1Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany. 2Department of Medicine, Division of Nephrology and Hypertension, Department of Medicine and the Immunology Research Group, Institute of Infection, Immunity and Inflammation, and 3Department of Medicine, Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada. 4Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, Missouri, USA.
Address correspondence to: Hans-Joachim Anders, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstr. 1, 80336 München, Germany. Phone: 49.89.218075855; Fax: 49.89.218075860; E-mail: hjanders@med.uni-muenchen.de.
Nephrocalcinosis, acute calcium oxalate (CaOx) nephropathy, and renal stone disease can lead to inflammation and subsequent renal failure, but the underlying pathological mechanisms remain elusive. Other crystallopathies, such as gout, atherosclerosis, and asbestosis, trigger inflammation and tissue remodeling by inducing IL-1β secretion, leading us to hypothesize that CaOx crystals may induce inflammation in a similar manner. In mice, intrarenal CaOx deposition induced tubular damage, cytokine expression, neutrophil recruitment, and renal failure. We found that CaOx crystals activated murine renal DCs to secrete IL-1β through a pathway that included NLRP3, ASC, and caspase-1. Despite a similar amount of crystal deposits, intrarenal inflammation, tubular damage, and renal dysfunction were abrogated in mice deficient in MyD88; NLRP3, ASC, and caspase-1; IL-1R; or IL-18. Nephropathy was attenuated by DC depletion, ATP depletion, or therapeutic IL-1 antagonism. These data demonstrated that CaOx crystals trigger IL-1β–dependent innate immunity via the NLRP3/ASC/caspase-1 axis in intrarenal mononuclear phagocytes and directly damage tubular cells, leading to the release of the NLRP3 agonist ATP. Furthermore, these results suggest that IL-1β blockade may prevent renal damage in nephrocalcinosis.
Kidney stone disease affects about 12% of men and 5% of women during their lifetimes in the United States (1). Unlike symptomatic urolithiasis, intrarenal nephrocalcinosis is often asymptomatic, but can lead to significant kidney injury and renal failure (1–3). Calcium oxalate (CaOx) stones account for the vast majority of calculi in primary and secondary forms of hyperoxaluria (4, 5). In addition, acute oxalosis (e.g., in ethylene glycol poisoning) can induce acute kidney injury as a result of widespread intrarenal CaOx deposition (6). Histopathology of human CaOx nephropathy as well as microarray analysis of rodent CaOx nephropathy document a substantial contribution of interstitial inflammation to tissue remodeling, but the molecular and cellular mechanisms of CaOx-induced renal inflammation remain largely unknown (7–11).
Inflammation and tissue remodeling in various crystallopathies, such as gout (caused by monosodium urate crystals), pseudogout (caused by calcium pyrophosphate crystals; ref. 12); atherosclerosis (caused by cholesterol crystals; ref. 13) silicosis (caused by silica crystals; ref. 14) and asbestosis (caused by asbestos; ref. 14), involve a unique intracellular signaling pathway for the secretion of the proinflammatory cytokine IL-1β: the NLRP3 inflammasome/apoptosis-associated speck-like protein containing CARD/caspase-1 (NLRP3/ASC/caspase-1) axis (15). The NLRP3 inflammasome is a large cytoplasmatic protein complex able to integrate multiple extracellular and intracellular danger signals into ASC binding and activation of caspase-1 (also known as pro–IL-1β converting enzyme; ref. 15). In contrast to other NF-κB–dependent cytokines that are readily secreted, the inactive pro-form of IL-1β requires cleavage (e.g., by caspase-1) as an additional enzymatic step before the bioactive form of IL-1β can be secreted from the cell (15).
Here, we hypothesized that CaOx crystals have a similar agonistic potential on NLRP3 and trigger IL-1β secretion and that this process contributes to CaOx-induced tissue inflammation, for example, in nephrocalcinosis and/or CaOx nephropathy.
CaOx crystals need NLRP3/ASC/caspase-1 to induce IL-1β in DCs. We hypothesized that CaOx crystals have the same potential to activate DCs as do crystals of uric acid, calcium pyrophosphate, or cholesterol (12, 13). CaOx crystals were of small size when used in vitro together with bone marrow–derived DCs (BMDCs; Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI63679DS1). CaOx crystal exposure activated murine BMDCs to release IL-1β in a dose-dependent manner, but only when cells were prestimulated with LPS, TNF, IL-1α, or histones (Figure 1A and Supplemental Figure 2). Western blotting of DC supernatants revealed that LPS prestimulation was a prerequisite for induction of pro–IL-1β expression (Figure 1A). LPS also induced pro–caspase-1 in BMDCs, and CaOx crystals induced caspase-1 activation similar to that by the NLRP3 agonist ATP (Figure 1A). To address a potential NLRP3 agonistic effect of CaOx crystals, BMDCs from Nlrp3–/–, Asc–/–, and Casp1–/– mice were also prepared; LPS/CaOx crystals were unable to induce IL-1β secretion in these cells (Figure 1, B and C). Although LPS induced pro–IL-1β and pro–caspase-1 expression, CaOX crystals were unable to induce caspase-1 activation in Nlrp3–/–, Asc–/–, and Casp1–/– BMDCs, as was the case with LPS/ATP stimulation (Figure 1, B and C). Additionally, the caspase inhibitor Z-VAD-FMK had the same effect on LPS/CaOx crystal–induced IL-1β secretion in BMDCs (Figure 1D). Thus, CaOx crystals induced IL-1β secretion in DCs via the NLRP3/ASC/caspase-1 axis.
