Endothelial cell plasticity in kidney fibrosis and disease

Renal endothelial cells demonstrate an impressive remodeling potential during angiogenic sprouting, vessel repair or while transitioning into mesenchymal cells. These different processes may play important roles in both renal disease progression or regeneration while underlying signaling pathways of different endothelial cell plasticity routes partly overlap. Angiogenesis contributes to wound healing after kidney injury and pharmaceutical modulation of angiogenesis may home a great therapeutic potential. Yet, it is not clear whether any differentiated endothelial cell can proliferate or whether regenerative processes are largely controlled by resident or circulating endothelial progenitor cells. In the glomerular compartment for example, a distinct endothelial progenitor cell population may remodel the glomerular endothelium after injury. Endothelial‐to‐mesenchymal transition (EndoMT) in the kidney is vastly documented and often associated with endothelial dysfunction, fibrosis, and kidney disease progression. Especially the role of EndoMT in renal fibrosis is controversial. Studies on EndoMT in vivo determined possible conclusions on the pathophysiological role of EndoMT in the kidney, but whether endothelial cells really contribute to kidney fibrosis and if not what other cellular and functional outcomes derive from EndoMT in kidney disease is unclear. Sequencing data, however, suggest no participation of endothelial cells in extracellular matrix deposition. Thus, more in‐depth classification of cellular markers and the fate of EndoMT cells in the kidney is needed. In this review, we describe different signaling pathways of endothelial plasticity, outline methodological approaches and evidence for functional and structural implications of angiogenesis and EndoMT in the kidney, and eventually discuss controversial aspects in the literature.

such as vascular permeability and leukocyte rolling/adhesion during inflammatory processes. 9Thereby, ECs structure and facilitate vascular transport of oxygen, nutrients, and various blood cells. 2 ECs display a versatile cell population with a remarkable remodeling potential.4][15] Therefore, it is crucial to take a specific look at each organ and its individual EC-subpopulations, respectively, if one is to fully understand EC plasticity and its respective impact in health and disease.
4][15] It can be assumed that these subpopulations demonstrate distinct potential for cell plasticity. 14he circulation in the kidney resides in two distinct capillary beds that are connected in series.As distinctive capillary beds and EC subpopulations are facing different physiological conditions 22 such as changes in osmolarity, oxygenation, large molecule flux, perfusion, or blood pressure, they potentially adapt differently to environmental changes.][25][26][27][28][29] This review serves to summarize endothelial remodeling in the kidney, with the focus on angiogenic potential in renal disease and renal endothelial-to-mesenchymal transition (EndoMT).We outline what is already known and what might yet to be elucidated to understand EC plasticity in the kidney and its contribution to disease and regenerative processes.

| EC PLASTICITY-AN OVERVIEW
ECs hold a remarkable plasticity potential in prenatal development 24,[30][31][32][33] as well as postnatally. 14,19Several different pathways are described, which lead to different cellular outcomes (Figure 1).The best understood EC-remodeling process is probably the principle of angiogenesis, a highly orchestrated process leading to the branching of emerging vessels. 34,35

| ANGIOGENESIS
Angiogenesis in vessels is driven by the lack of oxygen or nutrients 36,37 -an environment which can occur in health, e.g. during growth, as well as in disease. 36Angiogenesis is hence closely linked to inflammatory processes 38 or tumor progression. 391][42] Led by proangiogenic signals, tip cells are the spearhead in angiogenic sprouting. 36,41Stalk cells follow upon tip cell migration and actively elongate the new vessel through proliferation, thereby generating the lumen of the new vessel. 40,41,43However, the determination of tip cells and stalk cells is never ultimately settled, 36,44 but rather a very dynamic process in which the heading cells are continuously competing about the tip cell position. 36,44his competition examples the dynamic potential of EC plasticity. 45n preparation for the migratory sprouting process, the basement membrane is disrupted, which causes the loss of cellular polarity in future tip cells. 46,47Angiogenesis further requires an extensive cytoskeletal rearrangement, especially in tip cells, 36,43 which incorporate actin, vimentin, and microtubules. 43Upon the cytoskeletal changes, tip cells extend multiple filopodia at distal tips for guidance and migration, 41 while maintaining endothelialspecific cell adhesion marker CD31/Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1). 44,48,49The tip cell then advances through 3-D extracellular matrix (ECM) by extending and retracting their peripheral processes. 43,50his process is supported by disintegrin, metalloproteinase 17 (ADAM17), 43,50 and other matrix metalloproteinases (MMPs). 51,52Eventually, sprouting subsides when metabolic tissue needs are fulfilled, and proangiogenic signals diminish. 36Nascent sprouts can also form a new circuit by fusing with other tip or stalk cells 36,40 and form new anastomoses with resting ECs. 40If a new sprouted vessel is not perfused or experiences low shear stress, it can also prune. 53n contrast to the above-described sprouting angiogenesis, intussusceptive angiogenesis describes the duplication of the lumen via splitting. 45,54Both, sprouting angiogenesis and intussusceptive angiogenesis participate in tissue repair. 45,55,56During the maturation of nascent vessels, also perivascular cells, such as vascular smooth muscle cells are recruited. 12,57At this point it is important to mention that postnatal angiogenesis is not carried out routinely in all organs. 45There are selected organs like the placenta, in which angiogenesis takes place regularly.In many other organs, however, postnatal angiogenesis occurs rather upon stimuli such as muscle growth 58 or memory acquisition including neurogenesis in the brain. 59In pathophysiological settings, also injury 45,60 or tumor growth 61 can induce angiogenesis.

