidneys in Ectopic Sites antibodies at least 1 hour at room temperature or overnight at 4 C. After three washes in PBS-T, sections were incubated with Alexa Fluor 488 or 594 secondary antibodies (Thermo Fisher, Pittsburgh, PA) for 20 minutes. Sections were washed three times, and the nuclei were counterstained with Hoechst 33342 (Life Technologies, Frederick, MD). A solution of 50% glycerol in Hanks’ balanced salt solution was used as mounting medium. Fluorescent Immunocytochemistry Staining CD45-depleted stromal cells from omenta were cytospun onto glass slides (see below for isolation), fixed with acetone for 8 minutes, and stained with specific antibodies according to the same protocol described for frozen sections. Chromogenic Immunohistochemistry Staining of ParaffinEmbedded Tissue Sections Paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidases were blocked with the use of 3% hydrogen peroxide for 10 minutes. Antigen unmasking was performed with 10 mmol/L sodium citrate buffer pH 6.0. Nonspecific binding was prevented by incubating the slides in 3% bovine serum albumin in PBS-T for 30 minutes. The slides were exposed to primary antibodies overnight at 4 C, incubated with biotinylated secondary antibody, and later with streptavidinhorseradish peroxidase (R.T.U. Vectastain Kit; Vector, Burlingame, CA). 3-Amino-9-ethylcarbazole (HK092-5KE; BioGenex, Fremont, CA) was used as a chromogen. Immunostained sections were counterstained with Mayer’s hematoxylin (Dako, Carpinteria, CA) and mounted in permount mounting medium (SP15-500; Thermo Fisher). A list of antibodies used is provided in Table 1. Isotype½T1 matched antibodies were used as negative controls. Images were obtained with the Olympus IX71 inverted fluorescence microscope.

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Fluorescent Quantifications Areas of LN and intraomental kidney grafts were quantified by measuring pixel counts of GFP signal with the use of Photoshop CS5 (Adobe, San Jose, CA). The mean fluorescence intensity (MFI) of podoplanin (PDPLN) and the fluorescence intensities of CD31, CD105, and Ly-76 were measured among regions of interest drawn around the Bowman capsule with the use of ImageJ version 1.52a (NIH, Bethesda, MD; https://imagej.nih.gov/ij), as previously described.22 Glomerular areas were quantified at the time of PDPLN MFI measurement. The corrected total glomerular fluorescence for CD31, CD105, and Ly-76 was later calculated with the following formula: Integrated density e (area of selected glomerulus  mean fluorescence of background readings). Length of lotus tetragonolobus lectin (LTL)þ tubules was obtained with ImageJ version 1.52a by drawing a line over the LTLþ stain (circular tubular structures were not

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Table 1

List of Reagents Used in This Study

Antibody

Catalog number

Supplier

CD105 CD31 CD34 CDH1 CIV EFNB2 ER-TR7 Ki-67 LT-b LTbR LTL Ly-76 NIK PAI-1 PDPLN

550546 ab28364 MA1-82073 sc-7870 1340-01 ab131536 ab51824 ab15580 ab64835 ab70063 B-1325 ab91113 ab216409 ab66705 NBP1-90211

BD Biosciences (San Jose, CA) Abcam (Cambridge, MA) ThermoFisher (Pittsburgh, PA) Santa Cruz Biotechnology (Dallas, TX) SouthernBiotech (Birmingham, AL) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Vector Laboratories (Burlingame, CA) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Abcam (Cambridge, MA) Novus Biologicals (Centennial, CO)

CDH1, cadherin 1; CIV, collagen IV; EFNB2, ephrin B2; ER-TR7, erasmus university rotterdamethymic reticulum antibody 7; LT-b, lymphotoxin-b; LTbR, lymphotoxin-b receptor; LTL, lotus tetragonolobus lectin; NIK, nuclear factor kBeinducing kinase; PAI-1, plasminogen activator inhibitor 1; PDPLN, podoplanin.

considered) and later converting the obtained numbers into microns. Glomeruli and tubules from at least five sections per sample were considered. ER-TR7 MFI was measured in 6 sections per sample with the use of ImageJ version 1.52a. The number of NIKþ cells in LN and intraomental grafts was obtained by analyzing glomeruli from 7 to 13 microscopic fields depending on the experiment. The number of plasminogen activator inhibitor 1 (PAI-1)þ tubules in LNs and intraomental grafts was obtained by analyzing 12 to 20 microscopic fields. Histologic Quantifications Quantitative measurement of glomerular diameters was performed by taking the maximum diameter from the hilum to the end of the Bowman capsule of 18 H&E-stained glomeruli derived from three sections per mouse, using the measurement tool in cellSens Standard 1.9 software (Olympus). Ki67þ nuclei were counted in at least five random sites within the cortex across two separate stained sections per mouse. The number of Ki-67þ nuclei was finally expressed as a percentage of the total number of nuclei. All sections were randomly selected and analyzed under Q11 blinded conditions.

Stromal Cell Isolation and Flow Cytometry Stromal cells were isolated from GFPþ C57BL/6 mouse mesenteric LNs (n Z 10) or omenta (n Z 10) according to the method described by Turley laboratory.23 Briefly, tissues were collected and digested with an enzyme mix comprised of RPMI 1640 medium that contained 0.8 mg/mL dispase (17105-041; Gibco, Gaithersburg, MD), 0.2 mg/mL collagenase P (11213857001; Sigma-Aldrich, St. Louis, MO),

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and 1 mg/mL DNase I (89836; Thermo Scientific, Pittsburgh, PA). After digestion, anti-CD45 immunomagnetic beads (130-052-301; Miltenyi Biotec, Auburn, CA) were used to deplete hematopoietic cells from the bulk population. CD45-depleted stromal cells were then stained with Sytox Blue (S11348; Molecular Probes, Eugene, OR), phycoerythrin-cyanine 7 CD45 (552848; BD Biosciences, Bedford, MA), phycoerythrin Podoplanin (127407; BioLegend, San Diego, CA), and peridinin chlorophyll protein/ cyanine 5.5 CD31 (102419; BioLegend). Flow cytometric analysis was performed with a BD FACSAria cytometer and FlowJo software version 9.6 (TreeStar, Ashland, OR), according to standard procedures. Isotype antibodies were used as negative controls. Table 2

