Glomerular Capillary Endothelial Cells Research Articles

#246 Understanding the impact of antiangiogenic drug-induced proteinuria on kidney function in cancer patients

Abstract Background and Aims Proteinuria is a common adverse event associated with the use of vascular endothelial growth factor (VEGF) inhibitor drugs, which are widely employed in oncology for treating various tumors. The incidence of proteinuria of all degrees ranges from 10% to 90%, with proteinuria in the nephrotic range occurring in 1-5% of cases. VEGF plays a crucial role in maintaining the integrity of the glomerular capillary wall. Podocytes, constitutively express VEGF, and VEGF receptors are present on both the surface of glomerular capillary endothelial cells and podocytes. Any disruption in the podocyte-endothelial VEGF axis can lead to the loss of endothelial fenestrations in glomerular capillaries, proliferation of glomerular endothelial cells, loss of podocytes, and subsequent proteinuria. The most common pathological findings include focal segmental glomerulosclerosis and minimal change nephropathy, typically associated with tyrosine kinase inhibitors (TKIs), as well as thrombotic microangiopathy (TMA), more frequently observed in pts treated with anti VEGF. Proteinuria is a recognized risk factor for progressive renal damage and end-stage renal disease. However, it's worth noting that the average treatment duration for patients on anti-angiogenic therapy is considerably shorter than the time indicated by clinical studies as necessary to induce renal damage from proteinuria in healthy individuals. However, the long-term impact of drug-induced proteinuria on renal function remains uncertain. The aim of the study is to investigate the association between proteinuria induced by anti-VEGF/VEGFR drugs and its long-term effects on renal function in cancer patients. Method A retrospective study was conducted on 124 patients undergoing anti-VEGF/VEGFR therapy for ≥6 months. 45% of the patients enrolled in the study were on TKI monotherapy, 36% received TKI plus immune checkpoint inhibitors (ICIs), 10% received anti- VEGF treatment, 7% received TKI + chemotherapy (CT), and 2% received anti-VEGF + ICIs. Among patients, 51% were undergoing treatment for kidney cancer, 25% for thyroid cancer, 14% for colon cancer, 7% for liver cancer, and 5% for lung cancer. The study assessed the time course of estimated glomerular filtration rate (eGFR), the influence of baseline eGFR on subsequent variations, the association between proteinuria and renal function, and the identification of risk factors for proteinuria or renal failure. Proteinuria was quantified as proteinuria/24 h. Results Among the 124 patients, 53 developed proteinuria <1 g/24 h, 41 between 1-3 g/24 h, and 30 >3 g/24 h. Specific data are outlined in Table 1. A trend toward a reduction in eGFR of −5 ml/min/1.73 m2 was observed in all patients after 2 years of treatment. The high proteinuria group exhibited a greater percentage change in eGFR compared to the other groups (−1.5% vs −8.5% vs −12.3%) as depicted in Fig. 1A, along with a time-dependent decrease in eGFR. There was no significant difference in the development of eGFR <30 ml/min/1.73 m2 between groups, and no patients permanently discontinued treatment due to nephrotoxicity. No differences in eGFR reduction were noted among the three groups categorized by baseline eGFR (Fig. 1B). Importantly, renal function after the end of therapy was found to be reversible. Conclusion The study did not find a correlation between the degree of proteinuria secondary to anti-VEGF/VEGFR and significant reductions in renal function. Therefore, treatment can be continued irrespective of the degree of proteinuria. Clinical decisions regarding the continuation of therapy with these drugs should weigh the benefits in terms of survival against the potential risks of toxicity, including a modest reduction in renal glomerular filtration rate.

Neutrophil Extracellular Traps Aggravate Contrast-Induced Acute Kidney Injury by Damaging Glomeruli and Peritubular Capillaries.

