Glomerular Microvascular Endothelial Cells Research Articles

P02-39 Doxorubicin induced change in filtration permeability in podocytes and glomerular microvascular endothelial cells in co-culture at physiological oxygen tensions

P02-39 Doxorubicin induced change in filtration permeability in podocytes and glomerular microvascular endothelial cells in co-culture at physiological oxygen tensions

Modeling complement activation on human glomerular microvascular endothelial cells.

Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Sodium Butyrate as Key Regulator of Mitochondrial Function and Barrier Integrity of Human Glomerular Endothelial Cells.

The gut microbiota has emerged as an important modulator of cardiovascular and renal homeostasis. The composition of gut microbiota in patients suffering from chronic kidney disease (CKD) is altered, where a lower number of bacteria producing short chain fatty acids (SCFAs) is observed. It is known that SCFAs, such as butyrate and acetate, have protective effects against cardiovascular diseases and CKD but their mechanisms of action remain largely unexplored. In the present study, we investigated the effect of butyrate and acetate on glomerular endothelial cells. Human glomerular microvascular endothelial cells (hgMVECs) were cultured and exposed to butyrate and acetate and their effects on cellular proliferation, mitochondrial mass and metabolism, as well as monolayer integrity were studied. While acetate did not show any effects on hgMVECs, our results revealed that butyrate reduces the proliferation of hgMVECs, strengthens the endothelial barrier through increased expression of VE-cadherin and Claudin-5 and promotes mitochondrial biogenesis. Moreover, butyrate reduces the increase in oxygen consumption induced by lipopolysaccharides (LPS), revealing a protective effect of butyrate against the detrimental effects of LPS. Taken together, our data show that butyrate is a key player in endothelial integrity and metabolic homeostasis.

Circulating Permeability Factors in Focal Segmental Glomerulosclerosis: In Vitro Detection

The recurrence of proteinuria after kidney transplantation in patients with focal segmental glomerulosclerosis (FSGS) is considered proof of the presence of circulating permeability factors (CPFs). The aim of this study is to demonstrate the presence of plasma CPFs using series of invitro assays. Podocytes and endothelial cells (glomerular microvascular endothelial cells [GMVECs]) were incubated with plasma from FSGS patients with presumed CPFs in relapse and remission and from steroid-resistant nephrotic syndrome (SRNS), steroid-sensitive nephrotic syndrome (SSNS), membranous nephropathy (MN), and healthy controls (hCtrls). Cell viability, podocyte actin cytoskeleton architecture, and reactive oxygen species (ROS) formation with or without ROS scavenger were investigated by Cell Counting Kit-8 assay, immunofluorescence staining, and CM-H2DCFDA probing, respectively. Presumed CPF-containing plasma causes a series of events in podocytes but not in GMVECs. These events include actin cytoskeleton rearrangement and excessive formation of ROS, which results in podocyte loss. These effects were solely observed in response to CPF plasma collected during relapse, but not in response to plasma of hCtrls, or patients with SRNS, SSNS, and MN. The copresence of dimethylthiourea, a scavenger of ROS, abolished the aforementioned effects of CPF plasma. We provide a panel of invitro bioassays to measure podocyte injury and predict the presence of CPFs in plasma of patients with nephrotic syndrome (NS), providing a new framework for monitoring CPF activity that may contribute to future NS diagnostics or used for disease monitoring purposes. Moreover, our findings suggest that the inhibition of ROS formation or facilitating rapid ROS scavenging may exert beneficial effects in patients with CPFs.