CaOx crystals activate DCs to secrete mature IL-1β. (A) WT BMDCs were stimulated with increasing doses of CaOx crystals with or without LPS prestimulation (1 μg/ml), and IL-1β secretion was measured by ELISA. ATP served as a positive control of NLRP3 activation. Western blotting of supernatants showed induction of pro–IL-1β by LPS; only CaOx crystals and ATP induced mature IL-1β secretion. This process involved caspase-1 activation (cleavage product, 10 kDa). (B) DCs from WT, Nlrp3–/–, and Asc–/– mice. CaOx crystal– and ATP-induced IL-1β secretion depended on the presence of NLRP3 and ASC regardless of pro–IL-1β induction. (C) Similar results were obtained in DCs isolated from Casp1–/– mice. (D) WT BMDCs were stimulated with LPS/CaOx with or without the caspase inhibitor Z-VAD-FMK. Data are means ± SD from 3 separate experiments. *P < 0.05 vs. medium; ***P < 0.001 vs. respective WT (B and C) or PBS (D).
CaOx crystal–induced NLRP3 activation depends on phagocytosis and potassium efflux. Whether agonists directly interact with NLRP3 remains unknown, but various mechanisms are involved in NLRP3 activation by extracellular danger signals: particle uptake into the cell by phagocytosis, lysosomal cathepsin leakage, potassium efflux from the cell, P2X7 channel activation, and intracellular oxidative stress (15). We therefore performed a series of experiments to determine which of the aforementioned factors contributes to CaOx crystal–induced NLRP3 activation. Cytochalasin D, a phagocytosis-blocking agent that inhibited actin polymerization and crystal uptake into intracellular endosomes (Figure 2A), significantly reduced LPS/CaOx crystal–induced IL-1β secretion in BMDCs, whereas the cathepsin inhibitor CA074Me and the antioxidant N-acetyl cysteine had no effect (Figure 2B). Furthermore, blocking potassium efflux by a high concentration of potassium in the extracellular fluid had the same effect (Figure 2C), which suggests that potassium efflux is required for CaOx-induced NLRP3 activation. Comparison of LPS/CaOx crystal–induced IL-1β secretion between WT and P2x7–/– BMDCs did not support a contribution of P2X7 in this context (Figure 2D). Thus, CaOx crystals induced NLRP3-mediated IL-1β secretion in DCs via phagocytic uptake of CaOx crystals into intracellular endosomes as well as via inducing potassium efflux from the cell, a process known to activate NLRP3 (16).
Mechanisms of CaOx crystal–induced IL-1β secretion. (A) BMDCs were primed with LPS (1 μg/ml) and stimulated with CaOx crystals in the presence or absence of cytochalasin D. TEM showed that CaOx crystals appeared outside the cell (asterisks) and inside intracellular endosomes (arrowheads), whereas cytochalasin D treatment inhibited their uptake in DCs. (B) Incubation with cytochalasin D, CA074Me, and N-acetyl cysteine (NAC) affected IL-1β secretion, measured by ELISA in cell culture supernatants after 6 hours. (C) Similar experiments were performed using 75 mM KCl in cell culture medium to block potassium efflux from the DCs. NaCl of an identical molarity was used as a molarity control. High extracellular potassium prevented CaOx-induced IL-1β secretion. (D) DCs were isolated from WT and P2x7–/– mice. ATP-induced, but not CaOx-induced, IL-1β secretion depended on the presence of P2X7. Data are means ± SD from 3 independent experiments. (E) Renal ECs, MCs, TECs, and renal DCs were primed with LPS and exposed to CaOx crystals, and IL-1β secretion was measured. ATP was used as a positive control for NLRP3 activation. (F) Western blotting. LPS priming induced pro–IL-1β expression only in DCs. (G) Primary TECs were isolated from WT mice and cultured with increasing concentrations of CaOx. ATP release was quantified in cell culture supernatant upon 18 hours of CaOx crystal exposure with and without increasing concentrations of apyrase. (H) Coculture of LPS-primed DCs with CaOx (100 μg/ml) damaged TECs with or without apyrase (1 U/ml). IL-1β secretion was measured by ELISA after 24 hours. Data are means ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. medium (B, C, and E); vs. WT (D); vs. 1,000 μg/ml CaOx alone (G); or as indicated by brackets.
CaOx crystals induce IL-1β secretion in renal DCs, but not in renal parenchymal cells. To determine whether CaOx crystals have a similar potential to activate renal DCs or renal parenchymal cells, we stimulated primary renal CD45+CD11c+ DCs, ECs, mesangial cells (MCs), and tubular epithelial cells (TECs) with LPS/CaOx crystals as above. LPS/CaOx crystals induced IL-1β secretion in renal DCs in a manner similar to that of LPS/ATP, whereas none of the renal parenchymal cell types responded to the stimulation (Figure 2E). Western blotting confirmed this finding and also showed that LPS stimulation only induced pro–IL-1β expression in renal DCs (Figure 2F). Hence, CaOx crystals induced secretion of mature IL-1β only in renal DCs, not in renal parenchymal cells.
CaOx crystals induce ATP release from dying TECs. Consistent with previous reports (10), CaOx crystal exposure killed TECs (as demonstrated by lactate dehydrogenase [LDH] release) in a dose-dependent manner, independent of Nlrp3 genotype (Supplemental Figure 3). Dying cells release intracellular damage-associated molecular pattern molecules (DAMPs) that have the potential to activate pattern recognition receptors of other cells (17). ATP is one such intracellular DAMP, reported to drive sterile inflammation upon cell death via activation of NLRP3 (18, 19). Thus, we tested whether CaOx crystal–induced death of cultured TECs is associated with marked ATP release. In fact, CaOx crystal exposure induced ATP concentrations in cell culture supernatants in a dose-dependent manner (Figure 2G). In addition, extracellular ATP levels were readily degraded by adding apyrase at concentrations of 0.25 U/ml or higher (Figure 2G). Accordingly, coculture of BMDCs with CaOx-damaged primary tubular cells — with or without apyrase treatment — significantly reduced their capacity to secrete IL-1β (Figure 2H). Thus, CaOx crystals activated and destabilized TECs, a process that promoted the release of ATP, which activated DCs to release IL-1β.
