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.

Peroxisome proliferator-activated receptor ɣ agonist mediated inhibition of heparanase expression reduces proteinuria.

BACKGROUND: Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor É£ (PPARÉ£) agonists can ameliorate proteinuria. Since a recent study showed that PPARÉ£ regulates HPSE expression in liver cancer cells, we hypothesized that PPARÉ£ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. METHODS: Regulation of HPSE by PPARÉ£ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARÉ£ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARÉ£ agonist pioglitazone. FINDINGS: Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARÉ£ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARÉ£ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. INTERPRETATION: PPARÉ£-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. FUNDING: This study was financially supported by the Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships).

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.

Motile Cilia on Kidney Proximal Tubular Epithelial Cells Are Associated With Tubular Injury and Interstitial Fibrosis.

It is well established that mammalian kidney epithelial cells contain a single non-motile primary cilium (9 + 0 pattern). However, we noted the presence of multiple motile cilia with a central microtubular pair (9 + 2 pattern) in kidney biopsies of 11 patients with various kidney diseases, using transmission electron microscopy. Immunofluorescence staining revealed the expression of the motile cilia-specific markers Radial Spoke Head Protein 4 homolog A, Forkhead-box-protein J1 and Regulatory factor X3. Multiciliated cells were exclusively observed in proximal tubuli and a relative frequent observation in human kidney tissue: in 16.7% of biopsies with tubular injury and atrophy (3 of 18 tissues), in 17.6% of biopsies from patients with membranous nephropathy (3 of 17 tissues) and in 10% of the human kidney tissues derived from the unaffected pole after tumour nephrectomy (3 of 30 tissues). However, these particular tissues showed marked tubular injury and fibrosis. Further analysis showed a significant relation between the presence of multiciliated cells and an increased expression of alpha-smooth-muscle-actin (p-value < 0.01) and presence of Kidney-injury-molecule-1 (p-value < 0.01). Interestingly, multiciliated cells co-showed staining for the scattered tubular cell markers annexin A2, annexin A3, vimentin and phosphofructokinase platelet but not with cell senescence associated markers, like (p16) and degradation of lamin B. In conclusion, multiciliated proximal tubular cells with motile cilia were frequently observed in kidney biopsies and associated with tubular injury and interstitial fibrosis. These data suggest that proximal tubular cells are able to transdifferentiate into multiciliated cells.

Kidney tubule iron loading in experimental focal segmental glomerulosclerosis.

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

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.

Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo.

Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose-response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time.

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Increased Plasma Heparanase Activity in COVID-19 Patients.

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

Angiotensin-neprilysin inhibition confers renoprotection in rats with diabetes and hypertension by limiting podocyte injury.

OBJECTIVES: Combined angiotensin receptor--neprilysin inhibition (ARNI) reduces glomerulosclerosis better than single angiotensin receptor blockade (ARB) in diabetic, hypertensive rats. The renoprotective mechanism remains unknown, but may depend on superior blood pressure control, improved renal hemodynamics, suppressed renal inflammation or prevention of podocyte loss. METHODS: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic for 12 weeks and treated with vehicle (n = 10), valsartan (ARB; n = 7) or sacubitril/valsartan (ARNI; n = 8) for the final 3 weeks. Arterial pressure was measured via radiotelemetry. RESULTS: Sacubitril/valsartan lowered mean arterial pressure by -50 ±â€Š4 mmHg and valsartan by -43 ±â€Š4 mmHg (P = 0.3). Both treatments lowered albuminuria, but only sacubitril/valsartan maintained high urinary atrial natriuretic peptide, improved glycemic control and protected podocyte integrity, reflected by increased nephrin expression and suppression of transient receptor potential canonical 6 and regulator of calcineurin 1. This resulted in markedly reduced glomerulosclerosis (P < 0.05 vs. control and valsartan). Despite higher effective renal plasma flow and glomerular filtration rates, sacubitril/valsartan did neither improve filtration fraction nor renal immune cell infiltration. CONCLUSION: Sacubitril/valsartan offers drug-class-specific renoprotection in a preclinical model of diabetes and hypertension. Renoprotection is unrelated to antihypertensive efficacy, renal hemodynamics or inflammation, but may be related to protective effects of natriuretic peptides on podocyte integrity.

Heparanase Accelerates Obesity-Associated Breast Cancer Progression.

