Kidney Endothelial Cells Research Articles

Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells.

Vascular endothelial cell (EC) function depends on appropriate organ-specific molecular and cellular specializations. To explore genomic mechanisms that control this specialization, we have analyzed and compared the transcriptome, accessible chromatin, and DNA methylome landscapes from mouse brain, liver, lung, and kidney ECs. Analysis of transcription factor (TF) gene expression and TF motifs at candidate cis-regulatory elements reveals both shared and organ-specific EC regulatory networks. In the embryo, only those ECs that are adjacent to or within the central nervous system (CNS) exhibit canonical Wnt signaling, which correlates precisely with blood-brain barrier (BBB) differentiation and Zic3 expression. In the early postnatal brain, single-cell RNA-seq of purified ECs reveals (1) close relationships between veins and mitotic cells and between arteries and tip cells, (2) a division of capillary ECs into vein-like and artery-like classes, and (3) new endothelial subtype markers, including new validated tip cell markers.

Abstract P266: Western Diet Impairs Small Vessel Relaxation and Initiates Kidney Endothelial Stiffening, Fibrosis and Tubulointerstitial Fibrosis Through the Endothelial Mineralocorticoidreceptor

Obesity enhances mineralocorticoid receptor (MR) activation, development of vascular stiffness and end organ injury. In this context, western diet (WD) activation of the endothelial mineralocorticoid receptor (ECMR) contributes to endothelial cell stiffening and promotes maladaptive inflammatory responses and fibrosis in cardiovascular tissue of female mice. However, the role of ECMR on kidney endothelial stiffening, inflammation and fibrosis remains unknown. We hypothesized that deletion of the ECMR would prevent WD-induced increases in endothelial cell stiffness, reductions in bioavailable nitric oxide (NO), increased perivascular and tubulointerstitial inflammation oxidant stress, and fibrosis in females. Four-week-old female ECMR knockout and wild-type mice were fed either a mouse chow or a WD high in saturated fat and refined carbohydrates for 16 weeks. Without blood pressure changes between groups, WD-feeding increased body weight and fat mass as well as indices of vascular stiffness (pulse wave velocity and kidney endothelial cell stiffness) and impaired endothelial-dependent vasodilatation. The WD-induced kidney endothelial cell stiffness was associated with attenuated endothelial NO synthase activation, increased oxidative stress, along with pro-inflammatory immune responses, alterations in extracellular matrix degradation pathways and tubulointerstitial fibrosis. ECMR deletion prevented these abnormalities through improvements in endothelial NO synthase and reductions in macrophage polarization, LARP6, TG2 and MMP2. Our data support that activation of ECMR contributes to endothelial dysfunction, increased permeability and stiffening in the kidney which, in turn, promotes macrophage infiltration, M1 polarization, inflammation and oxidative stress, resulting in alterations in matrix degradation that promote tubulointerstitial fibrosis in females consuming a WD.

Progenitor Renin Lineage Cells are not involved in the regeneration of glomerular endothelial cells during experimental renal thrombotic microangiopathy.

Endothelial cells (EC) frequently undergo primary or secondary injury during kidney disease such as thrombotic microangiopathy or glomerulonephritis. Renin Lineage Cells (RLCs) serve as a progenitor cell niche after glomerular damage in the adult kidney. However, it is not clear whether RLCs also contribute to endothelial replenishment in the glomerulus following endothelial injury. Therefore, we investigated the role of RLCs as a potential progenitor niche for glomerular endothelial regeneration. We used an inducible tet-on triple-transgenic reporter strain mRen-rtTAm2/LC1/LacZ to pulse-label the renin-producing RLCs in adult mice. Unilateral kidney EC damage (EC model) was induced by renal artery perfusion with concanavalin/anti-concanavalin. In this model glomerular EC injury and depletion developed within 1 day while regeneration occurred after 7 days. LacZ-labelled RLCs were restricted to the juxtaglomerular compartment of the afferent arterioles at baseline conditions. In contrast, during the regenerative phase of the EC model (day 7) a subset of LacZ-tagged RLCs migrated to the glomerular tuft. Intraglomerular RLCs did not express renin anymore and did not stain for glomerular endothelial or podocyte cell markers, but for the mesangial cell markers α8-integrin and PDGFRβ. Accordingly, we found pronounced mesangial cell damage parallel to the endothelial injury induced by the EC model. These results demonstrated that in our EC model RLCs are not involved in endothelial regeneration. Rather, recruitment of RLCs seems to be specific for the repair of the concomitantly damaged mesangium.