| Signaling in angiogenesis
The key signaling pathway of angiogenesis is regulated by vascular endothelial growth factor A (VEGF-A). 36,62fter its secretion from hypoxic tissue, VEGF-A binds to endothelial VEGF-receptor 2 (VEGFR2). 36,62Based on gradient and concentration of VEGF-A, VEGFR2 signaling induces migration in tip cells and proliferation in stalk cells. 41][65] Next to DLL4 also other Notch-ligands such as Jagged1 regulate cell fate during angiogenesis in mammals. 63,66agged1 binding to Notch, presumably Notch1, is opposing the effects of DLL4 binding to Notch and hence 2 different ligands of the same receptor can regulate angiogenic sprouting including the dynamic process of endothelial tip cell competition. 67Since Notch ligands and receptors are transmembrane proteins, this signaling pathway operates through intercellular signaling of physically adjacent cells. 68,69VEGFR1 is less investigated than VEGFR2, but also crucial for angiogenesis, especially during embryonic development and supposedly prevents overshooting vessel growth. 70,71Furthermore, VEGFR1 was also shown to decrease tumor growth. 70his could be due to modulation of VEGFR2-mediated proliferative effects by VEGFR1 signaling as cell division was found to be increased under VEGFR1 deletion leading to lethal developmental vasculature overgrowth 72,73 and effects of VEGFR1 loss could be rescued by truncated VEGFR1. 73Loss of VEGFR1 was furthermore reported to reduce sprouting of embryonic vessels in vivo and reduce vasculature branching in vitro because of decreased cell migration and defective sprout formation. 74aken together, angiogenesis is tightly regulated by several mediators and constant wavering of VEGFR1 & 2, NOTCH1 and DLL4 signaling in individual stalk cells promoting an ongoing tip cell competition. 36,44Finally, also coregulation of Notch target genes by bone morphogenetic protein (BMP) seems to orchestrate the tip and stalk cell competition. 75BMP signaling can be regulated by EC themselves or their surrounding intercellular environment during angiogenesis. 76Importantly, tip and stalk cells stay in physical contact 47,77 and continue vascular endothelial Cadherin (VE-Cadherin/Cdh5) expression, 78,79 which furthermore supports the fusion of filopodia of different tip cells during anastomosis. 80ransforming growth factor β (TGFβ)-signaling is also known to play a significant role in angiogenesis, vascular development, and maintenance of vascular physiology. 81,82Pro-and anti-angiogenic characteristics of TGFβ-signaling in ECs have been described, which are regulated by phosphorylation of different Smad proteins. 81,83Smad proteins support the signal transmission from the cell surface to the nucleus, 84 which then activates or represses the transcription of specific genes. 85This distinct gene regulation is known to be determined by two different activin receptor-like kinase (ALK) pathways: TGFβ/ALK1 and TGFβ/ALK5. 83The TGFβ/ALK1-pathway leads to the phosphorylation of Smad1/5, which induces cell migration and proliferation. 83In contrast, the TGFβ/ALK5-pathway leads to phosphorylation of Smad2, which inhibits cell migration and proliferation. 83ALK1-mRNA was only found in ECs, whereas ALK5-mRNA was also found in other cellular populations. 83This underlines the complex role of downstream TGFβ-signaling in EC plasticity such as angiogenesis.TGFβ binding can be further enhanced through the TGFβ co-receptor Endoglin (CD105), which is upregulated during tumor-and embryogenic angiogenesis. 86,87Furthermore, TGFβ-signaling also launches recruitment of vasculature surrounding mural cells such as pericytes and vascular smooth muscle cells. 12,81,88][91] Angiopoietins (Ang) like Ang1 and Ang2 are also known signaling players with complex impact on angiogenesis 60,92 and bind to the Tyrosine-protein kinase 2 (Tie2) receptor in ECs. 60,92Constitutive Ang1 signaling is fundamental for vascular development, 60 and maturation 92 and promotes EC survival during angiogenic stimuli. 12Ang1 also influences adhesion and migration of ECs 92,93 and is furthermore expressed by perivascular cells. 92,94Moreover, Ang1-deficient mice lacked perivascular cells. 92,95In contrast, Ang2 signaling may be antagonistic to Ang1 signaling and has been found in adolescent tissue that naturally undergoes vascular remodeling like the placenta or the uterus. 60,96Ang2 further promotes the breaching of intercellular connections between ECs and perivascular cells, and cell death of vessel regressions.Interestingly, in the presence of VEGF-A signaling, Ang2 enhances angiogenesis. 92,97Ang2 is hardly expressed in resting ECs but gets upregulated and secreted upon VEGF-A signaling. 92,98Furthermore, high levels of Ang2 prevent pericyte recruitment while ECs undergo tip/stalk cell competition. 92ther pro-angiogenic signaling pathways include involvement of Fibroblast growth factor receptor (FGFR), 86,99 Wingless-related integration sites (Wnts), 86,100 transcription factors Snail (SNAI1) and Slug (SNAI2), 101 and Hypoxia-inducible transcription factors (HIF). 12,102enetic knock-in and knock-out (KO) mutations of Wnts/ Frizzled/β-catenin pathway suggested a role of Wnts and their fellow players in angiogenesis. 86,100Thus, Wnt regulates transcriptional and posttranslational signaling during angiogenesis, such as VEGF-A. 103,104In vitro experiments suggested Snail and Slug expression in sprouting ECs 101 and their genetic deletion prevented EC sprouting and migration. 101This could be partly explained by Slugmediated expression of membrane type 1-MMP, 105 as MMPs are important for EC migration and sprouting. 51,52urthermore, DLL4 was found to be an important downstream effector of Slug signaling, 106 which is highly expressed in tip cells during angiogenesis. 64,92Consistently, Slug KO mice showed reduced Notch activation and down scaling of EC proliferation.This caused a transient delay of vascularization, which was followed by a compensatory increase of tip cell number and vessel density as compared to murine wildtypes. 106Lastly, the expression of proangiogenic receptors and cytokines, such as VEGF, TGFβ and others, are often promoted by HIF, which is upregulated in oxygen-deprived tissues. 12,1023.2 | Where do angiogenic ECs emerge from?
Stem cell-mediated vessel formation during embryogenesis is commonly referred to as "vasculogenesis."It is distinct from new vessel formation during adult life, where preexisting blood vessels may undergo "angiogenesis" through sprouting and differentiation into tip and stalk cells, respectively. 107However, first evidence for vasculogenesis in adult life emerged when hematopoetic stem cell marker expressing endothelial progenitor cells were isolated from human peripheral blood. 108This suggested the existence of "stem cell"-like EC precursor cells (EPCs) 89 within the blood-so-called circulating endothelial precursor cells 108 ; as well as within the vasculatureso-called resident endothelial stem cells. 25,45,109,110EPCs define as capable of differentiating into endothelial cells while displaying clonal and stemness features 111 and are commonly identified by double or triple combination of the cell surface markers CD34, VEGFR-2 and CD133. 107owever, the lack of a specific marker hampers a clear separation from other hematopoetic cells as well as from mature ECs, which also express CD34 in the kidney and several other organs. 112irculating EPCs are described to be bone-marrowderived. 89,113,114Their differentiation and chemotaxis is regulated by VEGF 89,113,115 and their integration into growing vessels may postnatally contribute to vascular repair as well as to abnormal neovascularization. 89In general, circulating EPCs from human plasma can differentiate into blood-island-like cell clusters in vitro, integrate into murine capillary vessel walls after in vivo injection post injury, 108 and play a pivotal role in tumorangiogenesis. 116,117umerous studies have pointed out a major regenerative role of resident EPCs as summarized by Pasut et al. 45 : Wakabayashi et al. identified a CD157 and CD200 expressing population of residing ECs with stem cell and clonal colony expansion potential in bigger vessels of numerous murine organs 45,110 and contributed to vessel repair through angiogenic program activation.Furthermore, resident EPCs were shown to have homogeneous PDGFRα expression, 45,109 a marker that also identifies mesenchymal precursor cells. 24,109It has been shown that endovascular progenitor cells can initiate different trajectories for ECs-slow proliferation as well as maturation. 45,109n addition to circulating or resident EPCs, quiescent ECs may also contribute to angiogenesis.In 2018, Manavski et al. delivered evidence for clonal expansion of resident endothelial cells using transgenic Cdh5-Cre-ERT2-Confetti reporter mice which enable distinguishing between individual ECs through the expression of multiple fluorescent proteins. 25Thus, the authors documented hypoxia-induced clonal expansion of resident Cdh5-lineage cells in several organs. 25,45Since Cdh5 is a marker of mature ECs, these results suggested a role of pre-existing ECs in vessel repair.However, the data did not provide final conclusions on whether clonal expansion might have derived from a distinct Cdh5-positive progenitor population or from potentially any resident EC in the investigated organs.In the same year, McDonald et al documented a clear regenerative role of differentiated ECs when studying regeneration of aortic walls using multicolor lineage tracing and single cell transcriptomic analysis.Thus, differentiated ECs flanking the injury site entered the cell cycle and proliferated, which was largely dependent on the stress response gene Activating transcription factor 3 (Atf3).In summary, this study demonstrated that quiescent ECs of large arteries can enter mitosis and drive vessel regeneration independent of a distinct circulating or resident EPC population. 118

