Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling.

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.

Human scattered tubular cells represent a heterogeneous population of glycolytic dedifferentiated proximal tubule cells.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Podocyte Injury in Diabetic Kidney Disease in Mouse Models Involves TRPC6-mediated Calpain Activation Impairing Autophagy.

SIGNIFICANCE STATEMENT: Autophagy protects podocytes from injury in diabetic kidney disease (DKD). Restoring glomerular autophagy is a promising approach to limit DKD. This study demonstrates a novel regulatory mechanism of autophagy that blocks this critical protection of the glomerular filtration barrier. We demonstrated that TRPC6 induced in podocytes in mouse models of diabetes mediates calpain activation, thereby impairing podocyte autophagy, causing injury and accelerating DKD. Furthermore, this study provides proof of principle for druggable targets for DKD because restoration of podocyte autophagy by calpain inhibitors effectively limits glomerular destruction. BACKGROUND: Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. The regulation of podocyte autophagy is unique because it minimally uses the mTOR and AMPK pathways. Thus, the molecular mechanisms underlying the impaired autophagy in podocytes in diabetic kidney disease remain largely elusive. METHODS: This study investigated how the calcium channel TRPC6 and the cysteine protease calpains deleteriously affect podocyte autophagy in diabetic kidney disease in mice. We demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux because of decreased cysteine protease calpain activity. Diabetic kidney disease was induced in vivo using streptozotocin with unilateral nephrectomy and the BTBR ob/ob mouse models. RESULTS: Diabetes increased TRPC6 expression in podocytes in vivo with decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacologic inhibition of calpain activity, normalized podocyte autophagic flux, reduced nephrin loss, and prevented the development of albuminuria in diabetic mice. In kidney biopsies from patients with diabetes, we further confirmed that TRPC6 overexpression in podocytes correlates with decreased calpastatin expression, autophagy blockade, and podocyte injury. CONCLUSIONS: Overall, we discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury, and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting TRPC6 and/or calpain to restore podocyte autophagy might be a promising therapeutic strategy for diabetic kidney disease.

A Putative Role for TRPC6 in Immune-Mediated Kidney Injury.

Excessive activation of the immune system is the cause of a wide variety of renal diseases. However, the pathogenic mechanisms underlying the aberrant activation of the immune system in the kidneys often remain unknown. TRPC6, a member of the Ca2+-permeant family of TRPC channels, is important in glomerular epithelial cells or podocytes for the process of glomerular filtration. In addition, TRPC6 plays a crucial role in the development of kidney injuries by inducing podocyte injury. However, an increasing number of studies suggest that TRPC6 is also responsible for tightly regulating the immune cell functions. It remains elusive whether the role of TRPC6 in the immune system and the pathogenesis of renal inflammation are intertwined. In this review, we present an overview of the current knowledge of how TRPC6 coordinates the immune cell functions and propose the hypothesis that TRPC6 might play a pivotal role in the development of kidney injury via its role in the immune system.

Establishment and characterization of a novel conditionally immortalized human parietal epithelial cell line.

Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.

Primary Focal Segmental Glomerulosclerosis Plasmas Increase Lipid Droplet Formation and Perilipin-2 Expression in Human Podocytes.

Many patients with primary focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but were never validated. We aimed to find proteins involved in the mechanism of action of CPF(s) and/or potential biomarkers for the presence of CPF(s). Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s) or healthy and disease controls. Podocyte proteomes were analyzed by LC-MS. Results were validated using flow cytometry, RT-PCR, and immunofluorescence. Podocyte granularity was examined using flow cytometry, electron microscopy imaging, and BODIPY staining. Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, and correlated with the capacity of plasma to induce podocyte granularity, identified as lipid droplet accumulation. Elevated podocyte perilipin-2 was confirmed at protein and mRNA level and was also detected in glomeruli of FSGS patients whose active disease plasmas induced podocyte perilipin-2 and lipid droplets. Our study demonstrates that presumably, CPF-containing plasmas from FSGS patients induce podocyte lipid droplet accumulation and perilipin-2 expression, identifying perilipin-2 as a potential biomarker. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism, which ultimately may result in novel leads for treatment.

Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis.

BACKGROUND: Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. METHODS: Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. RESULTS: Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. CONCLUSIONS: We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

Repurposing Riociguat to Target a Novel Paracrine Nitric Oxide-TRPC6 Pathway to Prevent Podocyte Injury.

Increased expression and activity of the Ca2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.

Heparanase in Kidney Disease.