Molecular Analysis RNA isolation was performed with the RNeasy Mini Kit (74106; Qiagen, Germantown, MD) according to the manufacturer’s instructions (including the optional oncolumn DNase digestion). RNA purity and concentration were measured with a NanoDrop 2000/c spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA). cDNAs were synthesized with the RT2 First Strand Kit (330401; Qiagen), and expression of 84 genes modulating the biological processes of angiogenesis were measured with the Mouse Angiogenesis RT2 Profiler PCR Array (PAMM-024Z; Qiagen) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA),

List of Primer Sequences Used in This Study NCBI reference

Gene ID

Sequence

ANGPT1

F: 50 -AGTGCGACAGAGCAGTACAA-30 R: 50 -TGGGCCATCTCCGACTTCAT-30 F: 50 -TGTACCGTGGAGAAGCCTGT-30 R: 50 -CCTCACGGAACTTGGGATTTGA-30 F: 50 -CAAATGGGTCTTTGGAGGGC-30 R: 50 -ACGTCTTGTTGGACCGTGAT-30 F: 50 -GCCCATTACCCTGCTGTTTG-30 R: 50 -CACAGCCGACTCTTTCTGC-30 F: 50 -AGTCTTCTGCTCGCCCTGCTC-30 R: 50 -CGGACTCCACCACAACAGTTCC-30 F: 50 -AACCTGTGGCTGTGGAACAAC-30 R: 50 -GGGAAGTGATGTCCAGGAAGT-30 F: 50 -GTGGAAGTGTCCTCCCTTGAG-30 R: 50 -GGAGCCTTCCGTTCTTAGGGT-30 F: 50 -CAGGTCCTAGCTGGGTTGG-30 R: 50 -CGTTGAAAGGCACCACTTCC-30 F: 50 -CGGACACATGCAGTCTCTGT-30 R: 50 -CCTTCTCCAATACAGCGTCCA-30 F: 50 -CAACCCGACAGAGACAATCCT-30 R: 50 -TGAAGGCGTCTCTTCCCACT-30 F: 50 -CCAAGGCAGGCAGACGAAT-30 R: 50 -TAGTGTACTCGGCCAGGTGT-30 F: 50 -CCGGCTTAGTTCTCTGTGGA-30 R: 50 -GCAATGCAGGTGAGGGATGT-30 F: 50 -CTCTGGGTACGTGGGTGTTC-30 R: 50 -CACCACTCACAGTGTTTGCG-30 F: 50 -GATCACCACAACCCACACCT-30 R: 50 -CCAGGTTGCGGAAGCAGTAA-30 F: 50 -TTGGCGACTGGAGCAATACA-30 R: 50 -GAGACGGAACCTACCACAGC-30 F: 50 -GATTGAGACCCTGGTGGACATC-30 R: 50 -CATCTCTCCTATGTGCTGGCT-30 F: 50 -TGGCTGGGTGTGGTATGAAC-30 R: 50 -GGTGACCTCCCTCATGTTCC-30 F: 50 -TGCTAAGTCTGGCACGAAGG-30 R: 50 -ACACCAAACTGCCCGATCAT-30

EFNA1 EFNB2 ENG JAG1 NOS3 PECAM1 PGF PTK2 SERPINE1 TBX1 TEK TGFA TGFB3 TIE1 VEGFA GUSB HSP90AB1

NM_009640.4 NM_010107.4 NM_010111.5 NM_007932.2 NM_013822.5 NM_008713.4 NM_008816.3 NM_008827.3 NM_007982.2 NM_008871.2 NM_011532.2 NM_013690.3 NM_031199.4 NM_009368.3 NM_011587.2 NM_001287056.1 NM_010368.2 NM_008302.3

Primer sequences were designed with National Center for Biotechnology Information’s (NCBI) Primer-BLAST (Basic Local Alignment Search Tool) (https:// www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed April 27, 2018). The NCBI Reference sequences that were entered in the Primer-BLAST submission form are indicated for each gene and were retrieved from NCBI’s Nucleotide database (https://www.ncbi.nlm.nih.gov/refseq). F, forward; R, reverse.

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according to the manufacturer’s instructions. The CT values from all plates and wells were then exported from the Real-Time PCR instrument and formatted into a new Excel spreadsheet (Microsoft, Redman, WA) (Supplemental Table S1). The data were uploaded in the RT2 Profiler PCR Array Data Analysis Webportal (https:// dataanalysis.qiagen.com/pcr/arrayanalysis.php, last accessed April 4, 2018) and analyzed. GUSB and HSP90AB1 were automatically selected as optimal housekeeping genes. Genes with fold changes >2 and P values < 0.05 were later validated with Real-Time RTPCR. Primers were designed with NCBI’s Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed April 27, 2018). Primers bridged an exoneexon junction or spanned introns. Free online Beacon Designer software (http://www.premierbiosoft. com/qOligo/Oligo.jsp?PIDZ1, last accessed April 27, 2018) was used to evaluate possible secondary structures such as dimers or hairpins. To check the

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559 primer specificity, before performing Real-Time 560 RT-PCR, conventional PCR was performed, and ampli561 cons were separated on agarose gels. A melting curve 562 analysis was performed after each Real-Time RT-PCR to 563 further confirm that each assay produced a single, specific 564 ½T2 565 product. Primers sequences are given in Table 2.

Unilateral Nx Wild-type (n Z 3) and LTbR/ (n Z 4) C57BL/6 mice underwent unilateral nephrectomy (Nx). After exposing the left kidney, the renal artery, vein, and ureter were tied off. The kidney was finally excised distal to the ligatures.

Statistical Analysis Statistical analysis was performed with the unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001,

Figure 1 Vascularization of glomeruli engrafted in the mouse lymph node (LN) might be driven by host lymphotoxin-b receptor (LTbR)/nuclear factor kB-inducing kinase (NIK) axis. A: Left column: Frozen sections of a representative green fluorescent protein (GFP)þ metanephric (embryonic day 14) kidney graft 3 weeks after transplantation. Merged GFP signal with Hoechst stain (blue; top panel), merged GFP signal with CD31 (endothelial cell) stain (red; middle panel), and merged CD31 stain (red) with Hoechst stain (blue; bottom panel). Boxed and circled areas are shown at higher magnification to the right and demonstrate the presence of a host (GFP) CD31þ cell in an engrafted glomerulus. Right panel: Cartoon depicting the localization of glomerular endothelial cells (image adapted from http://whatwhen-how.com/acp-medicine/glomerular-diseasespart-1). B: Representative immunofluorescence stain for CD34 (endothelial cell marker, red) and NIK (green) in an engrafted wild-type (GFP) glomerulus 3 weeks after transplantation. Arrows point to double positive cells. C: Representative immunofluorescence stain for LTbR (red), erasmus university rotterdamdthymic reticulum antibody 7 (ER-TR7; LN fibroblastic reticular cells, red), or lymphotoxin b (LT-b, red) in engrafted GFPþ glomeruli 3 weeks after transplantation. Data presented in this figure are representative of one technical replicate of a minimum of three technical replicates per sample. n Z 3. Scale bars: 50 mm (A); 25 mm (B and C).