Downregulation of thrombomodulin-thrombin-activated protein C pathway as a mechanism for SARS-CoV-2 induced endotheliopathy and microvascular thrombosis

There is emerging evidence of microvascular thrombosis and thrombotic microangiopathy (TMA) induced by COVID-19, presumably from endothelial injury. Thrombomodulin (TM) is an endothelial glycoprotein that plays a dual role in maintaining healthy endothelium-as a natural anticoagulant by binding thrombin to activate protein C (APC) and a negative regulator of the alternate complement pathway (AP). TM is shed into the plasma as soluble TM (sTM) during endothelial injury.We hypothesize that SARS-CoV-2 spike proteins cause direct microvascular endothelial injury, leading to TM shedding, decreased activation of PC, and consequently, microvascular thrombosis in COVID-19. We conducted this study twofold: 1) in vivo, we assessed endothelial injury (by measuring sTM) and AP activation by quantifying Ba (cleavage product of AP component Factor B) in a cohort of critically ill COVID-19 pediatric patients and the implications on clinical outcomes; and 2)in vitro, we investigated endothelial injury (TM shedding) by SARS-COV-2 spike proteins and the subsequent functional consequence in activated PC (APC) levels and Ba levels.sTM and Ba in plasma samples from SARS-CoV-2 positive patients admitted to Texas Children's Hospital Pediatric Intensive Care Unit (n = 33) and from healthy controls (n = 38) were measured by ELISA. In vitro, confluent glomerular microvascular endothelial cells (GMVECs) were incubated for 48 h in the presence or absence (control) of purified SARS-CoV-2 spike proteins, S1 and S2. TM from the cell lysates while Ba and APC from the cell supernatants were measured by ELISA. sTM and Ba levels were significantly higher in the COVID-19 pediatric patients compared to healthy controls (p < 0.01 and p < 0.001, respectively). Among the COVID-19 patients, elevated sTM was associated with increased vasopressor use (p = 0.01) and elevated Ba was associated with increased duration of mechanical ventilation (p = 0.04). In vitro, surface bound TM and soluble APC were significantly lower in GMVECs after addition of spike proteins (p < 0.05), while Ba was undetectable in both control and spike proteins exposed GMVECs.In conclusion, we provide evidence of endothelial injury in COVID-19 pediatric patients and demonstrate a potential pathway of SARS-CoV-2 induced thrombosis. Decreased surface-bound TM results in lower amount of thrombin-TM complex, hence lesser activation of PC, likely leading to a pro-thrombotic state. These findings in GMVECs could explain the vulnerability of kidneys to COVID-19-induced TMA.

MO618: Influence of Butyrate and Acetate on Behavior and Metabolism of Human Glomerular Microvascular Endothelial Cells

Abstract BACKGROUND AND AIMS The gut microbiota has emerged as an important modulator of cardiovascular and renal diseases, including diabetic nephropathy (DN), one of the most common complications caused by diabetes mellitus. Importantly, diabetic patients display a reduction in bacteria producing short chain fatty acids (SCFAs) when compared with healthy individuals. SCFAs are generally associated with beneficial effects on the vascular system, as they help to reduce both oxidative stress and pro-inflammatory stimuli [1]. Although SCFAs help to protect against DN, their mechanisms of action at cell level remain largely unknown. For this reason, we aimed to study the effect of two SCFAs, butyrate and acetate, on human glomerular microvascular endothelial cells (hgMVECs) that represent one of the main components of the glomerular filtration barrier. In case of DN, glomerular endothelium shows multiple signs of dysfunction, such as uncontrolled proliferation [2], mitochondrial fragmentation [3] and monolayer disruption [4]. METHOD hgMVECs have been exposed to different concentrations of butyrate and acetate for 24 h and/or 48 h. After treatment with SCFAs, cells have been collected for qPCR, stained for mitochondrial mass or fixated for immunocytochemistry. For the latter, we have used antibodies for the proliferation marker ki-67 and cell-to-cell junction proteins. To measure the resistance of the endothelial monolayer, electric cell-substrate impedance sensing (ECIS) was used. This system lets a current go over and between cells and the tighter the monolayer, the higher the resistance. For this type of experiment, hgMVECs have been cultured on a specific plate that is located in the ECIS machine throughout the whole experiments (before and after treatment with SCFAs). RESULTS Butyrate decreases the number of ki-67 positive nuclei in a concentration-dependent manner, which implies lower cellular proliferation. On the other hand, at the highest concentration tested (500 uM), butyrate increases mitochondrial mass and elongation, as opposite to acetate, which does not show any effect on mitochondrial mass. These results are also supported by qPCR analysis, where it is possible to observe a decrease of genes involved in mitochondrial fragmentation upon butyrate exposure. MOreover, butyrate increases the resistance of the endothelial monolayer, as we can see in the results of the ECIS experiments. After an initial decrease, butyrate induces a steady increase of the resistance of the monolayer, which does not happen with acetate. We believe that the increase in resistance is due to a higher expression of genes and proteins involved in the formation of tight junctions. For instance, in cells treated with butyrate, gene expression of ve-cadherin, ZO-1, claudin 5 and occludin is upregulated and especially for ve-cadherin this is also observed by immunocytochemistry. CONCLUSION Our results show that in hgMVECs, on the one hand butyrate especially can increase monolayer resistance and influence mitochondrial morphology and possibly its metabolism, while on the other hand it decreases cellular proliferation. Therefore, at cellular level, butyrate can help against several hallmarks of endothelial dysfunction Of DN.