| Potential and cellular sources of angiogenesis in kidney disease
Tanaka and Nangaku pointed out the importance of understanding protective and dysregulated angiogenic factors in patients suffering from chronic kidney disease (CKD). 119Hypoxia is a key driver of CKD and despite endogenous upregulation of proangiogenic factors such as soluble VEGFR1 in early disease stages, VEGF-A expression is reduced in progressed CKD stages and vascular rarefication displays a hallmark of CKD progression.However, VEGF achieved controverse results regarding tubular damage as nascent vessel formation is often associated with inflammation and edema due to leaky and tortuous lumina. 119Dysregulated angiogenesis in CKD patients may be caused by distinct elevated or reduced soluble angiogenic modulators 120 and must be taken into account when one is to understand the contribution of remodeling ECs in kidney disease.
Circulating EPCs may play a role in acute and chronic kidney disease as reviewed by Ozkok et al. in 2018. 107Circulating EPC counts correlated with glomerular filtration rate 121 and are decreased in CKD patients. 122However, there is conflicting data on whether or not bone marrowderived EPCs are homed into injured renal vasculature 123,124 and have been reported to participate generally seldomly in angiogenic events. 89,114Of note, circulating EPCs seem to be less abundant in murine models as compared to other experimental animals 125 or humans, 116 indicating the need for more non-murine studies.
A study from 2021 characterized renal glomerular EC (GEnC) precursor cells leveraging Cdh5-Cre-ERT2-Confetti reporter mice in combination with serial intravital microscopy of the same glomerulus in single cell resolution and over time.This study demonstrated rapid and clonal expansion of single GEnC precursors in response to hypertensive, hyperglycemic, and laser-induced glomerular injury.Independent of the injury type, clonal EC expansion originated from the terminal afferent and efferent arteriole segment, suggestive of resident EPCs in this distinct location. 19The existence of resident EPCs in juxtaglomerular position 19 would allow for controlled replacement of glomerular ECs through clonal expansion along existing glomerular capillaries and may demonstrate a novel therapeutic target for enhanced glomerular vascular regeneration. 126evertheless, the existence of a respective precursor subpopulations in other vascular compartments of the kidney remains undefined.Do resident EPCs exist within the renal peritubular capillary bed and scattered in between non-progenitor ECs?This would imply the existence of distinct peritubular EC subpopulations with different regenerative potential.Or can every resident capillary EC proliferate, thus displaying the same angiogenic potential?Which cells regenerate bigger arteries and veins within the kidney?These questions remain yet to be unknown and invite future investigations.As chronic kidney disease commonly comprises vascular rarefaction and thus declining tissue oxygenation and disease progression, 127 stimulation of angiogenic programs may provide an attractive therapeutic intervention to halt CKD onset and progression.Thus, answers to the above-mentioned questions will be needed to better understand the underlying mechanisms and potential of angiogenesis in the kidney and to develop adequate therapeutic approaches.

| EN DOT HEL IAL -T O -HEM ATOPOIETIC TRANSITION (EHT)
During embryonal development, ECs may further undergo endothelial-to-hematopoietic transition (EHT) 31 -a process generally also referred to as hemogenic endothelium. 128EHT has been documented through cell fate tracing in transgenic murine embryos 32,129 and in vivo imaging in transgenic zebrafish embryos. 33EHT is initiated through switching the EC transcriptional program to the hematopoietic program. 31,130,131This is followed by profound structural changes, such as cell rounding and the disruption of tight junctions to neighboring cells.Finally, EHT cells are released into the blood stream as hematopoietic cells. 31,333][134][135] Upon EHT, Notch, BMP and TGFβ signaling pathways are initially upand later downregulated. 31,128,136,137Also, the activation of Smad2/3 and Smad1/5 were found to support the initiation of EHT. 31,138,139In vitro, EHT can be forcefully stimulated also in mature ECs.Furthermore, hematopoietic cells emerging through EHT revealed hematopoietic stem cell characteristics and were able to perform adaptive immune function in an in vivo experimental set up. 45,140

| EHT in the kidney
EHT takes place in the embryonic development of the kidney. 141To our knowledge, EHT has never been detected in postnatal conditions in the kidney.