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****P < 0.0001). Statistical power analysis was also performed to assess error rates for tubule length measurements. For all studies, the power of a two-tailed t-test was >0.9, given an a level of 0.05 (5%). A volcano plot of RT2 Profiler PCR Array data was used to identify genes differentially expressed in kidneys engrafted in LTbR/ versus wild-type omenta. Correlation between RT2 Profiler PCR Array and Real-Time RT-PCR was calculated by linear regression and Pearson correlation. Both SABiosciences’ data analysis (Frederick, MD) and GraphPad Prism 7 software (GraphPad Inc., San Diego, CA) were used.

Results Vascularization of Glomeruli Engrafted in the Mouse LN Might Be Driven by Host LTbR/NIK Axis With the use of GFP-expressing mouse metanephric kidneys, successful nephron maturation inside a host mouse LN has been demonstrated.11,12 The LN-engrafted nephrons could rely on a remarkable vascular network originating from both host- and graft-derived vascular cells.11,12 Even more impressive were the data of human fetal kidney engraftment inside the LN, which clearly indicated that,

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Treatment with lymphotoxin-b receptor antagonist (LTbR-Fc) impaired expansion of kidneys engrafted in lymph node (LN) and caused a decrease in nuclear factor kB-inducing kinase (NIK)-expressing glomerular cells. A: Schematic representation of LN transplantation assay with the course of mouse treatments. Green K stands for green fluorescent protein (GFP)þ metanephric (embryonic day 14) kidney. B: Merged fluorescence/bright-field images of whole mount kidney-bearing LNs isolated from either control Ige or LTbR-Fcetreated mice (GFPþ areas indicate kidney engraftment). C: Bar graph showing fold changes of GFP intensity (pixel counts obtained with Adobe Photoshop) in LNs as in B (blue bars: Control Ig; green bars: LTbR-Fc). D: Immunofluorescence stain for NIK (red) in glomeruli of engrafted kidneys as in B. E: Line graph showing the percentage of NIK-expressing cells in engrafted glomeruli as in B. Glomeruli were divided into five groups based on the number of NIK-expressing cells (0; 1 to 3; 4 to 6; 7 to 9; or 10 positive cells/glomerulus). Data presented in D and E are representative of one technical replicate of a minimum of three technical replicates per sample. n Z 3 per group (D and E). **P < 0.01. Scale bars: 500 mm (B); 25 mm (D).

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although the donor and host endothelial cells could coexist in the early stages of engraftment, over time the grafts were entirely re-vascularized by host cells.24 ½F1 Figure 1A shows abundance of host (GFP) endothelial cells (CD31þ) in the interstitium of a GFPþ mouse metanephric kidney 3 weeks after transplantation in the LN, as

well as the presence of a host endothelial cell in an engrafted glomerulus. Most glomerular endothelial cells (CD34þ) expressed NIK (Figure 1B). Of interest, the number of NIKþ cells and/or NIK fluorescence intensity were significantly decreased in 6-week engrafted glomeruli (Supplemental Figure S1, A and B), suggesting that NIK

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Figure 3 Treatment with lymphotoxin-b receptor antagonist (LTbR-Fc) affected lymph node (LN) kidney graft architecture and vasculature. A: Representative immunofluorescence stains for podoplanin (PDPLN; podocytes and lymphatic endothelia, red), lotus tetragonolobus lectin (LTL; proximal tubules, red), CD31 (endothelial cells, red), CD105 (newly formed vessels, red), and Ly-76 (erythrocytes, red) on serial frozen sections of kidney grafts isolated from either Control Ig- or LTbR-Fcetreated mice. Insets show a representative glomerulus for each group. B: Bar graph showing PDPLN mean fluorescence intensity (MFI; plum bars) in glomeruli of kidney grafts as in A, measured with ImageJ version 1.52a (each bar represents a glomerulus). Gray bars indicate the area of each glomerulus considered (ImageJ). C: Scatter dot plot with bar graphs  SD showing the length of LTLþ tubules in kidney grafts as in A, measured across sections, using ImageJ. Each dot represents a tubule. DeF: Scatter dot plot with bar graphs  SD presenting the corrected fluorescence intensities of glomerular (CTGF) CD31, CD105, and Ly-76 in kidney grafts as in A, measured with ImageJ. Each dot represents a glomerulus. Data presented in this figure are representative of one technical replicate of a minimum of three technical replicates per sample (n Z 3 per group). *P < 0.05, ****P < 0.0001. Power.t.test Z 1, sig.level Z 0.05 (C). Scale bars: 100 mm (A).

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expression could mark activated host endothelial cells during the early stages of kidney vascular integration in the host LN. NIK was activated in response to stimulation of tumor necrosis factor receptor superfamily members such as LTbR.25 LTbR was widely expressed on the three major CD45 LN stromal cell populationsdfibroblastic reticular cells (FRCs), lymphatic endothelial cells, and blood endothelial cellsdand was important for several adult LN functions.15 LTbR signals on FRCs regulated LN vascular

endothelial growth factor levels and, in turn, LN endothelial cell proliferation.26 Likewise, LTbR triggering on LN endothelial cells affected vasculature development.27 We therefore hypothesized that host LTbR/NIK signaling facilitated glomerular vascularization inside the LN. In accordance with our hypothesis, engrafted glomeruli were found to be embedded in a host LTbR reactive cellular scaffold, compatible with ER-TR7þ FRCs (Figure 1C). Intraglomerular LTbR expression could also be observed, which, together with a rare intraglomerular expression of the