Oxalate nephropathy involves diffuse crystal deposition, renal inflammation, and failure. Given the potential of CaOx crystals to activate renal DCs via NLRP3/ASC/caspase-1 in both direct and indirect ways, we suspected they make a functional contribution to oxalate nephropathy. Direct DC activation would require CaOx crystals to also reach the renal interstitial compartment, where DCs and other antigen-presenting cells span a dense network around the tubules (20). Murine oxalate nephropathy was characterized by diffuse CaOx crystal deposition throughout the kidney, with focal crystal casts obstructing proximal and distal tubules within the outer medulla as well as within the cortex, which was absent in control mice (Figure 3A and Supplemental Figure 4). Single crystals or small clumps of crystals, similar in appearance to the ones we applied for cell culture (Supplemental Figure 1), not only adhered to the luminal membranes of TECs, but were also found intracellularly within endosomes (Figure 3, B and C, and Supplemental Figure 5A). Diffuse CaOx crystal deposits were also found in the renal interstitial compartment, in close proximity to and within renal interstitial cells (Figure 3, B and D, and Supplemental Figure 5B). In fact, transmission EM (TEM) imaging showed that interstitial cells occasionally engulfed and took up crystals (Figure 3D). Renal CaOx crystal deposition was associated with diffuse neutrophil infiltrates and tubular necrosis mainly at the inner stripe of the outer medulla, as demonstrated by the disintegration of TECs and granular casts in tubular lumen (Figure 4, A and B). The structural alterations of oxalate nephropathy were associated with renal failure, as shown by a transient increase in plasma creatinine and BUN levels, peaking at 24 hours, compared with those in vehicle-injected mice (Figure 4A and Supplemental Figure 6). Intrarenal mRNA expression of the kidney injury markers KIM-1 and L-FABP, as well as that of the proinflammatory cytokines IL-6 and TNF-α, was increased (Figure 4C). Together, these observations indicated that murine oxalate nephropathy was characterized by diffuse CaOx crystal formation inside the tubular and the interstitial compartments, which is associated with tubular injury, renal inflammation, and failure.
CaOx nephropathy in C57BL/6 mice. (A) Kidney sections showed CaOx crystals distributed throughout, especially in the tubular lumina (Pizzolato stain; left) or viewed under polarized light (right). Original magnification, ×100 (left); ×200 (right); ×400 (inset). (B) At higher magnification, Pizzolato stain showed smaller (granular) crystals inside TECs as well as in the renal interstitial compartment. Original magnification, ×400 (left), ×1,000 (right). (C) TEM (left) confirmed large CaOx crystal aggregates within the tubular lumen (black arrow) as well as in the interstitial space (white arrow). Freeze-fracture EM (right) identified CaOx crystals in the tubular lumen (black arrow) as well as inside cytoplasmic organelles, some of which appeared to be lysosomes (white arrows). Original magnification, ×3,000. (D) TEM also showed CaOx crystals within the interstitial compartment in close proximity to interstitial cells (left; black arrows). At higher magnification (right), an interstitial cell (white asterisk indicates nucleus) engulfed a CaOx crystal (black arrows denote outer cell membrane); neighboring TECs are denoted by black asterisks. Original magnification, ×7,000 (left); ×25,000 (right).
Oxalate nephropathy in C57BL/6 mice. (A) Oxalate feeding caused diffuse oxalate deposition within 24 hours and significantly increased plasma creatinine levels and BUN levels. (B) PAS staining illustrated tubular necrosis, which was quantified by semiquantitative scoring (loss of brush border, flattening of cells, disruption and detachment of tubular cells from basement membranes). Renal neutrophil counts were determined by Ly6G B1.2 immunostaining and quantified per 15 hpfs. Original magnification, ×100. (C) Renal mRNA expression was determined by real-time RT-PCR. Data are means ± SEM from 5–6 mice per group. *P < 0.05, ***P < 0.001.
Oxalate nephropathy depends on MyD88, IL-1R, and IL-18. Cytokine expression and neutrophil infiltration are suggestive of the involvement of innate immunity in oxalate nephropathy. To test this concept, we induced oxalate nephropathy in Myd88–/– mice, since MyD88 is a central signaling adaptor that translates innate pattern recognition into NF-κB activation and expression of proinflammatory cytokines, including pro–IL-1β and pro–IL-18 (21, 22). Unlike WT mice, Myd88–/– mice were almost entirely protected from acute kidney injury and failure, despite harboring identical CaOx crystal depositions (Figure 5). Il1r–/– mice were almost entirely protected, whereas Il18–/– mice displayed only partial protection (Figure 5). Thus, oxalate nephropathy depended on MyD88-mediated innate immunity, which also involved IL-1R and IL-18.
Role of MyD88, IL-1R, and IL-18 in CaOx nephropathy. (A) Oxalate deposition was similar in Myd88–/–, Il1r–/–, and Il18–/– mice, but plasma creatinine levels and BUN were not significantly increased, suggesting a protective role of these molecules in CaOX-induced renal damage. (B) PAS staining illustrated tubular necrosis. Original magnification, ×100. (C) Tubular injury score was determined by semiquantitative scoring, and renal neutrophil counts were determined by immunostaining and quantified per hpf. Data are means ± SEM from 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.