Obese women have higher risk of bearing breast tumors that are highly aggressive and resistant to therapies. Tumor-promoting effects of obesity occur locally via adipose inflammation and related alterations to the extracellular matrix (ECM) as well as systemically via circulating metabolic mediators (e.g., free fatty acids, FFA) associated with excess adiposity and implicated in toll-like receptor-mediated activation of macrophages-key cellular players in obesity-related cancer progression. Although the contribution of macrophages to proneoplastic effects of obesity is well documented, the role of ECM components and their enzymatic degradation is less appreciated. We show that heparanase, the sole mammalian endoglucuronidase that cleaves heparan sulfate in ECM, is preferentially expressed in clinical/experimental obesity-associated breast tumors. Heparanase deficiency abolished obesity-accelerated tumor progression in vivo. Heparanase orchestrated a complex molecular program that occurred concurrently in adipose and tumor tissue and sustained the cancer-promoting action of obesity. Heparanase was required for adipose tissue macrophages to produce inflammatory mediators responsible for local induction of aromatase, a rate-limiting enzyme in estrogen biosynthesis. Estrogen upregulated heparanase in hormone-responsive breast tumors. In subsequent stages, elevated levels of heparanase induced acquisition of procancerous phenotype by tumor-associated macrophages, resulting in activation of tumor-promoting signaling and acceleration of breast tumor growth under obese conditions. As techniques to screen for heparanase expression in tumors become available, these findings provide rational and a mechanistic basis for designing antiheparanase approaches to uncouple obesity and breast cancer in a rapidly growing population of obese patients. SIGNIFICANCE: This study reveals the role of heparanase in promoting obesity-associated breast cancer and provides a mechanistically informed approach to uncouple obesity and breast cancer in a rapidly growing population of obese patients.

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.

C3 glomerulopathy - understanding a rare complement-driven renal disease.

The C3 glomerulopathies are a group of rare kidney diseases characterized by complement dysregulation occurring in the fluid phase and in the glomerular microenvironment, which results in prominent complement C3 deposition in kidney biopsy samples. The two major subgroups of C3 glomerulopathy - dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) - have overlapping clinical and pathological features suggestive of a disease continuum. Dysregulation of the complement alternative pathway is fundamental to the manifestations of C3 glomerulopathy, although terminal pathway dysregulation is also common. Disease is driven by acquired factors in most patients - namely, autoantibodies that target the C3 or C5 convertases. These autoantibodies drive complement dysregulation by increasing the half-life of these vital but normally short-lived enzymes. Genetic variation in complement-related genes is a less frequent cause. No disease-specific treatments are available, although immunosuppressive agents and terminal complement pathway blockers are helpful in some patients. Unfortunately, no treatment is universally effective or curative. In aggregate, the limited data on renal transplantation point to a high risk of disease recurrence (both DDD and C3GN) in allograft recipients. Clinical trials are underway to test the efficacy of several first-generation drugs that target the alternative complement pathway.

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.

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.

Sialic Acid Blockade Suppresses Tumor Growth by Enhancing T-cell-Mediated Tumor Immunity.

Sialic acid sugars on the surface of cancer cells have emerged as potent immune modulators that contribute to the immunosuppressive microenvironment and tumor immune evasion. However, the mechanisms by which these sugars modulate antitumor immunity as well as therapeutic strategies directed against them are limited. Here we report that intratumoral injections with a sialic acid mimetic Ac53FaxNeu5Ac block tumor sialic acid expression in vivo and suppress tumor growth in multiple tumor models. Sialic acid blockade had a major impact on the immune cell composition of the tumor, enhancing tumor-infiltrating natural killer cell and CD8+ T-cell numbers while reducing regulatory T-cell and myeloid regulatory cell numbers. Sialic acid blockade enhanced cytotoxic CD8+ T-cell-mediated killing of tumor cells in part by facilitating antigen-specific T-cell-tumor cell clustering. Sialic acid blockade also synergized with adoptive transfer of tumor-specific CD8+ T cells in vivo and enhanced CpG immune adjuvant therapy by increasing dendritic cell activation and subsequent CD8+ T-cell responses. Collectively, these data emphasize the crucial role of sialic acids in tumor immune evasion and provide proof of concept that sialic acid blockade creates an immune-permissive tumor microenvironment for CD8+ T-cell-mediated tumor immunity, either as single treatment or in combination with other immune-based intervention strategies.Significance: Sialic acid sugars function as important modulators of the immunosuppressive tumor microenvironment that limit potent antitumor immunity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3574/F1.large.jpg Cancer Res; 78(13); 3574-88. ©2018 AACR.

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.

A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration-Associated Complement Factor H Variant 402H.

Purpose: Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established. Methods: Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA. Results: ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant. Conclusions: A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.