Contrast-induced acute kidney injury (CI-AKI) is considered to be the third leading cause of hospital-acquired kidney injury. Current studies mostly suggest that contrast agents mainly harm renal tubular epithelial cells, but we hypothesized that the development of CI-AKI should be the result of the interaction of renal vascular and tubular injury. First we constructed a CI-AKI mouse model and verified the success of the model by pathological injury and serum creatinine level. Immunohistochemistry, protein quantification and qRT-PCR were used to detect the location and level of expression of neutrophil extracellular traps (NETs) in the kidney. Then, we blocked the in vivo accumulation of NETs using GSK484 and DNase I and detected the expression of NETs and the damage of glomerular and peritubular capillaries. We first identified the presence of NETs in CI-AKI mice, and NETs were mainly accumulated in glomeruli and peritubular capillaries. The expression of NETs was positively correlated with the severity of CI-AKI kidney. After inhibition of NETs release or promotion of NETs degradation by drugs, renal vascular endothelial cell injury was reduced and renal pathological changes and creatinine levels were reversed in CI-AKI mice. In addition, inhibition of NETs reduced apoptosis and pyroptosis of renal cells and attenuated inflammation in vivo. These findings suggest that NETs are involved in the development of CI-AKI by damaging glomerular and peritubular capillary endothelial cells. This study will provide a new strategy for clinical prevention and treatment of CI-AKI.

Low-Dose Erythropoietin Amplifies Beneficial Effects of Angiotensin II Blockade on Glomerulosclerosis

Exogenous erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). Concerns about the possible adverse effect of EPO on the progression of CKD have been raised owing to nonerythroid cell effects. We investigated the effects of low-dose EPO, independent of correcting anemia, on existing glomerulosclerosis. Adult mice underwent 5/6 nephrectomy and were randomized into the following 4 groups at week 8 after surgery: vehicle (VEH), losartan (angiotensin II type 1 receptor blocker [ARB]), darbepoetin-α (DA), or combination (DA+ARB). Four weeks later, mice were euthanized, followed by evaluation of renal structure and function. Glomerular endothelial cells and podocytes were cultured to evaluate the effects of DA on cell migration, apoptosis, and Akt signaling. ARB reduced blood pressure, albuminuria, and the level of serum creatinine and increased hematocrit compared with VEH, whereas low-dose DA only reduced the level of serum creatinine. Combination treatment showed a trend to increase hematocrit and survival compared with ARB alone. Combination treatment but not ARB alone significantly reduced the progression of glomerulosclerosis compared with VEH. Low-dose DA resulted in more preserved glomerular and peritubular capillary endothelial cells with increased p-Akt and even further endothelial cell preservation in combination with ARB. In cultured glomerular endothelial cells, angiotensin II induced more apoptosis, reduced migration, and decreased p-Flk1, a receptor for the proangiogenic vascular endothelial growth factor. DA counteracted these injuries and increased p-Akt, a key factor in angiogenesis and cell survival. DA also protected cultured podocytes against transforming growth factor β–induced apoptosis and synaptopodin loss. Low-dose EPO directly protects glomerular and peritubular endothelial cells via Akt phosphorylation. Therefore, treatment using a combination of low-dose EPO and ARB results in less progression of glomerulosclerosis in an experimental CKD model.

15-keto-Prostaglandin E2 exhibits bioactive role by modulating glomerular cytoarchitecture through EP2/EP4 receptors

AimsProstaglandins are important signaling lipids with prostaglandin E2 (PGE2) known to be the most abundant prostaglandin across tissues. In kidney, PGE2 plays an important role in the regulation of kidney homeostasis through its EP receptor signaling. Catabolism of PGE2 yields the metabolic products that are widely considered biologically inactive. Although recent in vitro evidence suggested the ability of 15-keto-PGE2 (a downstream metabolite of PGE2) to activate EP receptors, the question whether 15-keto-PGE2 exhibits physiological roles remains unresolved. Materials and methodsPharmacological treatment was performed in transgenic zebrafish embryos using 500 μM 15-keto-PGE2 and 20 μM EP receptors antagonists' solutions during zebrafish embryonic development. After the exposure period, the embryos were fixed for confocal microscopy imaging and glomerular morphology analysis. Key findingsHere, we show that 15-keto-PGE2 can bind and stabilize EP2 and EP4 receptors on the plasma membrane in the yeast model. Using lipidomic analysis, we demonstrate both PGE2 and 15-keto-PGE2 are present at considerable levels in zebrafish embryos. Our high-resolution image analysis reveals the exogenous treatment with 15-keto-PGE2 perturbs glomerular vascularization during zebrafish development. Specifically, we show that the increased levels of 15-keto-PGE2 cause intercalation defects between podocytes and endothelial cells of glomerular capillaries effectively reducing the surface area of glomerular filtration barrier. Importantly, 15-keto-PGE2-dependent defects can be fully reversed by combined blockade of the EP2 and EP4 receptors. SignificanceAltogether, our results reveal 15-keto-PGE2 to be a biologically active metabolite that modulates the EP receptor signaling in vivo, thus playing a potential role in kidney biology.