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Figure 4 Metanephric kidney engraftment in omentum: similarity with the lymph node (LN) system. A: Frozen section of a representative green fluorescent protein (GFP)þ glomerulus 3 weeks after transplantation in the omentum. Merged GFP signal with Hoechst stain (blue; left panel), merged GFP signal with CD31 (endothelial cell) stain (red; middle panel), and merged CD31 stain (red) with Hoechst stain (blue; right panel). Circled areas are shown at higher magnification below and demonstrate the presence of a host (GFP) CD31þ cell in the engrafted glomerulus. B: Merged GFP signal with CD31 (red, left column) or nuclear factor kB-inducing kinase (NIK; red, right column) stain in serial sections of GFPþ kidney graft in the omentum (3 weeks after transplantation). Top row shows expressions of CD31 and NIK in glomeruli; bottom row, expressions of CD31 and NIK in a vessel. Arrows point to a host (GFP) CD31þ/NIKþ cell. C: Flow cytometric profiles of CD45 cells isolated from LN (left panel) or omentum (right panel). From the podoplanin (PDPLN) and CD31 expression, distinct stromal cell populations can be identified: fibroblastic reticular cells (FRCs; PDPLNþ/CD31), lymphatic endothelial cells (LECs; PDPLNþ/ CD31þ), and blood endothelial cells (BECs; PDPLN/CD31þ). D: Representative immunofluorescence stain for lymphotoxin-b receptor (LTbR; red) and PDPLN (green) on CD45 omental cells. White asterisks indicate LTbRþ/PDPLN cells and yellow asterisks indicate LTbRþ/PDPLNþ cells. Nuclei were counterstained with Hoechst (blue). Data presented in A and B are representative of one of three technical replicates per sample. n Z 3 (A and B). Flow cytometric profiles and LTbR/PDPLN stain were generated by pooling cells from 10 mice. Scale bars: 25 mm (A); 50 mm (B); 100 mm (D). Cy, cyanine; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

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membrane-anchored LTbR ligand LT-b (Figure 1C), strengthened the idea that the LTbR/NIK axis could play a role in glomerular vascularization inside the LN.

Inhibition of LTbR Signaling Decreases Glomerular Vascularization/Angiogenesis in the LN To determine the role of LTbR signaling on kidney engraftment in the LN, 2 days before metanephric kidney

1055 transplantation, recipient mice were treated with a recom1056 binant LTbR-Fc fusion proteindwhich antagonized LTbR1057 mediated effects by engaging soluble and cell surface forms 1058 of LTbR ligandsdor control Ig (day 1). Mice were treated 1059 again at day 8 and 15, and LNs were collected at day 22 1060 (Figure 2A). LTbR-Fc treatment resulted in reduced ½F2 1061 expansion of transplanted GFPþ metanephric kidneys 1062 (Figure 2, B and C) and decreased numbers of NIKþ 1063 glomerular cells (Figure 2D). Indeed, although glomeruli 1064

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Growing Kidneys in Ectopic Sites

Defective lymphotoxin-b receptor (LTbR) signaling in the omentum resulted in reduced kidney graft expansion. A: Merged fluorescence/brightfield (left panels) or dissecting scope (right panels) images of whole mount omenta from wild-type (WT) or LTbR/ mice 2 to 6 weeks after transplantation of green fluorescent protein (GFP)þ metanephric (embryonic day 14) kidneys. Grafts from 3 mice are shown at 6 weeks after transplantation in comparison with an adult mouse kidney. Red arrows point to the engrafted kidneys in WT or LTbR/ mice. B: Bar graph showing fold changes of GFP intensity (pixel counts obtained with Adobe Photoshop) in transplanted omenta as in A (blue bars: WT; green bars: LTbR/). Dotted lines separate the samples based on the time (weeks) elapsed from transplantation (2 weeks: n Z 1 per group; 3 weeks: n Z 4 per group; 4 weeks: n Z 1 per group; 5 weeks: n Z 1 per group; 6 weeks: n Z 3 per group). Scale bars Z 500 mm (A).

