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.

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.

Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (µm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.

Serial intravital imaging captures dynamic and functional endothelial remodeling with single-cell resolution.

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

Essential role and therapeutic targeting of the glomerular endothelial glycocalyx in lupus nephritis.

Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN.

A Porthole over AKI: Cell Dynamics in Damage and Repair.

Acute kidney injury (AKI) is associated with an increased risk of CKD. Injury-induced multifaceted renal cell-to-cell crosstalk can either lead to successful self-repair or chronic fibrosis and inflammation. In this mini-review, we will discuss critical renal cell types acting as victims or executioners in AKI pathology and introduce intravital imaging as a powerful technique to further dissect these cell-to-cell interactions.

The Role of Tubule-Interstitial Crosstalk in Renal Injury and Recovery.

Renal epithelial cells show remarkable regenerative capacity to recover from acute injury, which involves specific phenotypic changes, but also significant profibrotic tubule-interstitial crosstalk. Tubule-derived profibrotic stimuli and subsequent myofibroblast activation and extracellular matrix deposition have been linked closely with decline of renal function and nephron loss. However, recent data have questioned the view of purely detrimental effects of myofibroblast activation in the injured kidney and even suggested its beneficial role for epithelial regeneration. This article reviews the current understanding of the underlying mechanisms of tubular cell turnover, new suggested pathways of proregenerative tubular-interstitial crosstalk, and relevant insights of proliferation-enhancing effects of myofibroblasts on epithelial cells in nonrenal tissues.

Correction to: Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy.

The original version of this chapter was inadvertently published without a proper acknowledgement. The authors informed to insert the following acknowledgement in this chapter.

Long-Term Cell Fate Tracking of Individual Renal Cells Using Serial Intravital Microscopy.

Intravital multiphoton microscopy of the kidney is a powerful technique to study alterations in tissue morphology and function simultaneously in the living animal and represents a dynamic and developing research tool in the field. Recent technological advances include serial intravital multiphoton microscopy of the same kidney regions over several weeks and combined with ex vivo histology for cellular biomarker expression of the same cells, which had been subject to serial imaging before. Thus, serial intravital multiphoton microscopy followed by ex vivo histology provides unique tools to perform long-term cell fate tracing of the same renal cells during physiological and pathophysiological conditions, thereby allowing the detection of structural changes of the same renal cells over time. Examples include renal cell migration and proliferation while linking these events to local functional alterations and eventually to the expression of distinct cellular biomarkers. Here, we provide a detailed step-by-step protocol to facilitate serial intravital multiphoton microscopy for long-term in vivo tracking of renal cells and subsequent ex vivo histology for immunohistological staining of the same cells in the fixed tissue.

Novel fluorescence techniques to quantitate renal cell biology.

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Correction to: Phenytoin inhibits necroptosis.

The name of the one of the authors was misspelt. The author's surname is Rodriguez, not Rodriquez as originally published. This has been corrected in both the PDF and HTML versions of the Article.

Phenytoin inhibits necroptosis.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Renal Interstitial Platelet-Derived Growth Factor Receptor-ß Cells Support Proximal Tubular Regeneration.

BACKGROUND: The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-ß (PDGFRß) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined. METHODS: In mice with fluorescent protein expression in renal tubular cells and PDGFRß-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration. RESULTS: Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRß-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRß-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRß-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRß impeded PDGFRß-positive cell migration to the tubular injury site and compromised the recovery of tubular function. CONCLUSIONS: Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRß-mediated communication between the renal interstitium and the tubular system.

Deep insights: intravital imaging with two-photon microscopy.

Intravital multiphoton microscopy is widely used to assess the structure and function of organs in live animals. Although different tissues vary in their accessibility for intravital multiphoton imaging, considerable progress has been made in the imaging quality of all tissues due to substantial technical improvements in the relevant imaging components, such as optics, excitation laser, detectors, and signal analysis software. In this review, we provide an overview of the technical background of intravital multiphoton microscopy. Then, we note a few seminal findings that were made through the use of multiphoton microscopy. Finally, we address the technical limitations of the method and provide an outlook for how these limitations may be overcome through future technical developments.

Just Look! Intravital Microscopy as the Best Means to Study Kidney Cell Death Dynamics.

Kidney cell death plays a key role in the progression of life-threatening renal diseases, such as acute kidney injury and chronic kidney disease. Injured and dying epithelial and endothelial cells take part in complex communication with the innate immune system, which drives the progression of cell death and the decrease in renal function. To improve our understanding of kidney cell death dynamics and its impact on renal disease, a study approach is needed that facilitates the visualization of renal function and morphology in real time. Intravital multiphoton microscopy of the kidney has been used for more than a decade and made substantial contributions to our understanding of kidney physiology and pathophysiology. It is a unique tool that relates renal structure and function in a time- and spatial-dependent manner. Basic renal function, such as microvascular blood flow regulation and glomerular filtration, can be determined in real time and homeostatic alterations, which are linked inevitably to cell death and can be depicted down to the subcellular level. This review provides an overview of the available techniques to study kidney dysfunction and inflammation in terms of cell death in vivo, and addresses how this novel approach can be used to improve our understanding of cell death dynamics in renal disease.

Intravital Imaging Reveals Angiotensin II-Induced Transcytosis of Albumin by Podocytes.

Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.