The PDE5 inhibitor, vardenafil, ameliorates progressive pathological changes in a focal segmental glomerulosclerosis mouse model

AimsPhosphodiesterase 5 inhibitors (PDE5is) inhibit the hydrolysis of cyclic guanosine 5′-monophosphate in smooth muscle cells and are a widely known treatment for erectile dysfunction. Accumulating evidence also suggests that PDE5is exhibit potential benefits in cardiovascular and chronic kidney diseases. In this study, we examined the therapeutic effects of a PDE5i, vardenafil (VAR), in a focal segmental glomerulosclerosis (FSGS) mouse model. Materials and methodsFSGS was induced in BALB/c mice by the intravenous administration of Adriamycin (AD, 11 mg/kg of body weight). After 24 h, VAR (at 12.5 μg/ml) was given in drinking water ad libitum until the animals were sacrificed. At the end of the experiment, plasma and kidney samples were harvested to evaluate clinical parameters, histopathological changes, and alterations in transcriptome and protein expressions. Key findingsIn this study, VAR treatment attenuated the deterioration of proteinuria, renal dysfunction, and hypercholesterolemia in AD-induced FSGS. Treatment with VAR exhibited reductions in the severity of both glomerulosclerosis and tubulointerstitial injury in the histological analysis. In addition to relieving AD-induced podocyte loss, VAR also preserved endothelial cells in glomerular capillaries and ameliorated the accumulation of collagen fibers in the mesangial area and Bowman's capsule basement membrane. In addition, VAR showed an ability to suppress transforming growth factor-β-induced fibroblast-to-myofibroblast transdifferentiation. SignificanceOur data suggest that VAR exhibited reno-therapeutic effects via attenuating podocyte loss, preserving the integrity of the glomerular vasculature, and ameliorating fibrotic changes. These findings suggest that PDE5is might be a promising treatment modality for nephrotic syndrome.

Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway

Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.

COVID-19 in Kidney Transplantation: Outcomes, Immunosuppression Management, and Operational Challenges.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2, has led to the death of hundreds of thousands of people worldwide. If infected, older individuals and those with diabetes, hypertension, cardiovascular disease, and compromised immune systems are at higher risk for unfavorable outcomes. These comorbidities are prevalent in patients with kidney disease, hence the significant burden of COVID-19 on kidney transplant programs. Multiple case series of kidney transplant recipients with COVID-19 have shown increased mortality compared to nontransplant patients. To date, we do not have high-level evidence to inform immunosuppression minimization strategies in infected transplant recipients. Most centers however have adopted early antimetabolite withdrawal in addition to other interventions. This review summarizes the published COVID-19 literature as it relates to outcomes and immunosuppression management in kidney transplant recipients. It also discusses challenges pertaining to pretransplant evaluation and wait-listed patients.

