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Background: Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease. As injury-related lymphangiogenesis is driven by signaling from the receptor VEGFR3 (vascular endothelial growth factor receptor 3) in response to the cognate growth factor VEGF (vascular endothelial growth factor)-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remain poorly defined., Methods: We generated a new mouse model expressing Vegfc under regulation of the nephron progenitor Six2Cre driver strain ( Six2Vegfc ). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and 3-dimensional imaging., Results: Six2Vegfc mice had reduced body weight and kidney function compared with littermate controls. Six2Vegfc kidneys demonstrated large peripelvic fluid-filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. Three-dimensional imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+ (lymphatic vessel endothelial hyaluronan receptor-1)/PDPN+ (podoplanin)/VEGFR3+ lymphatic capillaries extending alongside EMCN+ (endomucin) peritubular capillaries. There was no change in EMCN+ peritubular capillary density., Conclusions: Kidney lymphatic density was robustly increased in the Six2Vegfc mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR3. The model resulted in malformation of the lymphatic hilar plexus, resulting in severe hydronephrosis that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGFC signaling during kidney development and provides new insight into human renal lymphatic malformations., Competing Interests: S.E. Quaggin holds patents related to therapeutic targeting of the ANGPT-TEK (angiopoeitin-TEK receptor tyrosine kinase or Tie2) pathway in ocular hypertension and glaucoma and vascular diseases and owns stock in Mannin Research. S.E. Quaggin also receives consulting fees from AstraZeneca, Janssen, the Lowy Medical Research Foundation, Novartis, Pfizer, Janssen, Unity Biotechnology, and Roche/Genentech. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The other authors report no conflicts.

Lymphatic vessels are thought to arise from PROX1-positive endothelial cells (ECs) in the cardinal vein in response to induction of SOX18 expression; however, the molecular event responsible for increased SOX18 expression has not been established. We generated mice with endothelial-specific, inducible expression of an RAF1 gene with a gain-of-function mutation (RAF1(S259A)) that is associated with Noonan syndrome. Expression of mutant RAF1(S259A) in ECs activated ERK and induced SOX18 and PROX1 expression, leading to increased commitment of venous ECs to the lymphatic fate. Excessive production of lymphatic ECs resulted in lymphangiectasia that was highly reminiscent of abnormal lymphatics seen in Noonan syndrome and similar "RASopathies." Inhibition of ERK signaling during development abrogated the lymphatic differentiation program and rescued the lymphatic phenotypes induced by expression of RAF1(S259A). These data suggest that ERK activation plays a key role in lymphatic EC fate specification and that excessive ERK activation is the basis of lymphatic abnormalities seen in Noonan syndrome and related diseases.