Oxalate nephropathy is abrogated in Nlrp3–/–, Asc–/–, and Casp1–/– mice. To test the functional contribution of the NLRP3/ASC/caspase-1 axis, we induced oxalate nephropathy in Nlrp3–/–, Asc–/–, or Casp1–/– mice. Despite renal CaOx deposits identical to those of WT controls, all 3 groups were partially protected from kidney injury and failure, as evidenced by significant reductions in plasma creatinine, BUN, tubular damage, mRNA expression of tubular injury markers KIM-1 and L-FABP, and interstitial neutrophil infiltrates (Figure 6 and Supplemental Figure 7). Hence, NLRP3, ASC, and caspase-1 contributed to renal inflammation, damage, and dysfunction in CaOx nephropathy.
Role of NLRP3, ASC, and caspase-1 in CaOx nephropathy. (A) Nlrp3–/–, Asc–/–, and Casp1–/– mice showed CaOx deposits similar to those of WT mice, but decreased creatinine levels. (B) PAS staining illustrated reduced tubular necrosis compared with WT. Original magnification, ×100. (C) Tubular injury score and white cell counts were also reduced compared with WT. Data are means ± SEM from 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.
CaOx crystal–induced acute kidney injury depends on intrarenal mononuclear phagocytes. The results of our in vitro studies suggested that CaOx crystal–induced IL-1β secretion may selectively originate from the intrarenal network of mononuclear phagocytes, such as DCs and macrophages. Flow cytometry of renal cell suspension obtained after 24 hours of CaOx exposure revealed activation of CD45+CD11c+ intrarenal myeloid DCs, as evidenced by increased surface expression of CD40 and CD86 as well as by protrusions and dendrite expansion, visualized by scanning EM (Figure 7, A and B, and Supplemental Figure 8A). Next, we depleted monocytic phagocytes from the kidney by 2 injections of clodronate liposomes before inducing oxalate nephropathy. Depletion of CD11c+ cells was confirmed by flow cytometry of renal cell suspensions (Supplemental Figure 8B). This procedure reduced intrarenal IL-1β production, as determined by ELISA from kidney homogenates (Supplemental Figure 8C). This was associated with partial protection from acute kidney injury compared with control liposome–treated mice (despite identical amounts of CaOx deposits), as documented by a significant reduction of plasma creatinine level, BUN level, and tubular damage (Figure 7C). Similar results were obtained when cells were selectively depleted by diphtheria toxin in CD11c DTRg mice (Figure 7D and Supplemental Figure 8, B and C). Thus, intrarenal mononuclear phagocytes mediated cytokine signaling and subsequent renal inflammation, injury, and failure in oxalate nephropathy.
Role of DCs in CaOx nephropathy. CD45+CD11c+ cells were extracted from C57BL/6 by magnetic beads and sorted by flow cytometry using antibodies for the activation markers CD40 and MHC class II. (A) Percentage of cells with the specific markers. (B) CD45+CD11c+ cells were examined by scanning EM. DCs from CaOx mice showed marked cell surface disruption compared with the smooth bosselated surface of control cells. Scale bars: 2.5 μm. (C) CaOx nephropathy was induced in mice depleted of DCs (pretreated with clodronate liposomes) and compared with that in mice treated with control liposomes. (D) The same analyses were performed in CD11c DTRg mice in which CD11c+ cells were depleted by diphtheria toxin (DT) injection. Data are means ± SEM from 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.
Degradation of extracellular ATP with apyrase protects mice from oxalate nephropathy. Our in vitro studies showed that dying TECs released ATP, an endogenous NLRP3 agonist that triggers IL-1β secretion in DCs (19). Apyrase injection degrades extracellular ATP and can block the NLRP3-agonistic effect of this endogenous danger signal during sterile inflammation (18). We therefore used apyrase injections to test whether extracellular ATP also contributes to renal inflammation and injury during oxalate nephropathy. A single intraperitoneal injection of 20 U apyrase partially reduced tubular injury and neutrophil recruitment during oxalate nephropathy and improved renal excretory function, as documented by a significant reduction of plasma creatinine and BUN levels compared with vehicle-treated control mice (Figure 8A). Systemic IL-6 and MCP-1 levels were also reduced (Supplemental Figure 9A). We concluded that extracellular ATP (e.g., as it is released from dying TECs) served as an additional trigger for renal inflammation and dysfunction in oxalate nephropathy.
Apyrase and anakinra treatment in CaOx nephropathy in C57BL/6 mice. Oxalate nephropathy was induced in C57BL/6 mice treated with or without apyrase (A) and anakinra (B), and oxalate deposition was quantified using Pizzolato stains. Plasma creatinine levels were determined as described in Methods. Tubular injury score was determined by semiquantitative scoring of PAS sections, and renal neutrophil counts were determined by immunostaining and quantified per hpf. Original magnification, ×100. Data are means ± SEM from 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. ND, not done.
The recombinant IL-1R antagonist anakinra prevents oxalate nephropathy. All of the above observations suggested a functional role of NLRP3/ASC/caspase-1–mediated IL-1β secretion as a previously unrecognized pathomechanism for inflammation and tissue damage in CaOx nephropathy. Thus, therapeutic blockade of IL-1β may be able to interfere with this pathomechanism and protect against renal failure. Anakinra, a recombinant version of the endogenous IL-1 receptor antagonist, is approved by the FDA for the treatment of rheumatoid arthritis (23, 24). Intraperitoneal injection of anakinra dose-dependently reduced tubular injury and neutrophil recruitment and improved renal excretory function during oxalate nephropathy (Figure 8B and Supplemental Figure 9B). We concluded that IL-1β mediated inflammation and tissue damage in kidney injury induced by CaOx crystals, and thus IL-1 blockade protected from renal failure in oxalate nephropathy.
Renal stone disease is widely considered to be a mechanical problem causing intra- or extrarenal urinary outflow obstruction, but crystal nephropathies are also associated with significant intrarenal inflammation that can cause acute kidney injury (3, 11). We hypothesized that the NLRP3 inflammasome — an intracellular pattern recognition platform that translates danger signaling into secretion of mature IL-1β — is involved in this process, because immune recognition of numerous crystals involves the NLRP3/ASC/caspase-1 axis (12–14).