Targeting Cathepsin C in PR3-ANCA Vasculitis.

The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes.

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Bevacizumab Increases Endothelin-1 Production via Forkhead Box Protein O1 in Human Glomerular Microvascular Endothelial Cells In Vitro.

Molecular mechanisms underlying the nephrotoxicity associated with bevacizumab are unclear. Endothelin-1 (ET-1) is involved in podocyte injury and proteinuria, and its level increases in most cases of kidney disorders. Forkhead box protein O1 (FoxO1), a transcription factor, is a major determinant of ET-1 promoter activation and is regulated by protein kinase B (Akt) phosphorylation-dependent nuclear exclusion. We evaluated the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells (hGECs). We analyzed the changes in the mRNA and protein levels of ET-1 in hGECs treated with bevacizumab using real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Changes in the protein levels and phosphorylation status of Akt and FoxO1 in hGECs treated with bevacizumab were analyzed by western blotting. After cell lysis, FoxO1 protein was isolated from the cytoplasmic and nuclear fractions. We also investigated the effects of AS1842856 (a FoxO1 inhibitor) on bevacizumab-induced ET-1 production. Bevacizumab significantly and dose-dependently increased the mRNA and protein levels of ET-1 in hGECs (p < 0.05). Bevacizumab treatment also led to a decrease in phosphorylated Akt protein levels. Inhibition of Akt activity by LY294002 promoted ET-1 production. Bevacizumab also induced an increase in FoxO1 protein levels in the nucleus. Inhibition of FoxO1 activity by AS1842856 resulted in decreased ET-1 levels in bevacizumab-treated hGECs. ET-1 axis activation, Akt inactivation, and FoxO1 nuclear localization are the molecular mechanisms underlying bevacizumab-induced nephrotoxicity. Therefore, inhibition of renal ET-1 production could be a promising approach to protect against or treat bevacizumab-induced nephrotoxicity.

The Shiga Toxin Receptor Globotriaosylceramide as Therapeutic Target in Shiga Toxin E. coli Mediated HUS.

In 90% of the cases, childhood hemolytic uremic syndrome (HUS) is caused by an infection with the Shiga toxin (Stx) producing E. coli bacteria (STEC-HUS). Stx preferentially binds to its receptor, the glycosphingolipid, globotriaosylceramide (Gb3), present on the surface of human kidney cells and various organs. In this study, the glycosphingolipid pathway in endothelial cells was explored as therapeutic target for STEC-HUS. Primary human glomerular microvascular endothelial cells (HGMVECs) and human blood outgrowth endothelial cells (BOECs) in quiescent and activated state were pre-incubated with Eliglustat (Cerdelga®; glucosylceramide synthase inhibitor) or Agalsidase alpha (Replagal®; human cell derived alpha-galactosidase) in combination with various concentrations of Stx2a. Preincubation of endothelial cells with Agalsidase resulted in an increase of α-galactosidase activity in the cell, but had no effect on the binding of Stx to the cell surface when compared to control cells. However, the incubation of both types of endothelial cells incubated with or without the pro-inflammatory cytokine TNFα in combination with Eliglustat resulted in significant decrease of Stx binding to the cell surface, a decrease in protein synthesis by Stx2a, and diminished cellular Gb3 levels as compared to control cells. In conclusion, inhibition of the synthesis of Gb3 may be a potential future therapeutic target to protect against (further) endothelial damage caused by Stx.