Figure 5

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Growing Kidneys in Ectopic Sites 1303 1241 devoid of NIKþ cells could be observed in both experipossibility that the observed effects could be due to LTbR/ 1304 1242 mental groups, the number of such glomeruli was doubled herpesvirus entry mediator inhibition on donor kidney cells 1305 1243 in grafts from LTbR-Fcetreated mice (Figure 2E). Moreor other cell types present in the donor tissue at the time of 1306 1244 over, although both the groups had similar percentages of transplantation, including stromal and hematopoietic cells. 1307 1245 þ / 13 glomeruli that contained one to three NIK cells, the mice did not have LNs, the omentum Because LTbR 1308 1246 number of glomeruli with four to six NIKþ cells was was used as an alternative site for transplantation. Similarly 1309 1247 to the LN, the omentum supported the maturation of GFPdrastically reduced after LTbR-Fc treatment (Figure 2E). 1310 1248 expressing metanephric kidneys. Intraomental kidney Overall, glomeruli engrafted in LTbR-Fcetreated mice had 1311 1249 þ grafts contained host-derived (GFP) glomerular endothe1312 1250 Q16 a mean of 1.48  0.25 NIK cells, whereas glomeruli 1251 engrafted in control mice had a mean of 2.88  0.33 NIKþ lial cells (CD31þ) (Figure 4A) as well as NIK-expressing ½F4 1313 1314 1252 endothelial cells (CD31þ) in renal glomeruli and blood cells (P Z 0.001). 1315 1253 When graft maturity was investigated through immunovessels (Figure 4B). We therefore hypothesized that LN and 1316 1254 fluorescence staining for PDPLN to highlight the glomeruli, omentum supported kidney organogenesis through a similar 1317 1255 or LTL to visualize the proximal tubules, no obvious cellular/molecular mechanism. Our hypothesis was champ1318 1256 structural differences could be found between treated and ioned by the finding that both sites shared similar FRC/ 1319 1257 lymphatic endothelial cell/blood endothelial cell flow cyto½F3 control mice (Figure 3A). Further analysis confirmed that 1320 1258 metric profiles (Figure 4C). Moreover, LTbRþ/PDPLNþ neither significant changes in total surface area of glomeruli 1321 1259 nor significant changes in PDPLN expression had occurred and LTbRþ/PDPLN cell subsets could be identified in 1322 1260 (Figure 3B). Conversely, LTbR-Fc treatment resulted in omental cell suspensions, indicating the existence of FRCs 1323 1261 decreased average LTLþ tubule length, calculated in cross and blood endothelial cells that mediated LT signals in this 1324 1262 1325 1263 site as well (Figure 4D). sections (Figure 3C). The extent of vascularization/angio1326 1264 genesis was also evaluated by staining for CD31 and the Consistent with our previous findings (Figure 2, B and þ 1327 1265 neo-angiogenic marker CD105 (Figure 3A). The intraC), expansion of GFP metanephric kidneys was affected in 1328 1266 / glomerular CD31 and CD105 fluorescence intensities were the LTbR omentum. Not only did the grafts expand less 1329 1267 quantified, and a significantly weaker intensity was found over a 6-week period (Figure 5), but they also were ½F5 1330 1268 for both markers in glomeruli of LTbR-Fcetreated mice significantly less vascularized (Figure 6A). The glomerular ½F6 1331 1269 (Figure 3, D and E). Nevertheless, no significant difference CD31, CD105, and Ly-76 fluorescence intensities were 1332 1270 in glomerular blood perfusion was observed (Ly-76 stain), quantified; significant changes could be observed at every 1333 1271 although fewer erythrocytes were located within blood time point analyzed (Figure 6B). Of note, no significant 1334 1272 vessels of treated grafts (Figure 3, A and F). Thus, LTbR 1335 1273 changes in Ly-76 expression had been observed in LN signaling might mediate vascularization/angiogenesis of the 1336 1274 kidney grafts isolated from mice undergoing LTbR-Fc 1337 1275 kidneys transplanted in the LN. treatment with respect to their control counterparts 1338 1276 (Figure 3F). The cause for this different outcome remains 1339 1277 unknown. Glomerular Vascularization/Angiogenesis Is Decreased 1340 1278 CD31 and CD105 fluorescence intensities were also in Kidneys Engrafted in the LTbR/ Omentum 1341 1279 compared over time (Figure 6, C and D). CD31 expression 1342 1280 was stable up to 4 weeks in glomeruli engrafted in the wildThe LTbR ligands included the heterotrimeric LT a1b2 1343 1281 type omentum and decreased afterward (Figure 6C). (LTa1b2)28 and LT-related inducible ligand.29 Although 1344 1282 Conversely, CD31 expression began falling soon after the LTa1b2 bound uniquely to LTbR, LT-related inducible 1345 1283 second week of transplantation in glomeruli engrafted in the 1346 1284 ligand may also interact with herpesvirus entry mediator.30 1347 1285 LTbR/ omentum (Figure 6C). CD105 expression had a To rule out the possibility that impaired graft angiogenesis 1348 1286 similar trend in both experimental groups, with the highest resulted from LTbR-Fc interference with signaling path1349 1287 expression registered at 2 weeks after transplantation ways other than those mediated by LTbR, the outcome of þ 1350 1288 (Figure 6D). At this stage, significant differences in NIK kidney transplantation in an LTbR defective environment, 1351 1289 / glomerular cell numbers could also be observed between the such as that offered by the LTbR mouse, was investi1352 1290 two kidney groups (Supplemental Figure S1C and gated. In addition, this approach allowed excluding the 1353 1291 1354 1292 1355 1293 Figure 6 Defective lymphotoxin-b receptor (LTbR) signaling in the omentum resulted in reduced kidney graft vascularization/angiogenesis and decreased numbers of nuclear factor kBeinducing kinase (NIK)-expressing glomerular cells. A: Representative immunofluorescence stains for CD31 (endothelial cells, 1356 1294 red), CD105 (newly formed vessels, red), and Ly-76 (erythrocytes, red) on serial frozen sections of intraomental kidney grafts from wild-type (WT) or LTbR/ 1357 1295 mice over the course of 6 weeks. B: Scatter dot plot with bar graphs  SD presenting the corrected fluorescence intensities of intraglomerular (CTGF) CD31, 1358 1296 CD105, and Ly-76 in kidney grafts as in A, measured with ImageJ version 1.52a. Each dot represents a glomerulus. C and D: Line graphs  SEM presenting CD31 1359 1297 / and CD105 CTGFs over the time course (blue line: WT; green line: LTbR ). E: Line graph showing the percentage of NIK-expressing cells in glomeruli 2 weeks 1360 1298 / after transplantation in WT (blue line) or LTbR (green line) omentum. Glomeruli were divided into five groups based on the number of NIK-expressing cells 1361 1299 (0; 1 to 3; 4 to 6; 7 to 9; or 10 positive cells/glomerulus). Data presented in this figure are representative of one of three technical replicates per sample 1362 1300 (n Z 10 per group; 2 weeks: n Z 1 per group; 3 weeks: n Z 4 per group; 4 weeks: n Z 1 per group; 5 weeks: n Z 1 per group; 6 weeks: n Z 3 per group). 1301 Q28 Q29 *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars Z 100 mm (A). 1302 1363

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Figure 6E). Indeed, the number of glomeruli devoid of NIKþ cells increased in the LTbR/ omentum (Figure 6E). Moreover, although almost an equal number of glomeruli had either one to three or four to six NIKþ cells in the wildtype omentum, the number of glomeruli with one to three NIKþ cells increased in the LTbR/ omentum, to the detriment of glomeruli with four to six NIKþ cells (Figure 6E). Finally, glomeruli with seven to nine NIKþ cells were more abundant in the wild-type omentum than in the LTbR/ omentum, and glomeruli with up to 10 NIKþ cells could only be observed in the wild-type omentum

(Figure 6E). Overall, glomeruli engrafted in the LTbR/ omentum had a mean of 2.04  0.23 NIKþ cells, whereas glomeruli engrafted in the wild-type omentum had a mean of 4.04  0.30 NIKþ cells (P < 0.0001). Over the time course, the number of glomeruli devoid of NIKþ cells progressively increased in both groups, and the number of NIKþ cells per positive glomerulus also decreased (Supplemental Figure S1, D and E). Four weeks after transplantation, no more than three NIKþ cells could be observed in glomeruli engrafted in the LTbR/ omentum, whereas a small percentage of glomeruli engrafted in the

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Figure 7 Defective lymphotoxin-b receptor (LTbR) signaling in the omentum affected kidney graft architecture. A: Representative immunofluorescence stains for collagen IV (CIV; glomerular basement membranes and mesangial cells, red), podoplanin (PDPLN; podocytes and lymphatic endothelia, red), and lotus tetragonolobus lectin (LTL; proximal tubules, red) on serial frozen sections of intraomental kidney grafts from wild-type (WT) or LTbR/ mice over the course of 6 weeks. B: Bar graph showing PDPLN mean fluorescence intensity (MFI; plum bars) in glomeruli 2 weeks after transplantation in WT or LTbR/ omentum, measured with ImageJ version 1.52a (each bar represents a glomerulus). Gray bars indicate the area of each glomerulus considered (ImageJ). C: Scatter dot plot with bar graphs  SD showing the length of LTLþ tubules 2 weeks after transplantation in WT or LTbR/ omentum, measured across sections with ImageJ. Each dot represents a tubule. Power.t.test Z 1, sig.level Z 0.05. Data presented in this figure are representative of one of three technical replicates per sample (n Z 10 per group; 2 weeks: n Z 1 per group; 3 weeks: n Z 4 per group; 4 weeks: n Z 1 per group; 5 weeks: n Z 1 per group; 6 weeks: n Z 3 per group). ****P < 0.0001. Scale bars Z 100 mm (A).