P0122THE MECHANISM OF DNA INJURY INDUCED COLLAGEN TYPE VI EXCRETION IN GLOMERULAR ENDOTHERIAL CELLS

Abstract Background and Aims Collagen deposition is observed in various part of kidney tissue and is serious pathological damage in kidney. Nodular glomerulosclerosis is one of a phenotype of collagen deposition in glomerular lesion, which affects the prognosis of renal function, but there is no effective treatment at present. Recently, we identified that glomerular endothelial cells with DNA double-strand breaks (DSB) are involved in the long-term accumulation of ECM molecules, especially collagen type VI(COL6). However, the mechanism of glomerular COL6 accumulation is still unclear. Method We examined the phospho-histone H2AX (γ-H2AX) of the DSB marker and COL6 accumulation in biopsied human kidneys to investigate their association. In addition, we investigated in vitro the relationship between DSB and COL6 excretion and the intracellular signal pathways in human glomerular endothelial cells (HRGEc) using mitomycin C (MMc)-induced DNA damage. In this study, we focused on the expression of DSB response signal pathways, i.e. ATM, ATR and DNA-PK using their specific kinase inhibitors (KU55933, VE-821, Nu7441). Results COL6 deposits and γ-H2AX expression in nuclei were detected in human glomerular capillary loop and glomerular nodular lesions. The multiple regression analysis showed that γ-H2AX positive area is the most independent factor for COL6 accumulation in glomeruli (β: 0.539, t=2.668, p=0.014). Furthermore, COL6 was the independent factor of nodular lesion in various pathologic diseases such as diabetic nephropathy, hypertensive glomerulosclerosis and the long-term transplanted allografts (β: 0.373, t=2.268, p=0.032). In vitro study, COL6 excretion detected by the decrease of COL6 positive cells was suppressed only in the ATR-inhibited group (control vs. kinase inhibitors; 2 h, 40.1 ± 23.5 vs. 59.0 ± 23.2%, p <0.01; 24 h, 18.8 ± 23.6 vs. 56.3 ± 16.5%, p <0.001). Conclusion This study suggested that nodular glomerulosclerosis is induced by DNA damage and COL6 deposition through ATR pathway in glomerular capillary endothelial cells. These findings will provide great insights into the development of new therapies that would suppress glomerularsclerosis in various kidney diseases.

The immunological background of glomerular injuries

The basic structural units of the renal filtration are the glomeruli, which, in addition to their passive hemodynamic function, also participate in complex immune-mediated mechanisms. The immune system as a double-edged sword maintains the physiological homeostasis of the glomeruli, but also plays a crucial role in the induction of glomerular damage. The immune-mediated chronic glomerular injures are the most common cause of end-stage renal diseases. The unregulated and overactive immune response can damage both the structural and the cellular components of the glomeruli, including the glomerular basal membrane, mesangial and capillary endothelial cells, podocytes, and parietal epithelium. The manuscript summarizes the role of the glomerular components and the natural and adaptive immune response in the pathomechanism of glomerular diseases. Orv Hetil. 2020; 161(24): 993-1001.

Platelet retention in inflamed glomeruli occurs via selective prolongation of interactions with immune cells

Platelet-leukocyte interactions promote acute glomerulonephritis. However, neither the nature of the interactions between platelets and immune cells nor the capacity of platelets to promote leukocyte activation has been characterized in this condition. We used confocal intravital microscopy to define the interactions of platelets with neutrophils, monocytes, and endothelial cells in glomerular capillaries in mice. In the absence of inflammation, platelets underwent rapid on/off interactions with immune cells. During glomerulonephritis induced by in situ immune complex formation, platelets that interacted with neutrophils or monocytes, but not with other intraglomerular cells, were retained in the glomerulus for prolonged durations. Depletion of platelets inhibited both neutrophil recruitment and activation. Inhibition of platelet activating factor reduced neutrophil recruitment withoutimpacting reactive oxygen species generation, while blocking CXC chemokine ligand 7 (CXCL7) reducedboth responses. In contrast, inhibition of the adenosine diphosphate and thromboxane A2 pathways inhibited neutrophil reactive oxygen species generation without affecting neutrophil adhesion. Thus, platelet retention in glomerular capillaries following immune complex deposition stems from prolongation of platelet interactions with immune cells but not other substrates. Pro-inflammatory mediators play divergent roles in promoting neutrophil retention and activation in glomerular capillaries.

Loss of mucin-type O-glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney

The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1-derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1-derived O-glycans is catalyzed by the glycosyltransferase core 1 β1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1-/-) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process-associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.

Low vascularization of the nephrogenic zone of the fetal kidney suggests a major role for hypoxia in human nephrogenesis.

CD31 reactivity is generally utilized as a marker of endothelial cells. CD31 immunoreactivity in the developing human kidney revealed that fetal glomerular capillary endothelial cells change their immunohistochemical phenotype during maturation. The aim of this study was to analyze CD31 reactivity in the fetal human kidney in the different stages of intrauterine development: We observed different distribution of CD31-reactive vascular progenitors in the different areas of the developing kidney. In particular, the nephrogenic zone and the renal capsule were characterized by a scarcity of CD31-reactive cells at all gestational ages. These data suggest the hypothesis that nephrogenesis does not need high oxygen levels and confirms a major role of hypoxia in nephrogenesis.

Review series: The cell biology of renal filtration.