ENDOTHELIAL-TO -MESEN-CHYMAL TRANSITION (EndoMT)
EndoMT was first described in the embryonic development of the heart valves 142 and may play physiological as well as pathophysiological roles.EndoMT refers to the transition of an EC into a mesenchymal cell type, which further associates with loss of cell-cell contacts to neighboring ECs. 143ndoMT shares many similarities with the related phenomenon of epithelial-to-mesenchymal transition (EMT)-the transformation of an epithelial cell into a mesenchymal phenotype cell. 20,47,144Since EMT is better understood, 20 EndoMT is often referred to EMT processes.
Plasticity upon EMT includes the disassembling of epithelial cell-cell contacts; either by degradation or relocalization.Suppression of polarity complex proteins leads to loss of epithelial cell polarity. 144,145Then, epithelial gene expression is repressed, whereas the expression of mesenchymal genes is activated. 144,146Next, the cortical actin cytoskeleton reorganizes itself: Formation of lamellipodia, filopodia, and invadopodia enables cell motility and invasive capacity. 144,147,148These cellular protrusions and extensions facilitate cellular elongation with directional, migrational and sensory capabilities and proteolytic function for ECM degradation by expression of MMPs. 144,149inally, the formation of stress actin fibers increases the cellular contractility under EMT. 144,150It is generally assumed that respective cellular modifications in EndoMT run likewise as in EMT. 47,151C-fate upon EndoMT may be multifaceted.Thus, differentiation into multiple cell types has been documented, including: fibroblasts, 152 smooth muscle cells, 152 pericytes, 24 adipocytes, 153 osteoblasts, 153 chondrocytes, 153 and myofibroblasts-known executor cells in fibrotic tissue remodeling. 23,26,154,155Thus, EndoMT may be involved in physiological as well as pathophysiological pathways.
EndoMT is defined by the loss of endothelial markers and the de-novo expression of mesenchymal markers: In early EndoMT, EC markers like CD31/PECAM1, 49 are partially downregulated 20,156 and mesenchymal markers like α-Smooth muscle actin (αSMA), Smooth muscle protein 22-α (SM22α), Fibroblast-specific-protein-1/ S100A (FSP1/S100A) and CD44 are upregulated. 20,157urthermore, EndoMT cells can be detected by disturbed cell junctions 20 whilst possibly being still close to their original cell cluster.In late EndoMT, classical EC markers are moderately or strongly downregulated. 20Furthermore, mesenchymal markers such as Calponin 20,152,158 ), Smoothelin, 20,152 Fibronectin, 20,152 Tenascin, 20,152 Collagen III, 20,152 Thymus cell antigen 1 (THY1), 20,152 Vimentin, 20,159 Notch3, 20,160 Stem Cells Antigen-1 (SCA1), 20,161 Zinc finger E-box-binding homeobox 2 (ZEB2), 20,162 and MMP2 & 9 20,163 are upregulated. 20raditionally, EndoMT processes were detected using histological co-staining of mesenchymal and endothelial markers.Alternatively, genetic EC cell fate tracing in combination with histology for mesenchymal markers was applied. 20,23,24][166][167] The loss of endothelial markers evokes challenges for the investigation of EndoMT.Without the use of genetic lineage tracing, identification of ECs, which underwent EndoMT is limited to early EndoMT stages and hampers accurate estimation of the extend and role of EndoMT in health and disease. 20any insights regarding renal EC-remodeling have been generated using constitutive Tie2-Cre mouse models. 23,26,27,29However, some limitations need to be taken into account when working with Tie2-Cre mice for ECspecific lineage tracing.First, Tie2 is not a strictly specific marker for ECs and was also found in pericytes 168 and pericyte progenitors. 169Of note, pericytes are also known precursor cells of myofibroblasts. 164,170Thus, the Tie2-Cre model may lead to confounded results, when investigating the role of ECs as myofibroblast precursors.Furthermore, Tie2-Cre mice also enabled Cre-recombinase in hematopoietic cells. 171Secondly, constitutive Tie2-Cre models do not allow for inducible Cre-mediated recombinase and facilitate recombination and thus expression of fluorescent reporter proteins (e.g., EGFP) in any cell, which ever expressed Tie2 before or after birth.In contrast, the gold standard for genetic lineage tracing experiments involves inducible and thus conditional genetic recombination of a defined cell type at a defined time point due to the presence of an inducer (e.g., Tamoxifen or Doxycycline).This approach results in generation of a defined labeled cell population that can then be fatetraced upon wash out of the inducer. 172,173nother common marker for genetic EC lineage tracing, is Cdh5, which was exploited for analysis of functional, 28 metabolic 174,175 and signaling aspects 27,28,[176][177][178][179] of EndoMT in the kidney.While Cdh5 identifies endothelial cells quite specifically, [180][181][182] it must be noted that Cdh5-mediated Cre-recombinase at E7.5 of embryonic development was also found suitable for fate mapping of embryonic hematopoiesis 129 and 2 studies detected low levels of Cdh5-positivity in monocytes. 183,184Also lymphatic vasculature is reported to be Cdh5-positive. 4hile the existence of EndoMT in vivo seems quite certain, there are still open questions about this phenomenon: Is EndoMT a one-way street or can EndoMT cells transition back to fully functioning ECs? 20,185 Is postnatal EndoMT a driver of endothelial dysfunction, maybe even leading to "fibrotic" disease?Or on the contrary, even part of a healing response to tissue damage?

| Signaling in EndoMT
The signaling pathways involved in EndoMT are complex.7][188][189][190][191][192] While we have learned a lot from these studies, the heterogeneity in the literature makes it difficult to describe generally applicable signaling pathways for EndoMT.However, several overlapping pathway activations have been identified, which are outlined below.
TGFβ-signaling is a key activator of EndoMT. 20,190,191ore precisely, this involves the FGFR-let7-TGFβ pathway. 20,190,191Inflammatory cytokines, including TNFalpha and IL1β, as well high shear stress may decrease endothelial FGFR1 expression density. 20,160Thus, decreased FGFR1-signaling inhibits let-7, 20,190,191 which plays a role in cell differentiation and proliferation 20,193 and serves as a suppressor of TGFβ-signaling. 47,190Through this pathway, decreased endothelial FGF signaling loosens EC cell-cell contacts and promotes vascular permeability with compromised vascular integrity. 194Dismantled intercellular contacts between ECs can be partly explained by increased phosphorylation of the primary EC-specific cell-cell junction molecule, Cdh5. 79In addition to facilitating EndoMTmediated cell-cell contact interruption and potentially cell migration, FGFR-let7-TGFβ signaling also favors lymphocyte enrolment during inflammation. 195urthermore, EndoMT can also be activated after deletion or inhibition of Ccm genes 1 or 2. 20,151,196 Ccm genes refer to 3 genes of which any loss of function mutation will cause cerebral cavernous malformations (CCM) that define vascular lesions in the CNS and the retina. 20,151,197oss or decrease of Ccm1 or Ccm2 gene expression leads to increased expression of MEK5 and ERK5.This causes an increase of transcriptional activity of Kruppel-like factor 4 (Klf4) which increases expression of BMP and Smad and can then initiate EndoMT. 20,196Eventually, both TGFβ-signaling and MEK5/ERK5/BMP-signaling lead to the activation of Smad-dependent transcription factors such as Snail, Slug, Twist, and ZEB 1&2, 47,151 which upregulate mesenchymal gene expression. 151,198,199urthermore, also Notch signaling has been described in EndoMT. 189Notch signaling suppresses or activates gene expression directly or through the Snail and Slug pathway. 47,200In EndoMT, it was found that Notch can directly upregulate Slug, but not Snail. 186This Notch mediated Slug activation leads to Cdh5 repression. 186For En-doMT, both transcription factors Snail and Slug are needed and also act as repressors for E-Cadherin in EMT. 201oreover, TGFβ2 signaling in EndoMT was also found to be associated with Dipeptidyl peptidase 4 (DPP4) and Integrin β1. 188DPP4 or Integrin β1 silencing inhibited TGFβ-receptor signaling and decreased Smad3 phosphorylation and EndoMT. 188ther signaling pathways involved in EndoMT are the Wnt/β-catenin pathway 20,187 and VEGF-A signaling. 188EGF-A signaling inhibits EndoMT through interaction with VEGFR2, 202 whereas its interaction with VEGFR1 stimulates EndoMT.188,203 Furthermore, microRNAs, like miR-21 and miR-23, 204 epigenetic regulations and histone modifications might also impact EndoMT initiation and upholding.47,205 In conclusion, multiple pathways seem to activate En-doMT, and this might make sense. EC ubpopulations are specialized to serve the physiological need of the respective tissues hosting them. Similary, different EC subpopulations might be subject to distinct stimulators of EndoMT and experience distinct pathomechanisms.