Bile Acids Increase Doxorubicin Sensitivity in ABCC1-expressing Tumour Cells

Tumour cells possess or acquire various mechanisms to circumvent the cytotoxic effects of chemotherapy drugs. One such mechanism involves the overexpression of ABC transporters that facilitate the extrusion of a variety of structurally distinct chemotherapy drugs from the cytoplasm into the extracellular space. While specific ABC transporter inhibitors have been developed, many affect other ABC transporters, particularly at elevated concentrations. It is also unclear whether they show clear efficacy for combatting drug resistance in cancer patients with minimal host toxicity. In this study, we demonstrate the ability of two bile acids [β-cholanic acid (urso-cholanic acid) and deoxycholic acid] to specifically inhibit ABCC1-mediated drug transport, augmenting doxorubicin accumulation in breast and lung tumour cells selected for doxorubicin resistance through overexpression of the ABCC1 (but not ABCB1) drug transporter. The bile acids could also restore uptake and sensitivity to doxorubicin in human endothelial kidney cells genetically engineered to overexpress the ABCC1 drug transporter. These observations suggest a previously unreported role for bile acids as ABCC1 inhibitors or regulators. Given its additional properties of minimal clinical toxicity in humans and its ability to inhibit aldo-keto reductases involved in anthracycline resistance and anthracycline-induced cardiotoxicity, β-cholanic acid merits further in vivo and clinical investigation.

The Optimal PEG for Kidney Preservation: A Preclinical Porcine Study.

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).

The influence of HK2 blood group antigen on human B cell activation for ABOi-KT conditions

BackgroundIt is well known that ABO blood group system incompatible kidney transplantation (ABOi-KT) is an effective strategy for end-stage renal disease. The main barrier for ABOi-KT is how to keep host B cell activation and blood group antibody titer in low levels. Moreover, the mechanism of B cell activation induced by blood group antigen was unclear in ABOi-KT.ResultsIn this study, HK2 cells were identified to express blood group B antigen when cocultured with lymphocytes of blood group A. Optical microscope observation demonstrated that HK2 cells in coculture group gradually decreased. Furthermore, flow cytometer assay identified that T cell phenotypes (CD3+, CD3+CD4+ and CD3+CD8+) had no significant change and B cell phenotypes (CD19+ and CD138+) were all significantly enhanced (3.07 and 3.02 folds) at day 4. In addition, immunoturbidimetry analysis demonstrated that blood group B antibody was significantly increased to 2.35 fold at day 4, IgG was significantly increased to 3.60 and 2.81 folds at days 4 and 8 respectively, while IgM had no significant change at the measured time points.ConclusionsTaken together, B cells were activated and secreted blood group B antibody after treatment with HK2 expressing blood group B antigen. The results of this study maybe useful for further determination of the mechanism of B cell activation after ABO incompatible kidney endothelial cells stimulation.

Efficient uptake of blood-borne BK and JC polyomavirus-like particles in endothelial cells of liver sinusoids and renal vasa recta.

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells that mediate high-capacity clearance of soluble waste macromolecules and colloid material, including blood-borne adenovirus. To explore if LSECs function as a sink for other viruses in blood, we studied the fate of virus-like particles (VLPs) of two ubiquitous human DNA viruses, BK and JC polyomavirus, in mice. Like complete virions, VLPs specifically bind to receptors and enter cells, but unlike complete virions, they cannot replicate. 125I-labeled VLPs were used to assess blood decay, organ-, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and -cytochemistry. BK- and JC-VLPs rapidly distributed to liver, with lesser uptake in kidney and spleen. Liver uptake was predominantly in LSECs. Blood half-life (∼1 min), and tissue distribution of JC-VLPs and two JC-VLP-mutants (L55F and S269F) that lack sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. Liver uptake was not mediated by scavenger receptors. In spleen, the VLPs localized to the red pulp marginal zone reticuloendothelium, and in kidney to the endothelial lining of vasa recta segments, and the transitional epithelium of renal pelvis. Most VLP-positive vessels in renal medulla did not express PV-1/Meca 32, suggesting location to the non-fenestrated part of vasa recta. The endothelial cells of these vessels also efficiently endocytosed a scavenger receptor ligand, formaldehyde-denatured albumin, suggesting high endocytic activity compared to other renal endothelia. We conclude that LSECs very effectively cleared a large fraction of blood-borne BK- and JC-VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. In addition, we report the novel finding that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism.

Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation

BackgroundMortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. MethodsWild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. ResultsAICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. ConclusionsIn this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

An enhanced immune response against G250, induced by a heterologous DNA prime‑protein boost vaccination, using polyethyleneimine as a DNA vaccine adjuvant.

The heterologous DNA prime‑protein boost vaccination approach has been widely applied as an immune treatment for carcinoma. However, inefficient delivery of DNA remains a major issue. In the present study, polyethyleneimine (PEI) was used as a DNA vector carrier to improve the transfection efficiency of the DNA vaccine and stimulate the humoral and cellular immunity against the renal carcinoma‑associated antigenG250. A protein vaccine was included in the immunization strategy in order to produce a prime‑boost effect. A DNA plasmid encoding an antigen G250 fragment was constructed and complexed with PEI. A protein vaccine against G250 was expressed in BL21(DE3) Escherichiacoli cells, by transformation with a pET28a(+)/C‑G250 plasmid. The protein was purified using a nickel‑nitriloacetic acid purification system. The invitro transfection efficiency of the DNA vaccine was analyzed in HEK293 human endothelial kidney cells. The invitro transfection efficiency in HEK293 cells was highest 48h after transfection. Furthermore, mice were primed with 200µg pVAX1/C‑250 plasmid complexed with PEI, and boosted using 50µg of purified C‑G250 protein. In order to evaluate the immune response the antibody titer, splenocyte response, and interferon‑γ levels from CD8+T‑cell splenocytes were analyzed using ELISA, lymphocyte proliferation or enzyme‑linked immunosorbent spot assays. Firstly, the pVAX1/C‑G250 plasmid was shown to be constructed successfully. As compared with the DNA group, the antibody titer, lymphocyte proliferation percentage, and cytokine production level induced by the DNA‑PEI and DNA‑PEI+C‑G250 groups were significantly higher. Furthermore, the DNA‑PEI+C‑G250 group exhibited the strongest humoral and cellular response. Owing to the adjuvant effect of PEI, the pVAX1/C‑G250‑PEI prime plus C‑G250 protein boost regimen could induce a strong immune response, and has been proved to be a potent vaccine candidate against renal cell carcinoma.

Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors.Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.

Coagulation Factor VIII Is Synthesized In Endothelial Cells

Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder resulting from mutations in Lman1. This gene encodes a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and factor VIII (FVIII) to the plasma. F5F8D is characterized by levels of both FV and FVIII reduced to ∼5-30% of normal. In contrast, Lman1 knockout mouse models of F5F8D exhibit FV and FVIII activities that are ∼50% of normal, relative to wildtype mice. Though FV and FVIII are synthesized at markedly different levels and potentially in different tissues, loss of the LMAN1 cargo receptor leads to parallel reductions in both FV and FVIII activity. FV is synthesized in hepatocytes (as well as megakaryocytes in the mouse). However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. We took advantage of the dependence of efficient FV and FVIII secretion on LMAN1 to examine the cellular source of each protein. FV and FVIII secretion profiles of conditional Lman1 knockout mice were characterized, relative to that of wildtype mice and ubiquitous Lman1 null mice (Lman1-/-). In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2-Cre or Albumin-Cre transgene was crossed into Lman1 conditional mice (Lman1fl). FV and FVIII activity levels were measured by functional coagulation activity assays. Though Lman1fl/fl/Tie2-Cre+ mice (endothelial-specific knockout) exhibit normal plasma FV activity (99.9%) relative to wildtype mice (set to 100%), FVIII activity is reduced to 53.5% (p < 2.5 x 10-6). In contrast, Lman1fl/fl/Alb-Cre+ (hepatocyte-specific knockout) mice demonstrate normal FVIII activity (89.0%) and reduced FV activity (37.0%) (p < 1.4 x 10-10). To confirm endothelial cells as the biosynthetic source of FVIII, we took advantage of the previously reported RiboTag mouse (Sanz et al., 2009. PNAS 106(33):13939-44) to isolate endothelial cell RNA for qPCR analysis from various murine tissues. RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre-recombinase expressing animal. qPCR analyses of endothelial cell RNA isolated from total liver lysates of five RiboTag/Tie2-Cre+ mice demonstrated 10-20 fold enrichment for gene transcripts that are known to be endothelial-specific, such as Cdhs (12.1 fold enrichment, p < 8.0 x 10-3), Vcam1 (13.4 fold enrichment, p <1.1 x 10-5), and Vwf (15.3 fold enrichment, p < 7.0 x 10-4), as well as for FVIII transcripts (11.4 fold enrichment, p < 4.0 x 10-5). In contrast, this analysis demonstrated a statistically significant depletion (5-10 fold) of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse. These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool. Disclosures:No relevant conflicts of interest to declare.