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1488 wild-type omentum had four to six NIKþ cells 1489 (Supplemental Figure S1, D and E). These results suggested 1490 a critical time window between 2 and 3 weeks after trans1491 plantation for LTbR/NIK actions on the vascular remodel1492 ing of the transplanted glomeruli. 1493 1494 Structural Changes in Kidneys Engrafted in the 1495 1496 LTbR/ Omentum 1497 1498 With respect to their control counterparts, the kidneys 1499 engrafted in the LTbR/ omentum also showed structural 1500 changes overtime, with complete loss of glomeruli and tu1501 ½F7 bules by 6 weeks (Figure 7A). 1502 The time point of 2 weeks was specifically studied. 1503 Although no significant changes in total surface area of 1504 glomeruli were observed, highly significant changes 1505 affected PDPLN expression (Figure 7B) and LTLþ tubule 1506 length (Figure 7C). 1507 Tubulointerstitial injury has been described as a mediator 1508 1509 of progression of kidney disease.31 It was therefore studied 1510 whether the observed glomerular and tubular loss in later 1511 stages of engraftment could be due to the expansion of renal 1512 interstitial fibroblasts. To test this hypothesis, staining was 1513 performed for ER-TR7, which besides outlining the various 1514 compartments of peripheral lymphoid organs by detecting 1515 the reticular fibroblasts, also identifies interstitial fibroblasts 1516 in the kidney (Supplemental Figure S2A).32 Significant 1517 changes were not observed between the kidneys engrafted in 1518 the wild-type omentum versus those engrafted in the 1519 LTbR/ omentum over the 6-week period (Supplemental 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549

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Figure S2B). Moreover, in each group, ER-TR7 levels were quite stable until the fifth week, after which time they decreased (Supplemental Figure S2C). Thus, the precise mechanism of glomerular and tubular loss in late-stage grafts remains unknown. Similar results were obtained when using newborn kidneys, instead of metanephric kidneys (Supplemental Figure S3), indicating that host-driven angiogenesis was uncoupled from the maturation stage of the transplanted tissue (ie, ongoing versus completed angiogenesis).Of interest, when using newborn kidneys, more pronounced structural changes were detected between the two mouse groups, starting as early as the second week after transplantation (Supplemental Figure S3). Thus, these results pinpointed the importance of host LTbR signaling for successful vascularization/angiogenesis and maturation of the kidney grafts in lymphoid sites.

Reduced Expression of Angiogenesis-Related Genes in Kidneys Engrafted in the LTbR/ Omentum PCR array profiles of angiogenesis-related genes confirmed that host LTbR signaling sustains ectopic kidney angiogenesis. Indeed, when six distinct transplants engrafted either in the LTbR/ omentum or in the wild-type omentum 3 weeks after transplantation were compared, several proangiogenic factors were found to be down-regulated in the absence of functional LTbR signaling (Figure 8 and Supplemental Figure S4). These factors included components of the angiopoietineTie signaling system (ANGPT1,

Figure 8 Down-regulation of pro-angiogenic genes in kidneys engrafted in the lymphotoxin-b receptor (LTbR)/ omentum. A: The volcano plot displays genes differentially expressed in the kidneys engrafted in the LTbR/ omentum as compared with the kidneys engrafted in the wildtype (WT) omentum. Plotted along the x axis is the mean of log2 fold-change; along the y axis is the negative logarithm to the base 10 of the P values. The vertical dashed lines and the horizontal line reflect the filtering criteria (fold change  2.0 and P values  0.05). Green dots mark genes with decreased expression. B: Heatmap of the 14 statistically significant differentially expressed genes. C: Correlation between RT2 Profiler PCR Array and real-time RT-PCR data. Relative expression levels determined by RT2 Profiler PCR Array (x axis) and real-time RT-PCR (y axis) are plotted for a subset of 16 genes (LTbR/ 3 versus WT 3). Solid line indicates linear regression; both the squared and the Pearson correlation coefficients are given. D: Representative scatter plot (LTbR/ 3 versus WT 3) showing the gene expression patterns (presented as fold change) of selected genes by RT2 Profiler PCR Array (PCR Array) and real-time RT-PCR (RT-qPCR).

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1674 TEK, and TIE1); the epidermal growth factor signaling 1675 system (TGFA); the Epheephrin signaling system (EFNA1 1676 and EFNB2); the transforming growth factor b signaling 1677 system (ENG and TGFB3); the vascular endothelial growth 1678 factor signaling system (PGF ); and targets shared among 1679 these systems (JAG1, NOS3, PTK2, SERPINE1, TBX1). 1680 Expression of EFNB2 and SERPINE1 products was later 1681 analyzed in sections of 3-week intraomental kidney grafts. 1682 EFNB2 was found to be expressed in glomerular cells 1683 1684 (presumably podocytes and parietal epithelial cells), and 1685 PAI-1, the product of SERPINE1, was expressed in renal tubules (presumably proximal tubules) (Figure 9A). ½F9 1686 1687 Although EFNB2 fluorescence intensity was similar in both 1688 groups, a significant lower number of tubules were positive 1689 / for PAI-1 in the kidneys engrafted in the LTbR omen1690 tum (Figure 9B). A similar trend could be observed in 1691 kidneys engrafted in LNs under LTbR-Fc treatment 1692 (Figure 9, C and D), confirming that a similar cellular/mo1693 lecular mechanism drove kidney organogenesis in LNs and 1694 omenta. These results also suggested that, although the 1695 1696 lymphoid microenvironment provided signaling cues that 1697 affected the vasculature of the maturing kidney, the kidney 1698 graft self-sustains by producing angiogenic molecules. 1699 Production of these molecules appeared to be facilitated by 1700 an environment that mediated LT signals.