| Partial vs. full EndoMT
EndoMT can be further subclassified into partial and full EndoMT, which discriminate EC remodeling processes with respect to either partial or full loss of endothelial identity and obtaining of mesenchymal attributes, respectively. 28,47As suggested by 2 review articles, partial EndoMT may be closely linked to angiogenesis. 47,206uring angiogenesis, tip cells undergo distinct morphological and functional changes, which also occur during EndoMT.They de novo express mesenchymal markers, undergo similar cytoskeletal rearrangements, and express MMPs for breakdown of the basal membrane. 47Of note, common plasticity events during angiogenic sprouting and EndoMT seem to also share similar signaling pathways, which are largely controlled by the transcription factors Slug and Snail, respectively. 47,206However, tip cells largely retain EC marker expression. 206,207Importantly, they maintain CD31 and thus cell-cell connections with stalk cells, when migrating in a connected "train" during the formation of an emerging vessel sprout. 206In contrast, loss of cell-cell contacts and migration of individual cells away from the capillary bed, are key events during full EndoMT. 47These observations suggest that angiogenic sprouting and full EndoMT may initially share a common pathway, which later separates based on critical determination of either EC cell-cell contact preservation or disruption.As outlined above, the transcription factor Slug plays an important role during general EndoMT processes.In 2020, Hultgren et al. proposed Slug as a master regulator of EC morphology, proliferation, and cell-cell adhesion during partial EndoMT.While Slug-deletion prevented tumor vascularization, Slug over-expression inhibited EC adhesion and cell-cell junction proteins. 106hese data suggested that Slug levels determine the extent of EndoMT progression.In addition to Slug, p21-activated kinase 4 (PAK4) may also act as a critical determent of differential EC and mesenchymal gene expression. 208In glioblastoma ECs, Ma et al. identified that PAK4 induced ZEB1 mediated downregulation of claudin14 and other adhesion associated genes. 208It remains however poorly understood, how Slug levels or other contributing factors may be fine-tuned to govern cell-cell adhesions towards either partial or full EndoMT, respectively. 206 study from 2019 exploited genetic lineage tracing of individually labeled ECs (Cdh5-Cre-ERT2-Confetti) and documented the clonal origin of cavernomas from few proliferating Ccm3-deficient ECs, which co-expressed mesenchymal and progenitor markers.As discussed above, EndoMT plays a pivotal role in CCM pathology.When implanted into a wild-type environment, Ccm3-deficient ECs not only formed cavernomas through clonal expansion but were further able to expand them by recruiting surrounding wild type ECs which underwent EndoMT. 180aken together, these data indicate a close association of EC proliferation and mesenchymal transition within a pathophysiological setting.0][211][212] Myofibroblasts are mesenchymal cells, which are commonly identified by αsmooth muscle actin (αSMA)-expression 213 and can descent from multiple cell types: Residing fibroblasts, 154,214 hematopoietic stem cells, 154,215 epithelial cells (EMT), 154,216,217 pericytes, 167,218,219 and ECs. 23,26enal interstitial fibrosis is a hallmark of CKD of different etiologies and its extent is closely associated with progression to end-stage kidney disease. 220,221Furthermore, fibrosis can also manifest upon acute kidney injury (AKI) and may contribute to AKI-CKD-transition. 222,223Progression of renal fibrosis is characterized by myofibroblast accumulation, increased ECM deposition 224 and closely associated with tubular and vasculature deprivation. 221Hence, myofibroblasts have been acknowledged key players in the progress of renal fibrosis. 154,224EndoMT may be one pathway through which myofibroblasts form, and many studies have been conducted to investigate this in vivo (Table 1).
In 2008, Zeisberg et al. first described the occurrence of EndoMT in the kidney and explored its role in renal fibrosis.Three different experimental animal models were investigated-Unilateral Ureter Obstruction (UUO), Streptozotocin (STZ)-induced diabetes type 1, and a model of Alport syndrome, induced by Col4a3 deficiency. 23To detect EndoMT, the authors combined two different techniques: Co-staining of (myo-)fibroblast-markers FSP1 and αSMA with EC-marker CD31 and staining of FSP1 and αSMA on tissue from Tie2-Cre-EYFP transgenic mice, which constitutively identified Tie2-positive cells by EYFP-expression. 23On average, 36% of the FSP1+ cells also expressed CD31 and 25% of αSMA+ cells expressed CD31. 23 Double-positivity of FSP1/EYFP and αSMA/EYFP confirmed that EndoMT derived myofibroblasts originated from the EC-Tie2-Cre cell lineage. 23In end-stage diabetic mice, approximately 40% of the FSP1+ cells and 50% of the αSMA+ cells also expressed CD31. 23 Similar numbers were obtained from Alport-syndrome mice. 23These results suggested for the first time that En-doMT may contribute to the myofibroblast population in the kidney.Through co-detection of αSMA and FSP1 with genetically labeled Tie2, also late stages of EndoMT were detectable in which EC marker expression might had been lost.Li et al., used αSMA staining on renal tissue from Tie2-Cre-EGFP mice to investigate EndoMT at different time points of diabetic nephropathy. 26EndoMTemerged myofibroblasts accounted for 10% of the total population after 1 month, and 24% after 6 months of hyperglycemia onset, compared to 0.2% in the respective control group. 26EndoMT derived myofibroblasts were already detectable before albuminuria manifested. 26n 2010, Li et al. linked EndoMT to fibrotic kidney remodeling when investigating the role of advanced glycation end products (AGEs) on EndoMT using a model of diabetic nephropathy.The authors show that AGEreceptor (RAGE) stimulation and downstream Smad3 signaling initiated EndoMT. 27Detection of constitutively labeled ECs under the Tie2-promotor and co-staining for αSMA suggested formation of myofibroblasts from EC origins.Importantly, Smad3 inhibition reduced αSMA+ Tie2-lineage cells, which was associated with a remarked reduction of fibrosis associated markers collagen IV and fibronectin. 27Srivastava et al. documented metabolic modulation of EndoMT formation alongside respective alterations in the extend of detectable renal fibrosis. 174ndothelial mitochondrial SIRT3 deficiency in diabetic nephropathy induced increased intracellular accumulation of glucose in ECs and further stimulated EndoMT through TGFβ/Smad3 signaling.EndoMT was detected using co-staining of αSMA/CD31 and FSP1/CD31.Consistent with increased EndoMT, the authors also detected increased fibrosis in SIRT3-KO using collagen detection with Masson trichrome, Sirius red, and PAS stainings.In contrast, endothelial SIRT3 overexpressing mice 174 displayed reduced EndoMT and fibrosis, suggesting an attenuating role of SIRT3 in EndoMT activation.Similar results were also obtained in an earlier study, which investigated the role of endothelial SIRT3 and EndoMT in a model of hypertensive renal injury. 175everal other studies documented a link between EndoMT and kidney fibrosis when investigating the role of different signaling pathways: Shi et al. investigated RNA-regulated EndoMT in diabetic nephropathy.Suppression of the long non-coding RNA-H19 reduced EndoMT through activation of mi-RNA29a and interruption of TGFβ/Smad3 signaling. 178Xavier et al. investigated endothelial specific knockdown of TGFβRII signaling (Tie2-Cre-TβRII). 179Reduction of TGFβ signaling scaled down EndoMT and tubulointerstitial fibrosis in CKD models of folic acid nephropathy and UUO. 179hao et al. investigated MMP-9 dependent Notch signaling and its role for EndoMT and renal fibrosis in an UUO model. 177MMP9-KO mice demonstrated reduced abundance of myofibroblasts.More specifically, also the number of EC-derived myofibroblasts in the UUO model was reduced by 30%. 177harmacologically, gliptines (DPP4-inhibitors) were found to reduce EndoMT 227 and renal fibrosis in kidney disease, such as diabetic nephropathy. 227,230DPP4 and TGFβ (TGFβ2 > TGFβ1) levels were elevated in diabetic kidneys 227 and particularly in ECs of diabetic nephropathy. 188DPP4 and its molecular interaction with Integrinβ1 seemed to have pro-fibrotic effects, 188 which could be attenuated by Linagliptin, a DPP4 inhibitor. 227Linagliptin inhibits EndoMT through blockage of TGFβ 188,227 and AGE-RAGE signaling in ECs, 231,232 which are both known T A B L E 1 In vivo studies on EndoMT in the kidney.