Fli-1 drives transcription from the MCP-1 gene promoter, which may provide a novel mechanism for cytokine and chemokine activation. (P6236)

Abstract Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). We have demonstrated that Fli-1 binds to three EBSs within the endogenous MCP-1 promoter by ChIP assay. Transient transfection assays of the murine MCP-1 promoter indicate that the Fli-1 gene actively drives transcription from the MCP-1 gene promoter in a dose-dependent manner. The Ets-1 transcription factor was also tested, but failed to drive transcription. Studies to determine the relationship between Fli-1 and Ets-1 in the transcriptional control of MCP-1 will be presented. Deletion studies to determine the most important regions of the MCP-1 promoter for Fli-1 gene regulation are being investigated. Together, these results demonstrate a novel pathway, separate from NFκB, for the transcriptional activation of MCP-1. Evidence suggests that Fli-1 may interact with the promoters of other chemokines and cytokines, providing a novel mechanism for activation of the inflammatory response.

Effect of D-Alpha Tocopherol Therapy towards Malondialdehyde Level and Histology of Kidney in Rattus norvegicus with MLD-STZ Induction

Diabetic Nephropathy is a kidney disease which occurs due to complication of diabetes mellitus as a consequence of the damage of the kidney endothelial cells. Hyperglicemia condition in patients with diabetes mellitus that induces an oxidative stress, were related to endothelial cell damage. Oxidative stress as a result of hyperglycemia will activate a number of signal transduction pathways resulting in increase of free radicals. D-alpha tocopherol as one of antioxidant substance, that can act as an inhibitor of free radical chain reactions, play an important role in the reduction of the oxidative stress effect. Effect of D-alpha-tocopherol in reducing oxidative stress is identified by the levels of malondialdehyde (MDA) in kidney and histology of kidney. This study used five groups mice; they were a control group, a diabetic group which was induced with MLD-STZ, and a therapeutic groups with a varieties doses of D-alpha tocopherol (100 mg/kgBW, 200 mg/kgBW and 300 mg/kgBW). The results showed that the D-alpha tocopherol was able to reduce the levels of malondialdehyde (MDA) and repair the histology of kidney of mice induced by MLD-STZ.

Getting the “Inside” Scoop on EphrinB2 Signaling in Pericytes and the Effect on Peritubular Capillary Stability

Progressive CKD develops from a lack of fidelity in tissue remodeling after acute injury. A lack of successful repair following even subtle injuries, regardless of their cause, suggests that complete resolution of function may be difficult to achieve, even if initiating deficits are corrected. This

Calprotectin has a pathogenic pro-inflammatory role in anti-neutrophil cytoplasmic antibody associated vasculitis and glomerulonephritis