The primary filtration of blood occurs in the glomerulus in the kidney. Destruction of any of the layers of the glomerular filtration barrier might result in proteinuric disease. The glomerular endothelial cells and especially its covering layer, the glycocalyx, play a pivotal role in development of albuminuria. One of the main sulfated glycosaminoglycans in the glomerular endothelial glycocalyx is heparan sulfate. The endoglycosidase heparanase degrades heparan sulfate, thereby affecting glomerular barrier function, immune reactivity and inflammation. Increased expression of glomerular heparanase correlates with loss of glomerular heparan sulfate in many glomerular diseases. Most importantly, heparanase knockout in mice prevented the development of albuminuria after induction of experimental diabetic nephropathy and experimental glomerulonephritis. Therefore, heparanase could serve as a pharmacological target for glomerular diseases. Several factors that regulate heparanase expression and activity have been identified and compounds aiming to inhibit heparanase activity are currently explored.

Glomerular Function and Structural Integrity Depend on Hyaluronan Synthesis by Glomerular Endothelium.

BACKGROUND: A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. METHODS: To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. RESULTS: Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. CONCLUSIONS: Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.

Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions.

Beside the classical flat parietal epithelial cells (PECs), we investigated proximal tubular epithelial-like cells, a neglected subgroup of PECs. These cells, termed cuboidal PECs, make up the most proximal part of the proximal tubule and may also line parts of Bowman's capsule. Additionally, a third intermediate PEC subgroup was identified at the junction between the flat and cuboidal PEC subgroups at the tubular orifice. The transgenic mouse line PEC-rtTA labeled all three PEC subgroups. Here we show that the inducible Pax8-rtTA mouse line specifically labeled only cuboidal and intermediate PECs, but not flat PECs. In aging Pax8-rtTA mice, cell fate mapping showed no evidence for significant transdifferentiation from flat PECs to cuboidal or intermediate PECs or vice versa. In murine glomerular disease models of crescentic glomerulonephritis, and focal segmental glomerulosclerosis (FSGS), intermediate PECs became more numerous. These intermediate PECs preferentially expressed activation markers CD44 and Ki-67, suggesting that this subgroup of PECs was activated more easily than the classical flat PECs. In mice with FSGS, cuboidal and intermediate PECs formed sclerotic lesions. In patients with FSGS, cells forming the tip lesions expressed markers of intermediate PECs. These novel PEC subgroups form sclerotic lesions and were more prone to cellular activation compared to the classical flat PECs in disease. Thus, colonization of Bowman's capsule by cuboidal PECs may predispose to lesion formation and chronic kidney disease. We propose that tip lesions originate from this novel subgroup of PECs in patients with FSGS.

Vascular bioengineering of scaffolds derived from human discarded transplant kidneys using human pluripotent stem cell-derived endothelium.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell -derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury.

The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury.

BACKGROUND: The hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive. METHODS: We tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes. RESULTS: Compared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects. CONCLUSIONS: We elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.

CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.

Interleukin-6 is essential for glomerular immunoglobulin A deposition and the development of renal pathology in Cd37-deficient mice.

Immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is characterized by IgA depositions in the kidney. Deficiency of CD37, a leukocyte-specific tetraspanin, leads to spontaneous development of renal pathology resembling IgAN. However, the underlying molecular mechanism has not been resolved. Here we found that CD37 expression on B cells of patients with IgAN was significantly decreased compared to B cells of healthy donors. Circulating interleukin (IL)-6 levels, but not tumor necrosis factor-α or IL-10, were elevated in Cd37-/- mice compared to wild-type mice after lipopolysaccharide treatment. Cd37-/- mice displayed increased glomerular neutrophil influx, immune complex deposition, and worse renal function. To evaluate the role of IL-6 in the pathogenesis of accelerated renal pathology in Cd37-/-mice, we generated Cd37xIl6 double-knockout mice. These double-knockout and Il6-/- mice displayed no glomerular IgA deposition and were protected from exacerbated renal failure following lipopolysaccharide treatment. Moreover, kidneys of Cd37-/- mice showed more mesangial proliferation, endothelial cell activation, podocyte activation, and segmental podocyte foot process effacement compared to the double-knockout mice, emphasizing that IL-6 mediates renal pathology in Cd37-/- mice. Thus, our study indicates that CD37 may protect against IgA nephropathy by inhibition of the IL-6 pathway.

Cleaved N-terminal histone tails distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation.

