Endothelial Cell Identity, Heterogeneity and Plasticity in the Kidney

Endothelial monolayers line the inner surface of the whole circulatory system, from heart chambers to arterial, capillary, venous, and lymphatic vessels. All endothelial cells share specific functional characteristics, for instance production of anticoagulant and vasoregulatory mediators. However, depending on the vessel type and physiologic environment, there also is substantial functional and structural endothelial cell heterogeneity.^1,2 In this issue of JASN, Dumas et al.³ provide evidence for at least 24 distinct kidney endothelial cell phenotypes and show robust gene expression changes induced by water deprivation in renal medullary endothelial cells.

The endothelial identity⁴ is first recognizable in mesoderm-derived hemangioblasts expressing the KIT protoconcogene and TAL1 (TAL bHLH transcription factor 1, erythroid differentiation factor). These give rise, via intermediate CD34 and TEK (TEK receptor tyrosine kinase)–expressing hemogenic endothelial cells, to both hematopoietic stem cells and endothelial progenitor cells. Cells of the hematopoietic lineage express PTPRC (protein tyrosine phosphatase receptor type C), which is absent from endothelial progenitor cells. In tissues, self-renewing pools of endothelial progenitor cells give rise to differentiated endothelial cells, which continue to express CD34, and endothelial cell markers² including vascular endothelial cadherin, KDR (kinase insert domain receptor), VWF (von Willebrand factor), PECAM1 (platelet-endothelial cell adhesion molecule 1) and NOS3 (nitric oxide synthase 3), among others.

During embryogenesis the renal vasculature develops, in part, from hemogenic and endothelial progenitor cells resident in the metanephric mesenchyme.⁵ These cells assemble, through homotypic interactions, into endothelial cell cords and undergo endothelial cell differentiation and lumen formation in situ, the process of vasculogenesis. There also is invasion of the developing kidney by extrarenal angiogenic sprouts that grow into the embryonic kidney alongside the ureteric bud and connect vessels formed in situ to the systemic circulation. So far, intrarenal processes that lead to endothelial differentiation have been studied most comprehensively for glomerular endothelial cells whose recruitment into developing glomeruli and phenotypic differentiation is driven by podocyte-derived vascular endothelial growth factor A and a complex interplay between cell guidance molecules, Angiopoietin, Notch, and TGF-β signaling.

Renal blood vessels are organized sequentially, in that glomerular capillaries lie between afferent and efferent arterioles, and the peritubular vascular bed arises exclusively from efferent glomerular arterioles.¹ Glomerular afferent arterioles control blood flow and pressure in glomerular capillaries, which in turn serve as permselective filters of plasma. The glomerular capillary bed is followed by contractile efferent arterioles which are critical for maintaining and regulating the intraglomerular hydraulic pressure. Peritubular capillaries arising from efferent arterioles allow for return of reabsorbed tubular fluid into the circulation. In the renal medulla, the postglomerular descending and ascending vasa rectae are essential for the countercurrent exchange that maintains the medullary concentration gradient. Blood from peritubular capillaries and the ascending vasa recta drains into venules whose endothelial cells display surface receptors required for immune surveillance.

The fully developed renal vasculature is characterized by significant structural and functional heterogeneity of endothelial cells.^1,5 Endothelial cell height, their orientation relative to blood flow, and the presence and absence of fenestrae all distinguish endothelial cells structurally. Endothelial cells overlying renin-producing juxtaglomerular cells in the afferent arteriole, glomerular capillary, peritubular capillary, and ascending (but not descending) vasa recta endothelial cells are all fenestrated. Even so, only the fenestrae of peritubular and ascending vasa recta endothelial are spanned by PLVAP (plasmalemma vesicle associated protein)-containing diaphragms. Fenestrae of glomerular and juxtaglomerular afferent arteriolar endothelial cells are not diaphragmed. A number of other phenotypic differences between endothelial cells—including distinct cell-surface molecules, distinct transcription factors, and differences in the abundance of characteristic endothelial cell proteins like KDR, VWF and NOS3—have also been described, but they have not been systematically characterized for kidney endothelial cells until now.³

Reversion of endothelial cells to the mesenchymal phenotype (endothelial-mesenchymal transition), required for formation of the endocardial cushion and heart valves, also occurs in fibrotic disorders and in transplant arteriopathy,⁶ and endothelial cell dedifferentiation is an observed response to endothelial cell injury in vivo. Endothelial cell repair mechanisms probably involve both proliferation and redifferentiation of injured endothelial cells and repopulation by resident and circulating endothelial progenitor cells, followed by differentiation. Hence, there is substantial endothelial cell plasticity⁶ with a range of possible endothelial cell phenotypes, although some renal endothelial cell subtypes may also be caught in a terminally differentiated state.

Dumas et al.³ evaluated RNA expression profiles for rigorously selected, single, viable endothelial cells from distinct pools of cortical, medullary, and glomerular endothelial cells. Using bioinformatic algorithms, they then grouped them into clusters and subclusters based on their RNA expression profile, relying on “canonical marker genes” for identification of arterial, venous, and capillary endothelial cells. Association of other previously identified endothelial cell–subtype markers—for instance plasmalemma vesicle–associated protein for endothelial cells with diaphragmed fenestrae and EHD3 (EH domain containing 3) for glomerular endothelial cells—along with profiles of the most highly expressed genes in each cell, allowed identification of subclusters within the larger groups. Intermediate phenotypes that displayed overlapping gene expression profiles, for instance arterial/fenestrated endothelial cells, and venous/fenestrated capillary endothelium were identified. In all, 24 distinct expression subclusters were cataloged with a rich data set that is available for mining. Data are still lacking for lymphatic endothelial cells and resident renal endothelial progenitor cells seem to have escaped detection.

This work implies that the simplistic notion of a few endothelial cell subtypes needs to be revised. The many intermediate endothelial cell phenotypes identified by Dumas et al.³ suggest that endothelial cell differentiation is modular, allowing functional gene groups to be expressed in various combinations. Their work also suggests that regional differences in gene expression result from stimuli derived from neighboring cells and through physiologic changes, and that these are not likely to be static. Furthermore, there seem to be no clear-cut boundaries between endothelial cells of distinct vascular segments; endothelial cells in related subclusters are probably intermingled in the boundary regions.

Dumas et al.³ also show, for the first time, adaptation of medullary endothelial cells to hypertonicity, with robust upregulation of transporters required for accumulation of cytoprotective organic osmolytes and induction of genes required for oxidative phosphorylation, implying an increase in energy need by these medullary endothelial cells in response to water deprivation. So, we have here the first critical evaluation of a physiologic response by medullary endothelial cells in vivo.

The approach taken by Dumas et al.³ should be extremely useful for interrogation of endothelial cell responses to disease, and it is hoped that it will be expanded to human disease. Exploration of mechanisms that lead to capillary rarefaction in renal fibrosis, the endothelial cell responses to diabetes and AKI, the phenotypic changes in peritubular capillary endothelial cells observed in chronic transplant nephropathy… the list of important future work is very long, and the harvest should be rich.

Disclosures

None.

Funding

Work in the author’s laboratory is supported by the Heart and Stroke Foundation of Canada (grant HSFC G-16-00013991) and by the Natural Sciences and Engineering Research Council (grant NSERC RGPIN-2016-05609) of Canada.