BackgroundCalprotectin, an endogenous toll-like receptor 4 (TLR 4) agonist that is expressed in neutrophils, monocytes, and infiltrating macrophages, promotes endothelial activation and transcription of pro-inflammatory cytokines. We investigated calprotectin in renal biopsy samples and serum of patients with anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) and in mice deficient in calprotectin (cal−/−), and we assessed the interaction of calprotectin with macrophages and endothelial cells in vitro. MethodsWe examined renal biopsy samples with immunohistochemistry. Serum calprotectin levels were measured with ELISA, and cell surface expression with flow cytometry. Accelerated nephrotoxic nephritis experiments were performed on both wild-type (WT) and cal−/− mice. Macrophages were isolated from WT, TLR4−/−, and cal−/− mice, and kidney endothelial cells from WT mice, and stimulated with calprotectin and supernatants harvested. Phagocytosis with opsonised beads was compared between WT and cal−/− macrophages. FindingsPatients with active AAV glomerular lesions demonstrated the most calprotectin positivity in renal biopsy samples, sclerotic lesions the least (p<0·05), linking calprotectin with disease activity. Serum levels in patients were significantly higher than in controls. In limited systemic disease, calprotectin levels assessed at 1 and 6 months after treatment predicted relapse (sensitivity 78·6%, specificity 92·3%). Patients had persistently higher monocyte and neutrophil cell surface calprotectin expression than did healthy controls suggesting a persistently activated state. Cal−/− mice were protected from renal disease with less macrophage and T-cell infiltration, less thrombosis, and preserved renal function. Calprotectin stimulation of WT macrophages and endothelial cells increased production of tumour necrosis factor α, interleukin 6, and interleukin 8 (p<0·05), an effect abrogated in TLR4−/− and cal−/− macrophages. Additionally, cal−/− macrophages had decreased phagocytosis ability compared with WT (p<0·005). Together these data demonstrate a positive amplification of inflammation mediated by calprotectin. InterpretationSerum calprotectin is a potential biomarker in AAV, and may predict relapse. Calprotectin contributes to pathogenesis by promoting leukocyte and endothelial cell activation in a positive feedback loop. FundingUK Medical Research Council.

Aberrant production of extracellular matrix proteins and dysfunction in kidney endothelial cells with a short duration of diabetes.

Diabetic nephropathy is the most common cause of end-stage renal disease and is a major risk factor for cardiovascular disease. In the United States, microvascular complications during diabetic nephropathy contribute to high morbidity and mortality rates. However, the cell-autonomous impact of diabetes on kidney endothelial cell function requires further investigation. Male Akita/+ [autosomal dominant mutation in the insulin II gene (Ins2)] mice reproducibly develop diabetes by 4 wk of age. Here, we examined the impact a short duration of diabetes had on kidney endothelial cell function. Kidney endothelial cells were prepared from nondiabetic and diabetic mice (4 wk of diabetes) to delineate the early changes in endothelial cell function. Kidney endothelial cells from Akita/+ mice following 4 wk of diabetes demonstrated aberrant expression of extracellular matrix proteins including decreased osteopontin and increased fibronectin expression which correlated with increased α5-integrin expression. These changes were associated with the attenuation of migration and capillary morphogenesis. Kidney endothelial cells from Akita/+ mice had decreased VEGF levels but increased levels of endothelial nitric oxide synthase(eNOS) and NO, suggesting uncoupling of VEGF-mediated NO production. Knocking down eNOS expression in Akita/+ kidney endothelial cells increased VEGF expression, endothelial cell migration, and capillary morphogenesis. Furthermore, attenuation of sprouting angiogenesis of aortas from Akita/+ mice with 8 wk of diabetes was restored in the presence of the antioxidant N-acetylcysteine. These studies demonstrate that aberrant endothelial cell function with a short duration of diabetes may set the stage for vascular dysfunction and rarefaction at later stages of diabetes.

Low Proliferative Potential and Impaired Angiogenesis of Cultured Rat Kidney Endothelial Cells

CKD is histologically characterized by interstitial fibrosis, which may be driven by peritubular capillary dropout and hypoxia. Surprisingly, peritubular capillaries have little repair capacity. We sought to establish long-term cultures of rat kidney endothelial cells to investigate their growth regulatory properties. AKEC or YKEC were isolated using CD31-based isolation techniques and sustained in long-term cultures. Although YKEC grew slightly better than AKEC, both performed poorly compared with endothelial cells of the rat adult PMVEC, PAEC, or HUVEC cells. PMVEC and PAEC contained a large percentage of cells with high colony-forming potential. In contrast, KECs were incapable of forming large colonies and most remained as single nondividing cells. KEC expressed high levels of mRNA for VEGF receptors, but were surprisingly insensitive to VEGF stimulation. KEC did not form branching structures on Matrigel when cultured alone, but in mixed cultures, KEC incorporated into branching structures with PMVEC. These data suggest that the intrinsic growth of rat kidney endothelial cells is limited by unknown mechanisms. The low growth rate may be related to the minimal intrinsic regenerative capacity of renal capillaries.