Cell Biological Responses after Shiga Toxin-1 Exposure to Primary Human Glomerular Microvascular Endothelial Cells from Pediatric and Adult Origin.

Hemolytic uremic syndrome (HUS) is characterized by a triad of symptoms consisting of hemolytic anemia, thrombocytopenia and acute renal failure. The most common form of HUS is caused by an infection with Shiga toxin (Stx) producing Escherichia coli bacteria (STEC-HUS), and the kidneys are the major organs affected. The development of HUS after an infection with Stx occurs most frequently in children under the age of 5 years. However, the cause for the higher incidence of STEC-HUS in children compared to adults is still not well understood. Human glomerular microvascular endothelial cells (HGMVECs) isolated and cultured from pediatric and adult kidney tissue were investigated with respect to Stx binding and different cellular responses. Shiga toxin-1 (Stx-1) inhibited protein synthesis in both pediatric and adult HGMVECs in a dose-dependent manner at basal conditions. The preincubation of pediatric and adult HGMVECs for 24 hrs with TNFα resulted in increased Stx binding to the cell surface and a 20–40% increase in protein synthesis inhibition in both age groups. A decreased proliferation of cells was found when a bromodeoxyuridine (BrdU) assay was performed. A trend towards a delay in endothelial wound closure was visible when pediatric and adult HGMVECs were incubated with Stx-1. Although minor differences between pediatric HGMVECs and adult HGMVECs were found in the assays applied in this study, no significant differences were observed. In conclusion, we have demonstrated that in vitro primary HGMVECs isolated from pediatric and adult kidneys do not significantly differ in their cell biological responses to Stx-1.

Heme as Possible Contributing Factor in the Evolvement of Shiga-Toxin Escherichia coli Induced Hemolytic-Uremic Syndrome.

Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R2 −0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86–720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA.

Von Willebrand Factor Activates the Alternative Complement Pathway In Vivo in a Mouse Model of Complement Thrombotic Microangiopathy