The function of the kidney, filtering blood and concentrating metabolic waste into urine, takes place in an intricate and functionally elegant structure called the renal glomerulus. Normal glomerular function retains circulating cells and valuable macromolecular components of plasma in blood, resulting in urine with just trace amounts of proteins. Endothelial cells of glomerular capillaries, the podocytes wrapped around them, and the fused extracellular matrix these cells form altogether comprise the glomerular filtration barrier, a dynamic and highly selective filter that sieves on the basis of molecular size and electrical charge. Current understanding of the structural organization and the cellular and molecular basis of renal filtration draws from studies of human glomerular diseases and animal models of glomerular dysfunction.

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

Advanced glycation end‑products affect the cytoskeletal structure of rat glomerular endothelial cells via the Ras‑related C3 botulinum toxin substrate1 signaling pathway.

The present study aimed to determine the molecular mechanisms leading to the production of advanced glycation end‑products (AGEs) and their effect on the morphology and function of rat glomerular capillary endothelial cells (GECs). Primary rat GECs were treated with AGE‑modified human serum albumin (AGE‑HSA) and divided into groups according to AGE concentration and treatment time. The structure and distribution of cytoskeletal protein F‑actin and the cortical actin binding protein, cortactin, were analyzed using immunofluorescence and confocal microscopy. As the Ras‑related C3 botulinum toxin substrate1 (Rac1) signaling pathway was previously identified to be involved in mediating the contraction of endothelial actin‑myosin activity, Rac1 was examined subsequent to treatment of the cells with the Rac1 agonist 2'‑O‑methyladenosine‑3',5'‑cyclic monophosphate (O‑Me‑cAMP) for 1h using a pull‑down assay. Cell permeability was determined by the leakage rate of a fluorescein isothiocyanate fluorescent marker protein. AGE‑HSA treatment resulted in alterations in the structure and distribution of F‑actin and cortactin in a dose‑ and time‑dependent manner, while no effect was observed with HSA alone. The effect of AGE on the cytoskeleton was inhibited by the addition of O‑Me‑cAMP. AGE‑HSA significantly reduced the level of Rac1 activity (P<0.05); however, no effect was observed on total protein levels. Furthermore, AGE‑HSA treatment led to a significant increase in the permeability of endothelial cells (P<0.01), which was inhibited by O‑Me‑cAMP (P<0.01). The Rac1 signaling pathway is thus suggested to serve an important function in mediating AGE‑induced alterations in GEC morphology and function.

Crystalloid glomerulopathy in monoclonal gammopathy of renal significance (MGRS).

We report a case of monoclonal gammopathy of renal significance in a 63-year-old man who presented with nephrotic-range proteinuria and renal insufficiency. The kidney biopsy showed a membranoproliferative glomerulonephritis pattern with extensive crystalloid deposits in the glomerular capillary endothelial cells and very few in the tubular epithelial cells. The immunoperoxidase staining showed kappa light chain restriction. Subsequently, the bone marrow showed 6% plasma cells which confirmed the diagnosis of monoclonal gammopathy of renal significance. He responded well to bortezomib treatment with resolution of the nephrotic syndrome and normalization of renal function after 7 months.

Cytomegalovirus Glomerulopathy and Cytomegalovirus Interstitial Nephritis on Sequential Transplant Kidney Biopsies

Cytomegalovirus (CMV) nephropathy may be seen in kidney transplant biopsy specimens. We report a CMV-negative patient who received a kidney transplant from a CMV-positive donor and subsequently developed CMV glomerulopathy and CMV-associated interstitial nephritis, as observed in 2 sequential kidney biopsies. The first biopsy specimen showed CMV-positive endothelial cells in glomerular capillaries and CMV-infected monocytes in glomerular capillary lumens. The second biopsy specimen showed CMV-positive cells in the interstitium with associated lymphoplasmacytic infiltrate and tubular injury, but no evidence of direct CMV infection in tubular epithelial cells. Moreover, the second biopsy specimen showed persistent monocytes with cytoplasmic viral particles within glomerular capillary loops by electron microscopy. Our case shows that CMV glomerulopathy can be caused by direct CMV infection of glomerular capillary endothelial cells. CMV-positive circulating monocytes may play an important role in the different histopathologic manifestations of CMV nephropathy in kidney transplant grafts.