Srivastava
pathways of EndoMT activation. 20,27,190,191Kanasaki et al. used co-staining of CD31/αSMA and CD31/FSP1 to detect EndoMT cells in diabetic mice and found a significant reduction of EndoMT in Linagliptin treated diabetic mice. 227f note, the authors linked the antifibrotic effects of gliptines to increasing miRNA29 levels. 227As outlined above, the same group later established a role of miRNA29 in regulation of EndoMT. 178Gliptines were suggested to have renoprotective effects. 230,231They increase GLP1 levels and decrease TGFβ1 in the whole kidney, 230 while not affecting hyperglycemia levels. 227,230,231However, while preclinical studies documented antifibrotic actions of gliptines in kidney disease, the clinical trial CARMELINA failed to display improved renal function in linagliptin-treated patients as compared to placebo. 233This challenged the assumption that EndoMT might substantially contribute to disease progression and renal fibrosis in clinical settings.

| EndoMT and EC proliferation
EndoMT may further hamper endothelial proliferation in the diseased kidney.Ren et al. investigated renal fibrosis, dysfunctional angiogenesis and EndoMT in UUO wildtype and endothelial-specific Yes-associated protein (YAP)knockout mice (Cdh5-Cre-ERT2-YAP1). 176 Endothelial YAP activation plays a critical role in vessel maturation, barrier function and sprouting angiogenesis. 234EndoMT was detected by performing co-stainings of αSMA/CD31 and FSP1/CD31. 176In this study, endothelial YAP deletion inhibited EndoMT as significantly less αSMA/CD31 and FSP1/CD31 double-positive cells were detectable in the KO model.Generally, Ren et al. found that EndoMT associated with dysfunctional angiogenesis in fibrotic UUO-kidneys, while both EndoMT and dysfunctional angiogenesis were ameliorated during EC specific YAP deletion. 176Of note, also kidney fibrosis was reduced under EC specific YAPdeletion, while systemic inhibition of YAP worsened kidney fibrosis. 176YAP is expressed in all kinds of different cell types, while downstream effects may vary based on the cell associated G protein-coupled receptor type. 235Hence, Ren et al. speculated that lacking tissue selectivity during systemic YAP inhibition demonstrated paradox effects, 176 as also another group pointed out the necessity of cell type specific targeting when regulating YAP pharmacologically. 235asile et al. associated EndoMT to impaired vascular regenerative capacity in a model of ischemia-reperfusion injury (IRI). 29EndoMT was detected using co-staining of either genetically labeled ECs (Tie2-Cre-EYFP model) or staining for different endothelial markers (CD31, Cablin and infused tomato lectin) with the mesenchymal marker FSP1. 29En-doMT was detected already 6 h after IRI and up to 7 days after IRI, with the highest prevalence at 6 h after IRI. 29 At Abbreviations: EC(s), endothelial cell(s); ECM, extracellular matrix; EndoMT, endothelial-to-mesenchymal transition; IRI, ischemia-reperfusion-injury; KO, knockout; STZ, streptozotocin; UUO, unilateral ureteral obstruction; WT, wildtype; tg, transgenic.
the same time, the authors found low evidence for EC proliferation in the first days after IRI.Thus, 7 days after IRI, only 1% of the labeled ECs stained positive for proliferation, marked by BrdU (Bromdesoxyuridin).The authors suggested that impaired vascular regenerative capacity might contribute to the progression of CKD. 29 However, another study found increased EC proliferation at 3 days after IRI in comparison to 1 day after IRI 236 indicating the need of more data for final conclusions.