In contrast to widely expressed Toll-like receptors or Rig-like helicases, the NLRP3/ASC/caspase-1 axis is restricted to antigen-presenting cells inside the kidney (25–27). We used clodronate liposome in WT mice or diphtheria toxin in CD11c DTRg mice to demonstrate that CaOx-induced intrarenal IL-1β secretion originated from the intrarenal network of interstitial mononuclear phagocytes, which includes several DC populations (28). CaOx crystals also formed in the interstitial compartment, where they were internalized by interstitial cells, a process demonstrated by us and others using TEM (present study and ref. 11). Our in vitro studies confirmed that phagocytic uptake was required for CaOx-induced IL-1β secretion in DCs, an observation consistent with the mode of NLRP3 activation by monosodium urate and calcium pyrophosphate crystals (12), silica (14), asbestos (14), and cholesterol crystals (13) in other disease contexts. Potassium efflux from the cell, occurring upon cell membrane disruption or pore formation by bacterial toxins, is another trigger of NLRP3 activation in DCs (15, 29). CaOx crystal–induced NLRP3 activation also depended on this mechanism, which was again consistent with previous reports on crystals of asbestos, monosodium urate (14), silica (30), cholesterol (31), and calcium phosphate (32). Intrarenal DCs and macrophages are important regulators of innate immunity and tissue remodeling in the kidney (28, 33). They express all known pattern recognition platforms that translate infectious and noninfectious dangers into local expression of proinflammatory cytokines and chemokines, causing subsequent immune-mediated pathology in the kidney (28, 33, 34). In addition, renal DCs express numerous negative regulators of innate immunity that limit overshooting renal inflammation and inappropriate kidney injury, e.g., in postischemic tubular necrosis (35–37). The present study is the first to our knowledge to demonstrate that acute kidney injury in crystal nephropathy largely depends on the capacity of intrarenal mononuclear phagocytes to trigger renal inflammation upon crystal recognition.
TEM and freeze-fracture EM demonstrated crystal uptake into intracellular compartments of TECs in vivo. TECs express NLRP3 but lack pro–IL-1β induction upon NF-κB priming (26, 38, 39). Therefore, CaOx crystals did not trigger mature IL-1β secretion in TECs. However, TECs internalize CaOx crystals, inducing oxidative stress and tubular cell necrosis (40–42). We confirmed this finding and showed that necrotic tubular cells released ATP, a potent NLRP3 agonist (19). Iyer et al. recently demonstrated that ATP is released upon acute tubular necrosis, which triggers NLRP3 activation and renal inflammation, a process that aggravates postischemic acute kidney injury (43). These results were supported by our present in vitro studies. In addition, the protective effects of apyrase treatment on CaOx nephropathy supported the functional contribution of ATP to renal inflammation and damage. It is noteworthy that apyrase injections also reduced plasma levels of IL-6 and MCP-1, implicating some systemic effects, which could be either causative or secondary for the improvement of the renal phenotype.
IL-1 blockade with appropriate antagonists is an established treatment of IL-1–dependent diseases, such as rheumatoid arthritis, Still's disease, and rare hereditary autoinflammatory disorders (24). Recently, IL-1 blockade also proved effective in crystal-mediated diseases, such as gout and pseudogout (44, 45). The dramatic therapeutic effects seem to directly relate to the fact that NLRP3 translates crystal recognition into IL-1β secretion as a central element of crystal-induced tissue inflammation and pathology (24). Our data demonstrated a similar therapeutic effect of the recombinant IL-1 receptor antagonist anakinra in CaOx nephropathy in mice, which suggests that IL-1 blockade may also reduce renal inflammation and kidney damage in other crystal nephropathies.
In conclusion, CaOx crystals triggered IL-1β–dependent innate immunity via the NLRP3/ASC/caspase-1 axis in DCs. Thus, intrarenal antigen-presenting cells promoted IL-1–dependent inflammation and kidney injury in renal stone disease. In addition, CaOx crystals indirectly activated NLRP3 by damaging TECs and releasing the NLRP3 agonist ATP. IL-1β blockade with anakinra prevented CaOx kidney injury disease, which could be a novel therapeutic approach to limit kidney damage in crystal nephropathies.
C57BL/6, NOD/ShiLt, NOD/Casp1–/–, Il1r1–/–, Il18–/–, and Myd88–/– mice were procured from Jackson Laboratories. Nlrp3–/– and Asc–/– mice were provided by J. Tschopp (University of Lausanne, Lausanne, Switzerland) and by V. Dixit (Genentech, San Francisco, California, USA). CD11c DTRg mice were provided by C. Kurts (University of Bonn, Bonn, Germany) and D. Engel (University of Bonn, Bonn, Germany). Mice were housed in groups of 5 in filter top cages with unlimited access to food and water. Cages, food, and water were sterilized by autoclaving before use. 6- to 8-week-old mice received a single intraperitoneal injection of 100 mg/kg sodium oxalate and 3% sodium oxalate in drinking water, and kidneys were harvested after 24 hours. DC depletion was achieved by 2 intravenous injections of liposomal clodronate (200 μl) at 48 and 24 hours before injection of sodium oxalate. Control liposomes were used as vehicle control. In another experiment, CD11c DTRg mice were given a single intravenous injection of 8 ng diphtheria toxin (Sigma-Aldrich) 6 hours before sacrifice. Urine samples were collected after 24 hours, before sacrifice by cervical dislocation and harvest of plasma and kidney tissue (46). Kidneys were kept at –80°C for protein isolation and in RNA later solution at –20°C for RNA isolation. One part of the kidney was also kept in formalin to be embedded in paraffin for histological analysis.
