to characterize lymphatic architecture in the healthy human kidney, we analyzed tissue samples from 4 deceased organ donors with minimal chronic damage ( < 10 % interstitial fibrosis or tubular atrophy, supplemental table 1; supplemental material available online with this article; https: / / doi. org / 10. 1172 / jci168962ds1 ) ( 36 ). intact tissue samples ( < 3 mm ) were immunolabeled using a d2 - 40 monoclonal antibod y targeting podoplanin ( pdpn ) ( 37 ) and imaged using confocal or light - sheet fluorescence microscopy ( lsfm ) ( 16, 38 ). pdpn vessel networks were visualized in the human kidney cortex ( figure 1a and supplemental video 1 ), and antibod y - omitted controls displayed minimal autofluorescence or nonspecific binding ( supplemental figure 1a ). mapping vessel radius revealed a hierarchical network, with small lymphatics ( radius ~ 3. 5 μm ) initiating in the cortex and converging into larger vessels ( radius ~ 50 μm ) at the corticomedullary junction ( figure 1b ). the cells lining these vessels expressed prospero homeobox protein 1 ( prox1 ) ( figure 1c ), a canonical lec transcription factor ( 39, 40 ) but showed sparse expression of lymphatic vessel endothelial hyaluronan receptor 1 ( lyve1 ) ( figure 1d ), a glyco protein important for leukocyte entry into lymphatics ( 41 ). to elucidate the microanatomical localization of lymphatics in the human kidney, autofluorescent tissue signals were captured alongside pdpn labeling. large caliber lymphatics were observed adjacent to arteries at the corticomedullary junction branching into smaller cortical vessels ( supplemental video 2 ). colabeling with lotus tetragonolobus lectin ( ltl, proximal tubules ) and uromodulin ( umod, loop of henle ) revealed pdpn blind - ended lymphatics in the renal cortex ( figure 1e and supplemental video 3 ) and their absence from the medulla ( figure 1f ). despite previous reports of subcapsular lymphatics ( 42, 43 ), these were not detected in 3d reconstructions ( figure 1g ) or optical z - sections ( supplemental figure 1b ), even with the kidney capsule intact. in the cortex, lymphatics followed uae - i arterioles toward glomeruli ( figure 1h ), extending terminal branches near megalin ( lrp2 ) proximal tubules ( figure 1i ) and calbindin 1 ( calb1 ) distal nephron epithelium ( figure 1j and supplemental video 4 ). lymphatics converged toward the kidney hilum, adjacent to medullary dolichos biflorus agglutinin ( dba ) collecting ducts ( figure 1k ) and umod medullary tubules ( supplemental figure 1c ). a model summarizing these findings is presented in figure 1 l. source paper: pmc12435838 because of the rarity of lymphatics in the human kidney relative to other cell types, isolating sufficient lecs for molecular profiling is challenging. to surmount this, we leveraged published scrna - seq data from 59 kidneys, supplemented with 5 new samples ( supplemental figure 2a ). this integrated dataset comprised 217, 411 human kidney cells, with 151, 038 control samples ( living donor biopsies or unaffected regions of tumour nephrectomies ) and 66, 373 cells from diseased samples ( chronic kidney disease, ckd ) and kidney allograft injury; covering both chronic rejection and non - alloimmune etiologies ) ( supplemental figure 2b ). all cell types were manually annotate d ( supplemental figure 2c ), revealing 38 clusters ( figure 2a ), including a transcriptionally distinc t lec cluster containing 700 cells. from control samples, we curate d a transcriptional signature of healthy kidney lymphatics ( supplemental data 1 ), comprising 227 differentially expressed genes ( degs ) from 295 lecs. these genes were enriched for gene ontology ( go ) terms associated with lymphatic fate commitment ( go: 0060838, fold - enrichment > 100, fdr = 1. 66 × 10 ) and lymphangiogenesis ( go: 0001946, fold - enrichment = 67. 4, fdr = 8. 