| EndoMT and vascular rarefaction
EndoMT has been further suggested to drive vascular rarefaction 29 whereas the reduction of TGFβ signaling scaled down EndoMT and improved renal blood flow and blood vessel preservation in CKD models of folic acid nephropathy and UUO. 179During IRI, ECs are significantly injured.Not only hypoxia itself but also post-ischemic formation of reactive oxygen species upon reperfusion may induce EC swelling and interfere with blood flow. 237Using intravital multiphoton microscopy Basile et al. described morphological changes, such as increased cell size, in ECs of blood vessels with altered flow indicative of beginning vascular rarefication at 14 days after IRI while also detecting En-doMT events. 29Of note, VEGF-A (isoform VEGF 121 ) administration after reperfusion reduced IRI-associated vessel loss over time, without interference of the proliferative capacity of ECs. 29 Similarly, Curci et al. documented EndoMT, beginning tissue fibrosis, and a reduction of peritubular capillary density, within only 24 h when investigating IRI in Large White Pigs. 228When detecting EndoMT, using co-stainings of CD31+/α-SMA+ and CD31+/FSP1+, the authors found that 20%-30% of the αSMA+ cell population also stained positive for CD31. 228Of note, ECs did not test positive for the apoptosis marker Caspase-3. 228However, ECs revealed pronounced morphological changes and appeared detached from the basal lamina at 24 h post IRI. 228Another study confirmed the rare event of EC-apoptosis after IRI, 236 and stated the difficulty to detect endothelial apoptosis due to a mechanism called anoikis 236 -a programmed cell death in ECs that is induced by EC detachment from surrounding ECM. 238These observations suggest a role of anoikis in injury induced EC loss and pinpoint that events such as EndoMT and apoptosis may not be sufficient to explain vascular rarefaction during kidney disease.However, Curci et al. found a reduction of both, EndoMT and vascular rarefication, after inhibition of the complement system, 228 emphasizing that inflammation favors EndoMT as well as vascular rarefication in kidney disease.Twist and Snail are known transcription factors of the mesenchymal lineage. 28,151Through combination of genetic EC-lineage tracing in an inducible model (Cdh5-Cre-ERT2-EYFP) and co-staining with CD31 and αSMA, Lovisa et al. further identified different stages of EndoMT.Thus, partial EndoMT was identified in YFP (Cdh5-lineage) cells that co-stained for CD31 and αSMA (13% after UUO).In contrast, full EndoMT was detected in YFP (Cdh5-lineage) cells that only stained for αSMA and not for CD31, suggesting loss of cell-cell connections (10% after UUO). 28In general, YFP (Cdh5lineage) cells that co-stained for αSMA (partial and full EndoMT) were significantly reduced in EC specific Twist and Snail KO models after UUO compared to wildtype UUO mice. 28Consistent with reduced vascular permeability in EC-Twist and EC-Snail KO-mice, also EC tight junctions were more preserved than in wildtype mice. 28ntercellular contacts are known to be essential for EC barrier formation 239,240 and tight junction proteins are found to regulate Cdh5, 239 which is crucial for cell-cell connections between ECs. 79 Cells undergoing EndoMT, loose the expression of connection proteins and thereby loosen their cell-cell-contacts. 144This might drive vascular permeability. 28Of note, EndoMT induced tissue hypoxia increased tubular expression of the transcription factor Myc and linked to tubule metabolic dysfunction and interstitial fibrosis.These findings emphasized an endothelialepithelial crosstalk in kidney disease, which may bear new potential for pharmacological intervention. 28 5.35,[241][242][243] In a nephrotic CKD model of Tensin2-deficiency, EndoMT detection occurred alongside a remarked decrease of GEnC.226 Also, another study linked EndoMT to a reduction of endothelial markers in the glomerular filter of diabetic mice and human patients.225 Concomitant to EndoMT, the authors also detected increased glomerular albumin permeability, which could be diminished upon EndoMT inhibition using a ROCK1 inhibitor.In summary, these studies point towards a role of EndoMT in malfunctional remodeling of the glomerular filter.However, a definite role of EndoMT in glomerulosclerosis cannot be concluded.244 Overall, it seems certain that renal ECs commit to de novo expression of mesenchymal markers upon different environmental stimuli. EndoMT, as detcted through immunohistology techniques and EC lineage tracing, was further highly associated with loss of functional and structural integrity, such as vascular rarefaction, increased vascular permeability and interstitial fibrosis (Table 1).229 Of note, sequencing data from 2020 unraveled a great variety of EC subtypes in the kidney, distributed in the 3 renal compartments: renal cortex, medulla, and glomeruli.They furthermore dicated a profound potential to transcriptomic changes in health and disease.When challenged with dehydration and the accompanying hyperosmolarity, especially medullary ECs upregulated gene expression for hypoxia response, glycolysis, and also for oxidative phosphorylation, probably due to very high osmolar stress in the renal medulla.13,14,245 This not only emphasizes general EC versatility but leaves room for the assumption that ECs may adapt to environmental changes that exceed osmolar stress, such as inflammation, cancer, tissue repair and/or tubule atrophy, through transcriptomic shifts that may or may not lead to permanent transformation.In fact, they may also occur only transiently. Consistent th such an assumption, Tombor et al. demonstrated in 2021 transient acquisition of mesenchymal traits of ECs during a model of myocardial infarct.165 EndoMT in the kidney may be reflective of adaptive responses of ECs to various injury stimuli that seem associated with endothelial dysfunction and fibrotic tissue remodeling.Even though the above outlined studies point towards a close link of EndoMT and renal fibrosis, it remains unclarified if EndoMT cells directly cause renal fibrosis by deposition of ECM.Several single cell RNA sequencing studies failed to provide evidence for the expression of fibrosis and ECM associated genes in ECs of the diseased heart 164,165,246 and kidney.167 Considering these findings, the role of EndoMT and renal fibrosis is controversial.If EndoMT does not directly contribute to renal fibrosis, what other functions or roles may it have in the diseased kidney? In he next chapter, we will therefore discuss this topic further.5.3.6 | Potential roles of EndoMT in kidney disease With its first discovery in 2007, EndoMT was suggested to contribute to myofibroblast generation and thus to fibrotic tissue remodeling.247 However, there is now evidence that not only myofibroblasts, but also other fibroblast subgroups participate in fibrotic tissue scarring and remodeling.164,167,248,249 Which fibroblast cell type eventually dominates, might possibly depend on the initial intensity or location of injury and therefore pointing out possible different needs of tissue remodeling and contraction.164 Myofibroblasts are contractile cells, denoted and defined as αSMA expressing fibroblasts 250,251 that have been linked to synthesis and deposition of ECM, such as collagens, 252,253 and have thus been generally accepted as the key source of ECM in fibrotic diseases.154,254 As a result, multiple studies facilitated αSMA staining for the detection of myofibroblasts and for drawing conclusions on ECM producing cells. Howver, considering evidence that also αSMA negative mesenchymal cells contribute to fibrotic disease, 164,167,248,255 this terminology may require adaptation.Using time-course single cell sequencing, Kuppe et al. identified PDGFRα and PDGFRβ double-positive fibroblasts and myofibroblasts as the main ECM producing cells in murine and human kidney fibrosis.Thus, ECM producing cells predominantly derived from fibroblasts and pericytes, which was further confirmed by genetic lineage tracing using PDGFRβ-Cre-ERT2 reporter mice.167 Importantly, the same study failed to provide solid evidence for a contributing role of ECs in kidney fibrosis.However, the authors did not use genetic lineage tracing to account for potentially fully transformed (complete loss of EC markers) scar-forming cells of EC origin.Of note, the same group later investigated origins of ECM producing cells in a model of myocardial infarction.164 Exploiting next generation sequencing in combination with genetic EC lineage tracing using Cdh5-Cre-ERT2 mice, Peisker et al. found no evidence for ECM deposition by cells of EC origin.164 These findings were consistent with other studies, which failed to document scar-forming fibroblast/myofibroblast differentiation from ECs. 165,246 It seems therefore controversial if EndoMT cells in the kidney may differentiate into ECM producing myofibroblasts or fibroblasts and future studies will be needed for clarification.The question arises which physiological or pathophysiological functions EndoMT cells otherwise undertake?And are de novo αSMA expressing EndoMT cells necessarily myofibroblasts or may these cells, either transiently or permanently, transform into other mesenchymal cell types instead?Albeit αSMA+ myofibroblasts not always contribute to ECM-deposition, 164 they are known drivers of wound contraction.256,257 Hence, EndoMT derived myofibroblasts could support tissue and vessel repair through acting as contractile bundles and counterpoises in the process of wound closure (Figure 2).In principle such functions could also be provided by transient EndoMT cells as detected in a model of myocardial infarct in 2021.165 A potential role of EndoMT in wound contraction would make sense as EndoMT is usually detected in models of acute and chronic kidney disease during which nephrons are lost and leave behind large tissue gaps that demand closure. Wou healing processes further also depend on vascular permeability to allow for immune cell recruitment, 258 such as macrophages, that are important for tissue degradation and wound healing.259 Consistently, EndoMT processes increase vascular permeability, 28 which may facilitate immune cell infiltration during wound healing.
When looking at the detailed spatial tissue distribution of EndoMT cells in published studies (Table 1), it becomes evident that mesenchymal marker expressing ECs are rarely detected offside of intact vessels.Zeisberg et al., 23 illustrated FSP1/CD31 or FSP1/Tie2-YFP doublepositive cells lined up next to each other and in close vicinity of peritubular vessels.Also in the study from Li et al., 26 αSMA+ cells that co-expressed CD31+ or Tie2-EGFP+, appeared embedded in intact peritubular vessels.While spatial resolution with confocal microscopy imposes limitations to draw ultrastructural conclusions, preserved CD31-expression indicated that EndoMT cells maintained cell-cell connections.Hence, observations from these studies pinpoint towards partial rather than full EndoMT.As outlined above, angiogenic sprouting shares many molecular aspects of EndoMT but preserves cell-cell contacts to neighbor cells. 47Thus, partial EndoMT could facilitate angiogenic sprouting in the diseased kidney that adapts to injury stimuli.
The vast majority of our in vivo knowledge on EndoMT in the kidney, is based on co-detection of EC markers or lineage with 2 distinct mesenchymal markers: αSMA and FSP1 (see Table 1).However, both markers are also expressed in other cells than myofibroblasts and fibroblasts: For example, it was found that αSMA+ cells co-expressed progenitor cell markers, such as PDGFRα, after bone fracture. 260αSMA is further a common marker of smooth muscle cells (mural cells). 261Of note, Chen et al. detected cardiac pericytes and smooth muscle cells of EC origins during embryonic heart development in 2016. 24Similarly, FSP1 is also expressed by smooth muscle cells, 213,262,263 hematopoietic cells 263 such as macrophages, 264 and injured tubular epithelial cells (without the change to an fibroblastoid morphology). 265Overall, the magnitude of distinct αSMAand FSP1 expressing cell types, demands a more in-depth molecular characterization of EndoMT cells as the potential cellular outcomes of EndoMT pathways might be of higher diversity than previously anticipated.