Kidney sections (2 μm) were stained with periodic acid–Schiff (PAS) reagent. Tubular injury was scored by assessing the percentage of necrotic tubules and presence of tubular casts. Ly6B.2+ neutrophils were identified by immunostaining (clone 7/4; Serotec). Pizzolato staining visualized CaOx crystals, and crystal deposit formation in the kidney was evaluated as described previously (47), using the following point values: 0, no deposits; 1, crystals in papillary tip; 2, crystals in cortical medullary junction; 3, crystals in cortex. When crystals were observed in multiple areas, points were combined. Plasma creatinine and BUN were measured using Urea FS kit (DiaSys Diagnostic Systems) according to the manufacturer's protocol. Both parameters were corrected for body weight. Neutrophil infiltrates were counted in 15 high-power fields (hpfs) per section.
Scanning EM. Kidney tissues were rinsed with PBS and postfixed with 1% aqueous osmium tetroxide for a total of 2.5 hours. Subsequently, tissues were rinsed and dehydrated through a graded series of ethanol to absolute ethanol and critical-point dried using liquid CO2. After mounting on stubs, the specimens were sputter-coated with a gold-palladium alloy for 1.5 minutes. DCs in suspension were rinsed in PBS and centrifuged to concentrate the cells. Then cells were dropped on coated coverslips and allowed to adhere; coverslips were fixed as above. Coverslips were cut into pieces prior to mounting on stubs. Images were viewed with a Hitachi 2600 electron microscope.
TEM. Kidneys were immersed in cold modified Karnovsky fixative containing 3% glutaraldehyde and 1% paraformaldehyde in sodium cacodylate buffer (pH 7.4) processed as previously described (48). In another set of experiments, BMDCs were stimulated with 1 μg/ml LPS and 100 μg/ml CaOx, with or without cytochalasin D pretreatment. After stimulation, cells were fixed with 2% paraformaldehyde with 2% glutaraldehyde in PBS buffer (pH 7.4) and processed. The samples were viewed with a JEOL model 1200EX electron microscope.
Quick-freeze deep-etch EM. 1.5-mm bread slices of nonfixed kidney or kidney sections fixed overnight in 2% glutaraldehyde in NaHCa (100 mM NaCl, 30 mM Hepes, and 2 mM CaCl2, pH 7.2) were used after rinsing in NaHCa. Tissues were then cooled to 4°K with liquid helium; mounted in a Balzers 400 vacuum evaporator, fractured, and etched for 2.5 minutes at –104°C; rotary replicated with approximately 3 nm platinum, as previously described (49); and viewed using a JEOL 1400 microscope with attached AMT digital camera.
Total RNA was isolated from kidneys using a Qiagen RNA extraction kit following the manufacturer's instructions. After quantification, RNA quality was assessed using agarose gels. From isolated RNA, cDNA was prepared using reverse transcriptase (Superscript II; Invitrogen). Real-time quantitative RT-PCR (TaqMan) was performed using SYBRGreen PCR master mix and analyzed with a Light Cycler 480 (Roche). All gene expression values were normalized using 18s RNA as a housekeeping gene. All primers used for amplification were from Metabion. The expression of KIM-1, IL-6, and TNF-α was analyzed using the following primers: Kim-1 forward, TCAGCTCGGGAATGCACA; Kim-1 reverse, TGGTTGCCTTCCGTGTCT; L-FABP forward, AGGCAATAGGTCTGCCCGAGGAC; L-FABP reverse, CCAGTTCGCACTCCTCCCCCA; IL-6 forward, TGATGCACTTGCAGAAAACA; IL-6 reverse, ACCAGAGGAAATTTTCAATAGGC; TNF-α forward, CCACCACGCTCTTCTGTCTAC; TNF-α reverse, AGGGTCTGGGCCATAGAACT.
Anti-mouse CD11c, CD40, CD86, and CD45 antibodies (BD Biosciences — Pharmingen) were used to detect activated DC and leukocyte populations in kidneys. F4/80, 7/4, and Ly6G antibodies (Abd Serotec) and CD3, NK1.1, CD11b, and CD103 antibodies (BD Biosciences — Pharmingen) were used to detect macrophage, neutrophil, T cell, natural killer cell, and mononuclear phagocyte populations in kidneys. Respective isotype antibodies were used to demonstrate specific staining of cell populations. Quantification of cell number was achieved using counting beads for FACS (Invitrogen).
BMDCs were established as described previously (50). Renal DCs were isolated from whole kidneys by magnetic bead separation (MACS) using CD11c MicroBeads (Miltenyi Biotech). The purity of CD11c+ renal cells was confirmed by FACS analysis using anti-CD11c antibody (BD Biosciences — Pharmingen). Cells were stimulated for 3 hours with 1 μg/ml LPS followed by 100 μg/ml CaOx crystals or stimulated with 5 mM ATP for 6 hours (all Sigma-Aldrich). Cell-free supernatants were analyzed for cytokine secretion by ELISA (BD Biosciences — Pharmingen) and immunoblot analysis. Primary TECs were isolated from the kidneys and grown on type IV collagen–coated tissue culture plates (Sigma-Aldrich). Primary renal ECs and MCs were isolated as described previously (26). The isolated primary cells were confirmed under light microscope and further characterized by FACS, immunostaining, Western blot, and RT-PCR. Cells were grown to confluency before use. 1 × 105 cells per well were used. Primary TECs were maintained in DMEM/F12 (Sigma-Aldrich) containing 10% FCS (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), 125 ng/ml prostaglandin E1 (Calbiochem), 25 ng/ml EGF (Sigma-Aldrich), 1.8 μg/ml l-thyroxine (Sigma-Aldrich), 3.38 ng/ml hydrocortisone (Sigma-Aldrich), and 2.5 mg/ml insulin–transferrin–sodium selenite supplement (I-T-SS; Sigma-Aldrich). Primary endothelia and MCs were maintained in DMEM (Gibco, Invitrogen) plus 10% FCS and 1% penicillin-streptomycin, and primary renal DCs were maintained in RPMI (Gibco, Invitrogen) and used immediately. All cells were cultured in an incubator at 37°C, 5% CO2. Supernatants were analyzed for LDH release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega) to evaluate cell death, measuring LDH after 18 hours. Results were expressed as percent of positive control. For ATP release, the system bioluminescence detection kit for ATP measurement was used (Promega), following the manufacturer's instructions.