Introduction Thrombotic microangiopathy (TMA) is a group of disorders presenting with microvascular thrombosis, microangiopathic hemolytic anemia, thrombocytopenia, and microvascular endothelial injury ultimately leading to end organ damage. Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated TMA (TA-TMA) are two types of TMA known as "complement TMA," as they are associated with dysfunction of the alternative complement pathway (AP), part of the innate immune system. Complement TMA episodes are frequently initiated during inflammation, and the role of inflammation in these TMAs may be in part related to the finding that the inflammatory cytokine TNF up-regulates complement components, down-regulates complement regulators, and causes AP activation in human glomerular microvascular endothelial cells (GMVECs) in vitro. However, the precise mechanism of AP activation in complement TMA is poorly understood. It has been demonstrated that von Willebrand factor (VWF) serves as a surface for AP activation in vitro. Inflammatory cytokines are stimulatory to endothelial cells, leading to excessive VWF secretion; in fact, patients with inflammatory disorders such as complement TMA demonstrate elevated plasma VWF levels, and this abundance of VWF may lead to excessive AP activation. We hypothesized that in complement TMA, VWF released from endothelial cells during inflammation activates the AP. The objective was to investigate whether VWF-mediated AP activation is initiating episodes of complement TMA in vivo in a complement TMA mouse model. Methods A mouse model of complement TMA was developed. To provoke complement TMA, wild-type (WT) C57BL/6J mice (purchased from Jackson Laboratories) were injected intraperitoneally with 5 mg/kg of the inflammatory endotoxin lipopolysaccharide (LPS). Twenty-four hours after injection of either LPS or saline (control), markers of complement TMA-lactate dehydrogenase (LDH), creatinine, platelet count, and complement activation by a rabbit erythrocyte (ER) lysis assay-were measured. ER specifically activate the AP in mammalian plasma in the presence of Mg/EGTA, and the resulting lysis is an indicator of the extent to which complement components have been consumed in vivo: low ER lysis in vitro indicates high complement activation in vivo. To assess the role of VWF in AP activation in vivo, VWF-/- mice (purchased from Jackson Laboratories on a C57BL/6J background) were injected with 5 mg/kg LPS to induce complement TMA. Twenty-four hours after injection, plasma ER lysis was measured and compared to ER lysis in LPS-injected WT C57BL/6J mice. Other markers of complement TMA, including LDH, creatinine, and platelet count, were also measured in both LPS-injected VWF-/- and WT C57BL/6J mice. Results Compared to saline-injected WT C57BL/6J mice, the LPS-injected mice demonstrated significantly: A) higher LDH (hemolysis); B) higher creatinine (renal dysfunction); C) lower platelet count; and D) lower ER lysis (increased AP activation) (Fig. 1). These results confirm the establishment of a complement TMA murine model. Furthermore, LPS-injected VWF-/- mice demonstrated significantly higher ER lysis values than the LPS-injected WT C57BL/6J mice, indicating less AP activation (Fig 2). Because VWF-/- mice do not produce any VWF, these data suggest that VWF contributes to AP activation in vivo in complement TMA. Finally, the LPS-injected VWF-/- mice had less severe TMA as demonstrated by lower LDH levels, lower creatinine levels, and higher platelet counts compared to the LPS-injected WT C57BL/6J mice (Fig. 3). The LPS-injected VWF-/- mice still had a mild degree of AP activation and TMA compared to saline-injected WT C57BL/6J mice, implying that there are complex mechanisms of AP activation in complement TMA that rely heavily on VWF. Conclusions In this study we demonstrated that VWF was important in complement activation and provocation of TMA in a complement TMA murine model. These data are the first step toward a better understanding of VWF's role in complement TMA and may lead to the development of therapeutics that inhibit inflammation, VWF release, or VWF-complement binding to curb AP activation in aHUS and TA-TMA, thereby preventing devastating outcomes. Further study is necessary to delineate the precise mechanisms of VWF-mediated AP activation and TMA development so that these treatment modalities may be explored. Disclosures No relevant conflicts of interest to declare.

Shiga Toxin Selectively Upregulates Expression of Syndecan-4 and Adhesion Molecule ICAM-1 in Human Glomerular Microvascular Endothelium.

Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies.

Protein kinaseC and protein kinaseA are involved in the protection of recombinant human glucagon-like peptide-1 on glomeruli and tubules in diabetic rats.

Aims/IntroductionBlockade or reversal the progression of diabetic nephropathy is a clinical challenge. The aim of the present study was to examine whether recombinant human glucagon‐like peptide‐1 (rhGLP‐1) has an effect on alleviating urinary protein and urinary albumin levels in diabetic rats.Materials and MethodsStreptozotocin‐induced diabetes rats were treated with rhGLP‐1 insulin and saline. Using immunostaining, hematoxylin–eosin, electron microscopy and periodic acid–Schiff staining to study the pathology of diabetic nephropathy, and we carried out quantitative reverse transcription polymerase chain reaction, western blot and immunohistochemistry to identify the differentially expressed proteins. The mechanism was studied through advanced glycation end‐products‐induced tubular epithelial cells.ResultsrhGLP‐1 inhibits protein kinase C (PKC)‐β, but increases protein kinase A (PKA), which reduces oxidative stress in glomeruli and in cultured glomerular microvascular endothelial cells. In tubules, rhGLP‐1 increased the expression of two key proteins related to re‐absorption – megalin and cubilin – which was accompanied by downregulation of PKC‐β and upregulation of PKA. On human proximal tubular epithelial cells, rhGLP‐1 enhanced the absorption of albumin, and this was blocked by a PKC activator or PKA inhibitor.ConclusionsThese findings suggest that rhGLP‐1 can reverse diabetic nephropathy by protecting both glomeruli and tubules by inhibiting PKC and activating PKA.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells

Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes C3, CFB, and C5 and decreased expression of CFD This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (CD46, THBD, CD55, CFI, and CFH) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases C3AR1 expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.