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Host NIK Is Not Required for Ectopic Kidney Organogenesis A significant reduction in the number of NIK-expressing glomerular cells had been observed in kidneys engrafted in both LNs of mice undergoing LTbR antagonist treatment (Figure 2, D and E) and omenta of LTbR/ mice (Figure 6E and Supplemental Figure S1, CeE). To elucidate whether a functional NIK was required for successful ectopic kidney organogenesis, GFPþ metanephric kidneys were transplanted in wild-type or NIK/ omenta. Significant differences in graft size, structure, vascularization, or angiogenesis were not observed at 3 weeks after Figure 9 Pro-angiogenic gene expression in kidneys engrafted in the omentum and lymph node (LN). A: Representative immunofluorescence stains for ephrin B2 (EFNB2; red) and plasminogen activator inhibitor-1 (PAI-1; red) on serial frozen sections of kidneys engrafted in wild-type (WT; top left panels) or lymphotoxin-b receptor knockout (LTbR/; bottom left panels) omenta (3 weeks after transplantation). Boxed areas are shown at higher magnification to the right. B: Scatter dot plot with bar graphs  SD presenting the number of PAI-1þ tubules per microscopic field of sections stained as in A. C: Representative immunofluorescence stains for EFNB2 and PAI-1 (red) on serial frozen sections of LN kidney grafts isolated from either mice treated with Control Ig (top left panels) or LTbRFc (bottom left panels) (3 weeks after transplantation). Boxed areas are shown at higher magnification to the right. D: Scatter dot plot with bar graphs  SD presenting the number of PAI-1þ tubules per microscopic field of sections stained as in C. Data presented in this figure are representative of one of three technical replicates per sample (n Z 3 per group). ***P < 0.001, ****P < 0.0001. Scale bars: 100 mm (A and C). GFP, green fluorescent protein.

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transplantation (Supplemental Figure S5, AeK), indicating that, although NIK could be activated by LTbR signaling in this system, it was dispensable for ectopic kidney organogenesis. Moreover, NIK-expressing cells could still be detected in kidneys engrafted in the NIK/ omentum 3 weeks after transplantation (Supplemental Figure S6), although NIK fluorescence intensity was much lower than that observed in kidneys engrafted in the wild-type omentum at the same stage. These cells were likely donor cells, which might have been trying to compensate for the missing host NIK function.

LTbR Plays a Role in Glomerular Adaptive Responses to Nx Demonstration that LTbR signaling was important for adult kidney repair would strengthen our hypothesis that kidney rebuilding approaches might benefit from the presence of a microenvironment that mediated LTbR signals. To our knowledge, there are no studies that investigated LTbR physiological role in kidney development or during compensatory growth. The structure of LTbR/ kidneys versus wild-type kidneys was first compared. LTbR did not appear to be essential for kidney development during embryogenesis because LTbR/ mice had normal kidneys with what appeared to be normal vascularization and structure (Supplemental Figure S7). This finding likely suggested

1798 differences between ectopic organogenesis and normal 1799 organogenesis. Indeed, although LTbR-angiogenetic sig1800 nals appeared to be important for ectopic kidney organ1801 ogenesis, in normal kidney organogenesis (which 1802 occurs mainly through vasculogenesis), LTbR was not so 1803 crucial. 1804 LTbR relative contribution to compensatory renal growth 1805 after removal of one kidney was therefore investigated to 1806 corroborate the idea that not only was LTbR signaling 1807 1808 beneficial for glomerular vascularization/angiogenesis in 1809 lymphoid sites, but it also might play a role in glomerular 1810 adaptation to renal mass reduction. 1811 Contralateral compensatory renal growth and increased 1812 renal function followed contralateral Nx in experimental 1813 33 models. Accordingly, although kidneys of wild-type 1814 and LTbR/ mice had similar weights before surgery, 1815 LTbR/ mice showed significantly lower weights of their 1816 remnant kidneys 10 days after Nx (Figure 10A). The ½F101817 contralateral kidneys in wild-type mice showed glomerular 1818 enlargement as assessed in H&E-stained sections 1819 1820 (Figure 10B). These glomeruli often contained more eryth1821 rocytes, indicating an increase of renal blood flow. Differ1822 ences in adaptive responses seemed to be confined to the 1823 glomerulus, because tubular epithelial cells from both 1824 mouse groups showed a similar widespread positivity for 1825 the Ki-67 proliferation marker (Figure 10C). These data 1826 indicated a more efficient glomerular cell response to uni1827 lateral Nx in the presence of functional LTbR signaling. 1828

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Growing Kidneys in Ectopic Sites

Figure 10 Lymphotoxin-b receptor (LTbR) played a role in renal responses to nephrectomy (Nx). A: Scatter dot plot with bar graph  SD showing kidney weights before and after Nx (PreNx and PostNx, respectively) in both wild-type (WT) and LTbR/ mice. Each dot represents a mouse. B: Left panel: Scatter dot plot with bar graph  SD showing glomerular diameters in WT and LTbR/ PostNx kidneys. Each dot represents a glomerulus. Right panel: Representative hematoxylin and eosin (H&E) stain in Sham, WT_PostNx, or LTbR/_PostNx kidney paraffin sections. C: Left panel: Scatter dot plot with bar graph  SD showing the percentage of Ki-67þ tubular cell nuclei from WT and LTbR/ PostNx kidneys. Each dot represents one stained section. Right panel: Representative immunohistochemistry for Ki-67 in WT and LTbR/ PostNx kidney paraffin sections (3-amino-9-ethylcarbazole, red). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 50 mm (B and C).