Mannose 6-Phosphate Receptor and Sortilin Mediated Endocytosis of α-Galactosidase A in Kidney Endothelial Cells

Prominent vasculopathy in Fabry disease patients is caused by excessive intracellular accumulation of globotriaosylceramide (GL-3) throughout the vascular endothelial cells causing progressive cerebrovascular, cardiac and renal impairments. The vascular lesions lead to myocardial ischemia, atherogenesis, stroke, aneurysm, thrombosis, and nephropathy. Hence, injury to the endothelial cells in the kidney is a key mechanism in human glomerular disease and endothelial cell repair is an important therapeutic target. We investigated the mechanism of uptake of α-galactosidase A (α-Gal A) in renal endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and 125I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α-Gal A binds to human M6PR with very high affinity, but M6PR also binds to sortilin in a way that prevents α-Gal A binding to sortilin. Taken together, our data provide evidence that sortilin is a new α-Gal A receptor expressed in renal endothelial cells and that this receptor together with the M6PR is able to internalize circulating α-Gal A during enzyme replacement therapy in patients with Fabry disease.

Endothelial Cells Bind Immune Complexes in the Kidney (172.39)

Abstract Inflammatory kidney diseases often involve the deposition of antibodies (Ab) or antibody-antigen immune complexes (IC). The glomerulus is a highly evolved vascular system which filters blood to produce urine. Ab-mediated glomerular injury can lead to bleeding into the urine, protein wasting, hypertension, decreased urine output, edema, and kidney failure. However, the mechanisms by which IC deposit and the means by which they are cleared remain unclear. In order to model endothelium-Ab interactions, kidney endothelial cells (KEnCs) were isolated from C57BL/6 mice and cultured in vitro. Immuno-phenotyping was performed to assess expression of CD31, VEGF receptors, von Willebrand factor (vWF), and alpha smooth muscle actin. CD31+ vWF+ VEGF-R2+ KEnCs displayed a phenotype in culture similar to other microvascular cells. Monomeric and heat aggregated Ig (HA-IgG) and peroxidase-antiperoxidase IC (PAP-IC) were used for binding and stimulation assays. Effects of stimulation of KEnCs were identified using PCR arrays and multiplex cytokine analysis. KEnCs stained brightly with HA-IgG and PAP-IC, but only dimly with monomeric IgG. KEnCs responded to IC with robust chemokine and cytokine responses in a dose dependent manner. Competitive binding studies mapped the interaction to the Fc-portion of IgG, but suggest that the KEnCs bind independently of classical Fc-receptors. Results support an important role for KEnCs in the pathogenesis of IC kidney diseases.

Blood cleansing cells in head kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi (Eigenmann), (Characidae: Teleostei)

The general structure and cell types in kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi, family Characidae, are described. The capability and capacity of these organs to clean foreign ferritin from the blood stream are analysed and compared. Head kidney was mainly composed of neutrophils, macrophages, lymphocytes and other white blood cells, whereas unmatured and matured red blood cells were few in number. Spleen often contained much red pulp, that is mainly matured red blood cells between splenic cords, often with some macrophages and neutrophils in the latter. Occasionally, this pulp contained large volumes of unmatured red blood cells, particularly in the periphery of the spleen. The splenic white pulp consisted of ellipsoids composed of an inner endothelial layer covered by a thick sheet of white blood cells, which in the periphery consisted mainly of macrophages. Erythrocytes occupied nearly the entire splenic volume in some specimens, whereas up to half of this volume was filled by ellipsoid macrophages, neutrophils, lymphocytes and other white blood cells in other specimens. The macrophages and sinusoidal endothelial cells in kidney and spleen from ferritin-injected specimens were tightly packed by yellow-brown granules or Prussian blue precipitations, in tissue treated with Mallory stain or acid ferrocyanide, respectively, suggesting a large uptake of foreign ferritin. In the present tetra large amounts of white blood cells are developed in head kidney, where macrophages and sinusoidal endothelial cells play important roles in the cleansing of scavenger and foreign molecules and particles from the blood stream. The spleen seems primarily to be a site for iron recycling and production and storage of red blood cells. Sometimes, however, it was rich in macrophages, neutrophils, lymphocytes and other white blood cells, suggesting functions like blood cleansing and non-specific and specific defence in such specimens.