2108 BLOOD GROUP ANTIGEN-TARGETING PEPTIDE SUPPRESSES ANTI-BLOOD GROUP ANTIBODY BINDING TO ANTIGEN IN RENAL GLOMERULAR CAPILLARIES AFTER ABO-INCOMPATIBLE BLOOD REPERFUSION

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation, Renal Vascular Surgery1 Apr 20132108 BLOOD GROUP ANTIGEN-TARGETING PEPTIDE SUPPRESSES ANTI-BLOOD GROUP ANTIBODY BINDING TO ANTIGEN IN RENAL GLOMERULAR CAPILLARIES AFTER ABO-INCOMPATIBLE BLOOD REPERFUSION Tohru Yoneyama, Shingo Hatakeyama, Yuki Tobisawa, Hayato Yamamoto, Kengo Imanishi, Teppei Okamoto, Noriko Tokui, Naoki Sugiyama, Yuichiro Suzuki, Shigemasa Kudo, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Noritaka Kamimura, Michiko N Fukuda, and Chikara Ohyama Tohru YoneyamaTohru Yoneyama Hirosaki, Japan More articles by this author , Shingo HatakeyamaShingo Hatakeyama Hirosaki, Japan More articles by this author , Yuki TobisawaYuki Tobisawa Hirosaki, Japan More articles by this author , Hayato YamamotoHayato Yamamoto Hirosaki, Japan More articles by this author , Kengo ImanishiKengo Imanishi Hirosaki, Japan More articles by this author , Teppei OkamotoTeppei Okamoto Hirosaki, Japan More articles by this author , Noriko TokuiNoriko Tokui Hirosaki, Japan More articles by this author , Naoki SugiyamaNaoki Sugiyama Hirosaki, Japan More articles by this author , Yuichiro SuzukiYuichiro Suzuki Hirosaki, Japan More articles by this author , Shigemasa KudoShigemasa Kudo Hirosaki, Japan More articles by this author , Takahiro YoneyamaTakahiro Yoneyama Hirosaki, Japan More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto Hirosaki, Japan More articles by this author , Takuya KoieTakuya Koie Hirosaki, Japan More articles by this author , Noritaka KamimuraNoritaka Kamimura Hirosaki, Japan More articles by this author , Michiko N FukudaMichiko N Fukuda La Jolla, CA More articles by this author , and Chikara OhyamaChikara Ohyama Hirosaki, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Antibody (Ab)-mediated rejection after ABO-incompatible kidney transplantation (ABO-I KTx) is a major barrier to transplantation success. The advent of immunosuppressive therapy has markedly improved graft survival in ABO-I KTx. However, compared with normal KTx, clinical conditions during ABO-I KTx are difficult to control because of overimmunosuppression. To reduce the need for immunosuppression, we aimed to develop a novel blood group antigen-neutralizing therapy. METHODS We screened for an ABO blood group antigen-targeting peptide (BATP) by screening of T7 phage-displayed peptide library. After screening, hemagglutination (HA) inhibition assays, ELISA and cytotoxicity assay were used to analyze the blood group antigen-blocking effect and toxicity of BATP. We also tested the inhibitory effects on anti-blood group Ab binding in normal human kidney tissues blocked with BATP and excised kidneys perfused ex vivo with BATP. RESULTS We identified 6 peptide sequences that efficiently suppressed HA of red blood cells by anti-A or -B antigen Abs. Most effective peptide is RPRNPNK and named as BATP. Cytotoxicity of BATP against primary culture of blood type O human kidney glomerular capillary microvascular endothelial cells (HGMEC) was very low. BATP bound to peritubular and microvascular endothelia in type A and B kidney tissues but not in type O kidney tissues. BATP significantly inhibited the reactivity of anti-A and B Abs against A and B histo-blood group antigens expressed in glomerular capillaries as well as peritubular and microvascular endothelia. Surprisingly, ex vivo perfusion of BATP in kidneys excised from renal cell carcinoma patients caused significant suppression of anti-blood group antibody binding to antigen, IgG and IgM deposition in renal glomerular capillaries after ABO-I blood reperfusion (shown in figure). CONCLUSIONS These data indicate that A/B blood group antigens on red blood cells and in kidney tissues may be neutralized by BATP. This approach may enable the development of a novel blood group antigen-neutralizing therapy to overcome the challenges of ABO-I KTx. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e864 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tohru Yoneyama Hirosaki, Japan More articles by this author Shingo Hatakeyama Hirosaki, Japan More articles by this author Yuki Tobisawa Hirosaki, Japan More articles by this author Hayato Yamamoto Hirosaki, Japan More articles by this author Kengo Imanishi Hirosaki, Japan More articles by this author Teppei Okamoto Hirosaki, Japan More articles by this author Noriko Tokui Hirosaki, Japan More articles by this author Naoki Sugiyama Hirosaki, Japan More articles by this author Yuichiro Suzuki Hirosaki, Japan More articles by this author Shigemasa Kudo Hirosaki, Japan More articles by this author Takahiro Yoneyama Hirosaki, Japan More articles by this author Yasuhiro Hashimoto Hirosaki, Japan More articles by this author Takuya Koie Hirosaki, Japan More articles by this author Noritaka Kamimura Hirosaki, Japan More articles by this author Michiko N Fukuda La Jolla, CA More articles by this author Chikara Ohyama Hirosaki, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression of Toll-Like Receptor 4 in Glomerular Endothelial Cells under Diabetic Conditions