Briefly, TECs and BMDCs were generated as described above. TECs were treated with or without 100 μg/ml CaOx for 18 hours, after which the supernatants were treated with 1 U/ml apyrase (Sigma-Aldrich). Next, LPS-primed (1 μg/ml) or unprimed BMDCs were cocultured with the TECs at a 4:1 ratio in RPMI 1640 (Gibco, Invitrogen) supplemented with 10% FBS. After 24 hours of coculture, the supernatants were collected, centrifuged, and analyzed for IL-1β concentration by ELISA (BD Biosciences — Pharmingen).
Upon completion of the 6-hour stimulation experiment, supernatants and cells were collected. Supernatants were concentrated using Amicon Ultra centrifugal filters (Millipore Billerica) and resolved on 15% SDS polyacrylamide gels. After electrophoresis, proteins were transferred to PVDF membrane. Primary antibodies were goat polyclonal anti–IL-1β (BAF401; R&D Systems) and rabbit polyclonal anti–caspase-1 (sc-514; Santa Cruz); this was followed by incubation with secondary antibody anti-biotin or anti-rabbit IgG labeled with HRP. Immunostained bands were detected using a chemiluminescence kit (ECL kit).
Data are presented as mean ± SEM. Comparison of groups was performed using ANOVA; post-hoc Bonferroni correction was used for multiple comparisons. A P value less than 0.05 was considered significant.
All experimental procedures were approved by the local government authorities.
The study was funded by the Deutsche Forschungsgemeinschaft (GRK 1202 and AN372/9-2). The expert technical support of Heni Eka Susanti, Dan Draganovici, Nuru Eltrich, and Jana Mandelbaum is acknowledged, as is the expert technical support of Jaclyn Lett (WUSTL Research Core Lab for Auditory and Vestibular Studies, funded by NIH/NIDCD grant no. P30DC04665 SEM), Karen Green (Pathology and Immunology Electron Microscopy Research Core lab, Washington University School of Medicine), and Robyn Roth (Heuser Lab, Department of Cell Biology, Washington University School of Medicine). We thank Christian Kurts and Daniel Engel for providing CD11c DTRg mice.
Reference information: J Clin Invest. doi:10.1172/JCI63679.
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text:
FEC: Campaigns Can Use Discounted Cybersecurity Services
The U.S. Federal Election Commission (FEC) said today political campaigns can accept discounted cybersecurity services from companies without running afoul of existing campaign finance laws, provided those companies already do the same for other non-political entities. The decision comes amid much jostling on Capitol Hill over election security at the state level, and fresh warnings from U.S. intelligence agencies about impending cyber attacks targeting candidates in the lead up to the 2020 election.
Current campaign finance law prohibits corporate contributions to campaigns, and election experts have worried this could give some candidates pause about whether they can legally accept low- to no-cost services from cybersecurity companies.
But at an FEC meeting today, the commission issued an advisory opinion (PDF) that such assistance does not constitute an in-kind contribution, as long as the cybersecurity firm already offers discounted solutions to similarly situated non-political organizations, such as small nonprofits.
The FEC's ruling comes in response to a petition by California-based Area 1 Security, whose core offering focuses on helping clients detect and block phishing attacks. The company said it asked the FEC's opinion on the matter after several campaigns that had reached out about teaming up expressed hesitation given the commission's existing rules.
In June, Area 1 petitioned the FEC for clarification on the matter, saying it currently offers free and low-cost services to certain clients which are capped at $1,337. The FEC responded with a draft opinion indicating such offering likely would amount to an in-kind contribution that might curry favor among politicians, and urged the company to resubmit its request focusing on the capped-price offering.
Area 1 did so, and at today's hearing the FEC said "because Area 1 is proposing to charge qualified federal candidates and political committees the same as it charges its qualified non-political clients, the Commission concludes that its proposal is consistent with Area 1's ordinary business practices and therefore would not result in Area 1 making prohibited in-kind contributions to such federal candidates and political committees."
POLICY BY PIECEMEAL
The decision is the latest in a string of somewhat narrowly tailored advisories from the FEC related to cybersecurity offerings aimed at federal candidates and political committees. Most recently, the commission ruled that the nonprofit organization Defending Digital Campaigns could provide free cybersecurity services to candidates, but according to The New York Times that decision only applied to nonpartisan, nonprofit groups that offer the same services to all campaigns.
Last year, the FEC granted a similar exemption to Microsoft Corp., ruling that the software giant could offer "enhanced online account security services to its election-sensitive customers at no additional cost" because Microsoft would be shoring up defenses for its existing customers and not seeking to win favor among political candidates.
Dan Petalas is a former general counsel at the FEC who represents Area 1 as an attorney at the law firm Garvey Schubert Barer. Petalas praised today's ruling, but said action by Congress is probably necessary to clarify the matter once and for all.
"Congress could take the uncertainty away by amending the law to say security services provided to campaigns to do not constitute an in-kind contribution," Petalas said. "These candidates are super vulnerable and not well prepared to address cybersecurity threats, and I think that would be a smart thing for Congress to do given the situation we're in now."