Effects of alloantibodies to human leukocyte antigen on endothelial expression and serum levels of thrombomodulin

Rationale Thrombomodulin (TBM) is an anticoagulant and anti-inflammatory transmembrane protein expressed on endothelial cells. Donor-specific alloantibodies, particularly those against human leukocyte antigen (HLA) class II, are associated with microvascular endothelial damage in solid allografts. Objective Our aim was to characterize the effects of anti-HLA antibodies on endothelial expression of TBM, and in particular, the differential effects of anti-HLA class I compared with those of anti-HLA class II. Methods We used human glomerular microvascular endothelial cells to examine TBM expression on anti-HLA-treated cells, and we tested sera from transplant recipients for soluble TBM. Results We found that whereas membrane TBM expression increased in a dose-dependent manner in the presence of anti-HLA class I antibodies, treatment with anti-HLA class II led to minimal TBM expression on the endothelial surface but to a cytosolic accumulation. Platelet adhesion studies confirmed the functional impact of anti-HLA class II. Quantitative densitometry of the membrane lysates further suggested that anti-HLA class II impairs TBM glycosylation. Furthermore, we found a significant association between the presence of circulating anti-HLA class II antibodies in transplant recipients and low serum levels of TBM. Conclusion These results indicate that ligation of anti-HLA class I and II antibodies produces different effects on the endothelial expression of TBM and on serum levels of TBM in transplant recipients. Anti-HLA class II antibodies may be associated with a prothrombotic state, which could explain the higher occurrence of microangiopathic lesions in the allograft and the poor outcomes observed in patients with these alloantibodies.

A kidney-selective biopolymer for targeted drug delivery.

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH2-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.

Distribution and Function of PACAP and Its Receptors in the Healthy and Nephrotic Kidney

Background/Aims: Plasma deficiency of pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated in children with nephrotic syndrome (NS). Previous studies have reported an important protective effect of PACAP on kidney proximal tubules. The aim of this study was to explore the expression of PACAP and its receptors PAC1, VPAC1 and VPAC2 in the healthy and nephrotic kidney and to determine if PACAP has an effect on renal proximal tubular cells exposed to albumin. Methods: Expression of PACAP and its receptors was studied using kidney tissue from healthy and nephrotic children, and in 3 human renal cell lines (glomerular microvascular endothelial cells, podocytes and proximal tubular epithelial HK-2 cells). The functionality of the VPAC1 receptor was tested in HK-2 cells, measuring cyclic adenosine monophosphate levels after PACAP exposure. The influence of PACAP on cell viability and transforming growth factor-β1 (TGF-β1) expression was measured in HK-2 cells exposed to albumin, mimicking proteinuria related damage. Results: VPAC1 expression was detected in the tubular proximal epithelial cells and in the glomerular podocytes of renal tissue from healthy and nephrotic children. Increased staining for PACAP was found in the proximal tubules of renal sections from children with NS compared to healthy renal sections. Expression and functionality of VPAC1 were demonstrated in HK-2 cells. Finally, PACAP did not alter cell viability or TGF-β1 expression of HK-2 cells exposed to albumin. Conclusion: VPAC1 is the predominant receptor in the human kidney. The enhanced presence of PACAP in proximal tubular epithelial cells in nephrotic kidneys points to the reabsorption of filtered PACAP. On short term, PACAP has no in vitro effect on cell viability and TGF-β1 expression of proximal tubular epithelial cells exposed to high concentrations of albumin.