37 × 10 ). canonical lec markers were identified, including prox1 ( log 2 fc = 2. 97 ), pdpn ( log 2 fc = 2. 65 ), neuropilin 2 ( nrp2, log 2 fc = 2. 73 ), and c - c motif ligand ccl21 ( log 2 fc = 7. 23 ) ( figure 2b ). we also identified genes previously linked to kidney disease ( 44 – 46 ), such as fatty acid binding protein 4 ( fabp4, log 2 fc = 5. 69 ), trefoil factor 3 ( tff3, log 2 fc = 5. 58 ), and angiopoietin 2 ( angpt2, log 2 fc = 2. 46 ) ( figure 2b ). given the frequent use of prox1 and lyve1 to identify or target kidney lymphatics in preclinical studies ( 11, 14 ), we examined their expression within the human kidney in more detail. prox1 was detected not only in lecs, but also in loop of henle and distal convoluted tubule clusters ( figure 2b ), a finding validated by prox1 and e - cadherin ( cdh1 ) immunolabeling of medullary tubules ( figure 2c ) ( 47 ). in contrast to mouse data ( 38, 48 ), prox1 was not detected in vasa recta at both the transcript ( figure 2b ) and protein level ( figure 2d ). lyve1, meanwhile, was expressed by macrophages ( figure 2e ) as reported in mouse ( 49 ) and human kidneys ( 50 ), and it was also detected in glomerular ( figure 2f ) and peritubular capillary endothelium ( supplemental figure 1, d – e ). to probe the phenotype of human kidney lymphatics, and whether the vessels we detected included smooth muscle – lined collecting vessels ( 51 ) as in the lungs ( 52 ) and skin and mesentery ( 53 ), we costained kidneys for pdpn and α - smooth muscle actin ( acta2 ). kidney lymphatics in both hilum and cortex lacked smooth muscle coverage ( figure 2, g and h ). we corroborated this using subclustering analysis of our scrna - seq atlas, combining 295 lecs from healthy kidney with 157 additional cells from a recent study ( 54 ). this revealed 2 transcriptionally distinc t lec subclusters ( figure 2i ), which expressed lec capillary markers prox1, pdpn, and ccl21 ( figure 2j ) with sparse lyve1 expression, consistent with our imaging data ( figure 1d ). only rare cells, not specific for either subcluster, expressed molecular markers of lymphatic valve endothelial cells, gata2 and foxc2 ( figure 2j ). differential expression analysis between the 2 capillary subclusters identified 129 degs ( supplemental data 2 ). one subcluster was enriched for adipose signaling peptide neurotensin ( nts, log 2 fc = 3. 60 ) ( 55 ), and the other expressed ccl2 ( log 2 fc = 3. 30 ), cxcl2 ( log 2 fc = 4. 22 ), and icam1 ( log 2 fc = 4. 15 ) ( figure 2k ), indicative of capillaries involved in immune cell egress. to investigate if, akin to their blood vascular counterparts, kidney lymphatics possess an organ - specific signature ( 56, 57 ), we created a multiorgan human lec atlas by integrating our 452 kidney lecs with scrna - seq data of lecs from other organs, including skin ( n = 4, 765 cells ) ( 58 ), breast ( n = 4, 991 ) ( 59 ), heart ( n = 432 ) ( 60, 61 ), lung ( n = 1, 891 ) ( 62 ), and small and large intestine ( n = 462 and 471, respectively ) ( 63 ). the final dataset encompassed 13, 454 lecs from 19 anatomical sites ( supplemental figure 3a ). we resolved 5 transcriptionally distinc t subclusters ( figure 3a and supplemental data 3 ). four subclusters expressed lymphatic capillary markers ccl21 and lyve1, while the fifth expressed lymphatic valve markers foxc2 and integrin alpha 9 ( itga9 ) ( supplemental figure 3b ) ( 64 ). visceral organ – derived lecs ( kidney, heart, lung, intestines ) were predominantly grouped within 1 subcluster ( lec 1 ), whereas breast lymphatics were found in lec 1, lec 2, and lec 3, and skin lymphatics in lec 2, lec 3, and lec 4 ( figure 3b ). this spatial segregation was reflected in predicted transcription factor activity ( supplemental figure 3c ). comparative analysis ( supplemental data 4 ) identified 118 degs upregulated in kidney lecs compared with other organs ( figure 3c ).