| CONCLUSION
ECs display a remarkable plasticity potential.They may engage in angiogenic sprouting, partial or full EndoMT and EHT.While the molecular pathways between different routes of EC plasticity partly overlap, the cellular identities ECs can potentially acquire are not fully elucidated.Similarly, there remains controversy regarding the (patho)physiological roles of postnatal EC plasticity.
For better understanding of renal EC plasticity in the future, it will be necessary to expand the methodological toolbox.As ECs may entirely lose endothelial surface markers, EC lineage tracing techniques are required.However, mere combination of EC lineage tracing with traditionally applied immunohistological techniques, does not seem sufficient to precisely determine cellular outcomes and especially the pathophysiological impact of those cellular changes.Especially the renal interstitium is an understudied compartment in the kidney.It homes peritubular vasculature and multiple mesenchymal cell types, which express various overlapping cellular markers that likely engage in different (patho)physiological mechanisms, of which several are still unknown.Thus, a more in-depth analysis of differentially expressed cellular markers is necessary to determine EC fate and demands for future use of modern -omics techniques that allow for an unbiased screening of cellular marker expression and thus better determination of cell identity.Especially when one is to define the functional and pathophysiological aspect of each EC remodeling event, a broader and more detailed definition of individual cells will be needed.To furthermore generate a better understanding of dynamic structural and functional changes of transdifferentiating ECs over time, more longitudinal data will be needed.Serial intravital microscopy analysis is a powerful technique to track individual cells in the kidney in vivo (Figure 3) over time and has already demonstrated important insights in spatial-temporal dynamics of remodeling kidney cells. 19,266,267Thus, combination of several state-of-the-art techniques would be desirable to shed more light on EC plasticity in the kidney and its actual impact in health and disease.

AUTHOR CONTRIBUTIONS
LP and IMS conceptualized the content of the work.LP reviewed the literature and drafted the manuscript, figures, and tables.LP and IMS wrote the manuscript.IMS supervised and funded the writing of the manuscript.

F I G U R E 1
Overview of different pathways of endothelial plasticity.(A) Scheme of small blood vessel, including cross-sectional view.(B) Endothelial-to-hematopoietic transition (EHT) (C) Angiogenic sprouting with stalk and tip cell differentiation.(D) Endothelial-tomesenchymal transition (EndoMT) with different potential cellular outcomes.Created with BioRe nder.com.AKI, Acute Kidney Injury; CKD, Chronic Kidney Injury.

F I G U R E 2
Pathophysiological roles of endothelial-to-mesenchymal transition (EndoMT) in kidney disease.Healthy conditions: peritubular capillaries composed of endothelial cells (ECs) and pericytes surround healthy tubule epithelium.Partial/full endothelialto-mesenchymal transition (EndoMT): Upon injury during acute kidney injury (AKI) or chronic kidney disease (CKD), injured tubule epithelium dedifferentiates and proliferates.Surrounding ECs undergo EndoMT and de novo express mesenchymal markers, such as αSMA.Tissue scarring: Upon failed tubule recovery, tubulointerstitial fibrosis and tubule atrophy establish.The role of EC-derived myofibroblasts in this process is controversial.Thus, they may contribute to deposition of extracellular matrix (ECM) or contribute to wound closure.Created with BioRe nder.com.

F I G U R E 3
In vivo visualization of renal microvasculature and hemodynamics using two-photon microscopy.Intravital twophoton image of a Cdh5-CreERT2-tdTomato mouse kidney cortex (Excitation λ = 820 nm).Tubule epithelium (Ep) demonstrates blue autofluorescence.Endothelial cells (red, arrowheads) are identified by endogenously expressed tdTomato.Plasma labeling with Alexa680albumin (gray) enables visualization of peritubular capillaries and assessment of renal hemodynamics: Red blood cells are excluded from Alexa680-albumin plasma staining (appear as black bands, arrows) and demonstrate laminar flow.L, Tubule lumen.