Diabetic conditions promote glomerulosclerosis by mesangial cells but the mechanisms are not fully elucidated. The present study evaluated the expression of toll-like receptor 4 in glomerular endothelial cells in the streptozotocin (STZ)-induced type 1 diabetic mouse (ICR-STZ) and the type 2 diabetic KK/TaJcl mouse which were fed a high fat diet feed (KK/Ta-HF). In the ICR-STZ and KK/Ta-HF almost glomeruli were immunostained with anti-TLR4 but there was no glomerulus immunostained by ani-TLR4 in the control ICR and KK/Ta. Laser-scanning confocal microscopy showed that the TLR4-positive region did not coincide with the podoplanin-positive region but coincide with the PECAM-1- and VE-cadherin-positive regions in the glomeruli of the ICR-STZ and KK/Ta-HF. The in situ hybridization showed that almost signals for TLR4 mRNA were present in the glomerulus of the ICR-STZ and KK/Ta-HF to a stronger extent than in the control ICR and KK/Ta. These suggest that glomerular endothelial cells usually express the TLR4 gene and hyperglycemia in the diabetic condition induces the TLR4 protein expression in the glomerular capillary endothelial cells. Cytokine productions through the TLR signaling pathway in glomerular endothelial cells may allow mesangial cells to produce extracellular matrix proteins in the diabetic milieu.

ENOS Deficiency Acts through Endothelin to Aggravate sFlt-1–Induced Pre-Eclampsia–Like Phenotype

Excess soluble fms-like tyrosine kinase 1 (sFlt-1) of vascular endothelial growth factor receptor 1 secreted from the placenta causes pre-eclampsia-like features by antagonizing vascular endothelial growth factor signaling, which can lead to reduced endothelial nitric oxide synthase (eNOS) activity; the effect of this concomitant decrease in eNOS activity is unknown. We tested whether the decrease in nitric oxide occurring in female mice lacking eNOS aggravates the pre-eclampsia-like phenotype induced by increased sFlt-1. Untreated eNOS-deficient female mice had higher BP than wild-type mice. Adenovirus-mediated overexpression of sFlt-1 increased systolic BP by approximately 27 mmHg and led to severe loss of fenestration of glomerular capillary endothelial cells in both eNOS-deficient and wild-type mice. However, only the eNOS-deficient sFlt-1 mice exhibited severe foot process effacement. Compared with wild-type sFlt-1 mice, eNOS-deficient sFlt-1 mice also showed markedly higher urinary albumin excretion (467±74 versus 174±23 μg/d), lower creatinine clearance (126±29 versus 452±63 μl/min), and more severe endotheliosis. Expression of preproendothelin-1 (ET-1) and its ET(A) receptor in the kidney was higher in eNOS-deficient sFlt-1 mice than in wild-type sFlt-1 mice. Furthermore, the selective ET(A) receptor antagonist ambrisentan attenuated the increases in BP and urinary albumin excretion and ameliorated endotheliosis in both wild-type and eNOS-deficient sFlt-1 mice. Ambrisentan improved creatinine clearance and podocyte effacement in eNOS-deficient sFlt-1 mice. In conclusion, reduced maternal eNOS/nitric oxide exacerbates the sFlt1-related pre-eclampsia-like phenotype through activation of the endothelin system.