Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction

Abstract

Background: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. Methods: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy) that abrogates its kinase ability. Vegfr3Chy/+ mice were examined for developmental abnormalities and kidney specific outcomes. Control and Vegfr3Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue clearing, light-sheet microscopy, and computational analyses. Results: In the kidney, VEGFR3 is expressed not only in lymphatic vessels, but also in various blood capillaries. Vegfr3Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in blood pressure, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low dose cisplatin (5 mg/kg), although Vegfr3Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length, but showed increased lymphatic density due to decreased cortical volume. Conclusions: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histological changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in chronic kidney disease requires assessment of changes to cortical volume.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity

“…We hypothesize that the absence of vascular congestion with MAZ51 treatment is due to the increased structural damage we observed and possible effects of MAZ51 on vascular injury and subsequent extravasation of red blood cells (26). Additionally, although VEGFR3 is restricted to the adult lymphatic endothelium, low levels of VEGF-R3 are also detectable in vascular endothelial cells, particularly in peritubular capillaries (22,81,82), and its expression may be upregulated in the blood vasculature during injury. In a study by Liu and colleagues (81), they showed that heterozygous mutation of VEGFR3 was required for the development of lymphatics in the kidney but did not have an adverse effect on kidney function in a model of low-dose Cp nephrotoxicity associated with mild injury.…”

“…Additionally, although VEGFR3 is restricted to the adult lymphatic endothelium, low levels of VEGF-R3 are also detectable in vascular endothelial cells, particularly in peritubular capillaries (22,81,82), and its expression may be upregulated in the blood vasculature during injury. In a study by Liu and colleagues (81), they showed that heterozygous mutation of VEGFR3 was required for the development of lymphatics in the kidney but did not have an adverse effect on kidney function in a model of low-dose Cp nephrotoxicity associated with mild injury. Genetic deletion of Vegfr3 (Flt4) increases vascular permeability through upregulation of VEGFR2 (55).…”

Lymphangiogenesis and Lymphatic Barrier Dysfunction in Renal Fibrosis

“…Although the authors proposed that renal fibrosis was not related to lymphatic proliferation in proteinuric situation, the relatively late start of treatment may have influenced the outcome. In another research, mouse model containing missense mutation in Vegfr3 did not exhibit change in renal function both in normal and low-dose cisplatin-mediated CKD [113]. However, they also proved cisplatin injury caused increased lymphatic density in renal cortex was due to decreased cortex volume, not due to changes in lymphatic volume or length.…”

Tissue Cytometry With Machine Learning in Kidney: From Small Specimens to Big Data

“…Technological advancements such as single cell RNA sequencing that provide high content information at the cell and molecular levels have enhanced our ability to further classify cells into subtypes, and study alterations in cell states, which could be linked to disease pathogenesis and outcomes (Park et al, 2018;Lake et al, 2019Lake et al, , 2021Wilson et al, 2019;Menon et al, 2020). Innovative approaches in high content and high-volume imaging of kidney tissue are also rapidly evolving (Winfree et al, 2017a(Winfree et al, , 2018(Winfree et al, , 2021Singh et al, 2019;Black et al, 2021a,b;Ferkowicz et al, 2021;Lipp et al, 2021;Liu et al, 2021;Melo Ferreira et al, 2021;Neumann et al, 2021), and these advancements are urgently needed to: (1) provide a platform of discovery based on imaging data, thereby delivering a unique context within an intact tissue environment and (2) anchor and interpret in situ emerging findings from technologies that lose the spatial context (Winfree et al, 2021). In the last decade, we saw an evolution of imaging kidney tissue from a qualitative toward a highly quantitative science (Winfree et al, 2017b(Winfree et al, , 2021Singh et al, 2019;Martins et al, 2020;Black et al, 2021a,b;Melo Ferreira et al, 2021;Neumann et al, 2021).…”

“…The ability to image multiple labels simultaneously (multiplexing) has significantly increased the depth of content acquired (Singh et al, 2019;Woloshuk et al, 2020;Ferkowicz et al, 2021;Melo Ferreira et al, 2021;Neumann et al, 2021). Furthermore, imaging in all 3 dimensions using optical sectioning has allowed faithful preservation of tissue architecture and spatial context (Puelles et al, 2016;Klingberg et al, 2017;Winfree et al, 2017b;Ferkowicz et al, 2021;Lake et al, 2021;Liu et al, 2021). These advancements were catalyzed by the availability of novel software tools that allow streamlined image processing and quantitative analysis (Dao et al, 2016;Winfree et al, 2017a;Czech et al, 2019;Stoltzfus et al, 2020).…”