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Discussion LNs and omenta can be used as bioreactors to grow organs. LN grafting is a unique approach pioneered by our laboratory,11,12,24,34,35 with no previous records in the literature. Conversely, the greater omentum has long been described as an ideal transplantation site mainly for its easy accessibility and large vascular network.36 Nevertheless, to our knowledge, cellular and molecular mechanisms that allow tissue and organ development within these locations have not been explored yet. By transplanting kidney rudiments either in the LNs of mice undergoing LTbR antagonist treatment or in the omenta of LTbR/ mice, it has been shown that host LTbR signals are crucial for obtaining a well-vascularized kidney graft. Indeed, defective host LTbR signaling correlated with decreased expression of endothelial and angiogenic markers in the transplanted tissues as well as structural alterations. Although the number of glomerular endothelial cells expressing the LTbR target NIK decreased in the absence of a functional LTbR, the results from the transplantation of kidney rudiments in the omenta of NIK/ mice indicated that host NIK is not required for successful ectopic kidney organogenesis. NIK and subsequent non-canonical NF-kB signaling have been described to regulate both inflammation-induced and tumor-associated angiogenesis.17 From this perspective, kidney angiogenesis in lymphoid tissues shares features with pathologic angiogenesis. However, the progressive reduction in NIK-expressing cells that was observed in glomeruli engrafted in LNs and omenta seems to suggest a tight regulation of angiogenesis in the ectopic kidney grafts, whereby NIK likely elicits a transient signal. In the wildtype omentum, for example, NIK and the neoangiogenic marker CD105 shared a similar pattern, with decreased expressions in engrafted glomeruli starting as early as the second week after transplantation, a pattern different from that of the endothelial cell marker CD31, which was quite stable for 4 weeks. These data seem to suggest that NIK is required during the early stages of engraftment, coinciding with early angiogenesis and tissue vascular integration, but is no longer needed in later stages of kidney maturation. NIK-mediated signals, however, are not necessary for kidney organogenesis in lymphoid sites, because overall vascularization/angiogenesis was not affected in NIK/ recipients. Besides activating non-canonical NF-kB pathway through NIK,16 LTbR could also trigger the canonical NF-kB route to stimulate endothelial cells.37 Moreover, other angiogenic signals elicited by the donor tissue itself must not be underestimated, as indicated by the data of PAI-1 production by the kidney tubular cells. How host LTbR signals can influence donor tissue production of PAI-1 and possibly of other angiogenetic molecules remains unknown.

16

Kidney tissue integration after transplantation likely involves extensive vascular remodeling, whereby several players are involved, including endothelial cells, smooth muscle cells, and fibroblasts. To record these dynamic processes is technically demanding, but not impossible. Transplanting GFPþ tissue in a wild-type recipient has been an effective method for visualizing the engrafted tissue later on, but the use of mice with constitutive or inducible endogenous fluorescent reporters, in combination with tracer injections and/or mosaic genetic recombination,38 would better allow characterizing the multicellular dynamics of angiogenesis and vascular remodeling in the transplanted tissues. Three-dimensional reconstruction of the engrafted kidneys that was recently performed on human fetal kidneys engrafted in the LN24 would also allow us to better quantify the differences in tubular elongation across the experimental groups. Indeed, the current adopted strategy of LTLþ tubule length quantification on two-dimensional sections cannot be considered accurate, because the apparent tubule length depends on the level at which the tissue was sectioned and observed. However, this strategy highlighted some important changes in the absence of a functional LTbR signal that warrant future study. Despite these limitations, the findings of host lymphoid stromal microenvironment contribution to ectopic kidney organogenesis seem to suggest implications for future development of new approaches to treat kidney disease. The renal sub-capsular space has served as the preferred site for kidney regeneration from embryonic metanephroi39,40 and recently kidney organoids.41e43 However, there is no convincing evidence that this approach can improve kidney function after a major injury. The inflammatory and fibrotic milieu of a host’s diseased kidney could constitute an adverse environment for cell and tissue engraftment and ultimately function. Finally, clinical progress in renal subcapsular transplantation has lagged behind other techniques because of the inelastic and tight nature of the human renal sub-capsular space.44 Additional strategies to engineer functional kidneys include repopulating synthetic/decellularized scaffolds with kidney-specific cells and transplanting these constructs orthotopically.45 Unfortunately, these approaches have often failed to recapitulate native kidney functions because of inadequate blood supply. Indeed, despite efforts to endow the scaffolds with an endothelium,45 it is still not possible to generate vascularized and perfused renal tissues that produce urine and/or have endocrine functions such as generation erythropoietin,46 necessary for erythropoiesis.47 Although reconstructing a fully functional kidney in the LN or the omentum for clinical purposes is challenging, stromal cells from these sites could be exploited to create an artificial scaffold for renal progenitor cells and thus provide an auxiliary aid during renal failure. A major concern about this approach might arise from the LTbR documented role in kidney disease.

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1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045

Growing Kidneys in Ectopic Sites Renal biopsies from lupus nephritis and IgA nephropathy patients showed elevated LT/LTbR levels, and LTbR inhibition improved renal function in a murine lupus model.48 LTbR-Ig was efficacious when given from week 3 to 7, whereas delaying the treatment until 5 weeks did not lead to significant results, highlighting a dynamic role for LTbR across different stages of disease.48 LTbR signaling in lupus likely results from the formation of tertiary lymphoid organs. Unlike secondary lymphoid organs such as the LN, tertiary lymphoid organs arise in response to nonresolving inflammation over the course of several years. Therefore, although tertiary lymphoid organ-mediated LTbR signaling leads to a prolonged, deregulated, and maladaptive response in injured kidneys that involves active inflammation and repeated unsuccessful attempts to repair the tissue, secondary lymphoid organ-mediated LTbR signaling might be exploited to support kidney development. The finding that glomerular adaptive responses to loss of renal mass were impaired in the absence of a functional LTbR indicated that LTbR can participate to repair responses after injury, although we cannot rule out that these responses might become maladaptive over time. In conclusion, although our results do not allow any unbiased conclusion on the precise cellular/molecular machineries that support kidney organogenesis in lymphoid sites, they point to an important function of the lymphoid stromal microenvironment in sustaining angiogenesis that warrants future investigation. Better understanding of the unique properties of this microenvironment might have implications for future engineering of functional organs.

Acknowledgments We thank Hannah Liu and Adam DeDionisio for technical support with tissue sectioning and immunofluorescence/ immunohistochemistry staining and Lynda Guzik (McGowan Institute Flow Cytometry Facility) for help with flow cytometric profiling of lymph node and omental stromal cell populations and for proofreading the manuscript. M.G.F. designed the study, performed most of the experiments, analyzed the data, prepared the figures, and wrote the manuscript; B.H. managed mouse colonies, performed the mouse surgeries, isolated omental stromal cells, and analyzed them by immunofluorescence; E.L. designed and supervised the study; all authors discussed the results and approved the final version of the manuscript.

Supplemental Data Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2019.08.018.

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