OBJECTIVES: Neutrophil extracellular traps (NETs) act in various rheumatic diseases. Although NET formation was originally described as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent pathway, it appears that there are also NOX-independent pathways of NET release. Currently, no tools are available that can discriminate between both NET-forming pathways. We aimed to develop a serological method allowing the discrimination between NETs generated through NOX-dependent or NOX-independent pathways. METHODS: Histones from in vitro generated NOX-dependent and NOX-independent NETs were characterised with a panel of lupus-derived antibodies against N-terminal histone tails using immunofluorescence microscopy, western blot and ELISA. NETs in patients with NET-associated diseases, that is, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriatic arthritis (PsA) and sepsis, were characterised in sandwich ELISAs employing antibodies against myeloperoxidase (MPO) and N-terminal histone tails as detecting and capturing antibodies, respectively. Functional responses of endothelial cells to NOX-dependent and NOX-independent NETs were assessed as well. RESULTS: Neutrophil elastase cleaves the N-terminal tails of core histones during NOX-dependent, but not during NOX-independent NET formation. Consequently, the detection of MPO-histone complexes with antibodies against N-terminal histone tails allows discrimination between NETs formed through a NOX-dependent or NOX-independent manner. Characterisation of in vivo circulating NETs revealed the presence of NOX-independent NETs in RA, SLE and sepsis, but NOX-dependent NETs in PsA. NOX-independent NETs displayed an increased capacity to activate endothelial cells when compared with NOX-dependent NETs. CONCLUSIONS: These results indicate heterogeneity in NET-forming pathways in vivo and highlight the need for disease-specific strategies to prevent NET-mediated pathology.

Systemic Monocyte Chemotactic Protein-1 Inhibition Modifies Renal Macrophages and Restores Glomerular Endothelial Glycocalyx and Barrier Function in Diabetic Nephropathy.

Inhibition of monocyte chemotactic protein-1 (MCP-1) with the Spiegelmer emapticap pegol (NOX-E36) shows long-lasting albuminuria-reducing effects in diabetic nephropathy. MCP-1 regulates inflammatory cell recruitment and differentiation of macrophages. Because the endothelial glycocalyx is also reduced in diabetic nephropathy, we hypothesized that MCP-1 inhibition restores glomerular barrier function through influencing macrophage cathepsin L secretion, thus reducing activation of the glycocalyx-degrading enzyme heparanase. Four weeks of treatment of diabetic Apoe knockout mice with the mouse-specific NOX-E36 attenuated albuminuria without any change in systemic hemodynamics, despite persistent loss of podocyte function. MCP-1 inhibition, however, increased glomerular endothelial glycocalyx coverage, with preservation of heparan sulfate. Mechanistically, both glomerular cathepsin L and heparanase expression were reduced. MCP-1 inhibition resulted in reduced CCR2-expressing Ly6Chi monocytes in the peripheral blood, without affecting overall number of kidney macrophages at the tissue level. However, the CD206+/Mac3+ cell ratio, as an index of presence of anti-inflammatory macrophages, increased in diabetic mice after treatment. Functional analysis of isolated renal macrophages showed increased release of IL-10, whereas tumor necrosis factor and cathepsin L release was reduced, further confirming polarization of tissue macrophages toward an anti-inflammatory phenotype during mouse-specific NOX-E36 treatment. We show that MCP-1 inhibition restores glomerular endothelial glycocalyx and barrier function and reduces tissue inflammation in the presence of ongoing diabetic injury, suggesting a therapeutic potential for NOX-E36 in diabetic nephropathy.

Neutrophil Extracellular Traps Drive Endothelial-to-Mesenchymal Transition.

OBJECTIVE: An excessive release and impaired degradation of neutrophil extracellular traps (NETs) leads to the continuous exposure of NETs to the endothelium in a variety of hematologic and autoimmune disorders, including lupus nephritis. This study aims to unravel the mechanisms through which NETs jeopardize vascular integrity. APPROACH AND RESULTS: Microvascular and macrovascular endothelial cells were exposed to NETs, and subsequent effects on endothelial integrity and function were determined in vitro and in vivo. We found that endothelial cells have a limited capacity to internalize NETs via the receptor for advanced glycation endproducts. An overflow of the phagocytic capacity of endothelial cells for NETs resulted in the persistent extracellular presence of NETs, which rapidly altered endothelial cell-cell contacts and induced vascular leakage and transendothelial albumin passage through elastase-mediated proteolysis of the intercellular junction protein VE-cadherin. Furthermore, NET-associated elastase promoted the nuclear translocation of junctional ß-catenin and induced endothelial-to-mesenchymal transition in cultured endothelial cells. In vivo, NETs could be identified in kidney samples of diseased MRL/lpr mice and patients with lupus nephritis, in whom the glomerular presence of NETs correlated with the severity of proteinuria and with glomerular endothelial-to-mesenchymal transition. CONCLUSIONS: These results indicate that an excess of NETs exceeds the phagocytic capacity of endothelial cells for NETs and promotes vascular leakage and endothelial-to-mesenchymal transition through the degradation of VE-cadherin and the subsequent activation of ß-catenin signaling. Our data designate NET-associated elastase as a potential therapeutic target in the prevention of endothelial alterations in diseases characterized by aberrant NET release.