'A RECIPE FOR DISASTER'
The FEC's decision comes as federal authorities are issuing increasingly dire warnings that the Russian phishing attacks, voter database probing, and disinformation campaigns that marked the election cycles in 2016 and 2018 were merely a dry run for what campaigns could expect to face in 2020.
In April, FBI Director Christopher Wray warned that Russian election meddling posed an ongoing "significant counterintelligence threat," and that the shenanigans from 2016 — including the hacking of the Democratic National Committee and the phishing of Hillary Clinton's campaign chairman and the subsequent mass leak of internal emails — were just "a dress rehearsal for the big show in 2020."
Adav Noti, a former FEC general counsel who is now senior director of the nonprofit, nonpartisan Campaign Legal Center, said the commission is "incredibly unsuited to the danger that the system is facing," and that Congress should be taking a more active roll.
"The FEC is an agency that can't even do the most basic things properly and timely, and to ask them to solve this problem quickly before the next election in an area where they don't really have any expertise is a recipe for disaster," Noti said. "Which is why we see these weird advisory opinions from them with no real legal basis or rationale. They're sort of making it up as they go along."
In May, Sen. Ron Wyden (D-Ore.) introduced the Federal Campaign Cybersecurity Assistance Act, which would allow national party committees to provide cybersecurity assistance to state parties, individuals running for office and their campaigns.
Sen. Wyden also has joined at least a dozen other senators — including many who are currently running as Democratic candidates in the 2020 presidential race — in introducing the "Protecting American Votes and Elections (PAVE) Act," which would mandate the use of paper ballots in U.S. elections and ban all internet, Wi-Fi and mobile connections to voting machines in order to limit the potential for cyber interference.
As Politico reports, Wyden's bill also would give the Department of Homeland Security the power to set minimum cybersecurity standards for U.S. voting machines, authorize a one-time $500 million grant program for states to buy ballot-scanning machines to count paper ballots, and require states to conduct risk-limiting audits of all federal elections in order to detect any cyber hacks.
BIPARTISAN BLUES
Earlier this week, FBI Director Wray and Director of National Intelligence Dan Coats briefed lawmakers in the House and Senate on threats to the 2020 election in classified hearings. But so far, action on any legislative measures to change the status quo has been limited.
Democrats blame Senate Majority Leader Mitch McConnell for blocking any action on the bipartisan bills to address election security. Prior to meeting with intelligence officials, McConnell took to the Senate floor Wednesday to allege Democrats had "already made up their minds before we hear from the experts today that a brand-new, sweeping Washington, D.C. intervention is just what the doctor ordered."
"Make no mistake," McConnell said. "Many of the proposals labeled by Democrats to be 'election security' measures are indeed election reform measures that are part of the left's wish list I've called the Democrat Politician Protection Act."
But as Politico reporter Eric Geller tweeted yesterday, if lawmakers are opposed to requiring states to follow the almost universally agreed-upon best practices for election security, they should just say so.
"Experts have been urging Congress to adopt tougher standards for years," Geller said. "Suggesting that the jury is still out on what those best practices are is factually inaccurate."
Noti said he had hoped election security would emerge as a rare bipartisan issue in this Congress. After all, no candidate wants to have their campaign hacked or elections tampered with by foreign powers — which could well call into question the results of a race for both sides.
These days he's not so sanguine.
"This is a matter of national security, which is one of the core functions of the federal government," Noti said. "Members of Congress are aware of this issue and there is a desire to do something about it. But right now the prospect of Congress doing something — even if most lawmakers would agree with it — is small."
Tags: Adav Noti, Campaign Legal Center, Dan Petalas, Defending Digital Campaigns, Department of Homeland Security, Eric Geller, FBI Director Christopher Wray, Federal Election Commission, Garvey Schubert Barer, Microsoft Corp., Politico, Sen. Ron Wyden, Senate Majority Leader Mitch McConnell, The New York Times
This entry was posted on Thursday, July 11th, 2019 at 4:41 pm	and is filed under A Little Sunshine. You can follow any comments to this entry through the RSS 2.0 feed. Both comments and pings are currently closed.
First to comment! I know, so what!
""The FEC is an agency that can't even do the most basic things properly and timely, and to ask them to solve this problem quickly before the next election in an area where they don't really have any expertise is a recipe for disaster," Noti said
This also sounds like Congress!
JCitizen
For SURE!!! =)
Made all the more evident by McConnell's suggesting that following well-known security best practices is some kind of political maneuver on the part of Democrats.
If anyone was still wondering which group has our best interests in mind…
Well, if nothing else, Mitch is consistent:
https://www.npr.org/sections/thetwo-way/2018/01/24/580171396/biden-mcconnell-refused-to-sign-bipartisan-statement-on-russian-interference
…consistently awful, but consistent.
Great article and great gumshoe work Brian, so don't let my following statement damper your news.
Americans are too smart to be swayed by Russian meddling, in fact they hardly pay attention to the billions of dollars of advertising and campaign finance all the parties spend on elections. How many times have we seen candidates win with hardly a penny spent compared to who they are running against?
Congress is wasting our time looking into all this, with one exception. and that would be cracking into election machines. That is the only subject I'm concerned with.
"Americans are too smart…"
The electoral college win for Trump suggests otherwise.
The founding fathers can be thanked for that – it was designed to protect the electorate from situations like the Weimar Republic disaster, where too many parties were running at the same time and no majority was able to reach enough of a quorum to effectively govern the state, setting up Germany's fragile democracy, for conditions that favored a coup d'état.
It is simple to look at the pie chart of how many people that voted in different parties for different candidates, and see that there actually isn't a verifiable majority by one single candidate. You have to count all the contest votes to get a picture of how freedom works in a Democratic Republic. One candidate may have a majority of votes when compared to other single candidates, but they don't necessarily have a majority when all opposing votes are compared to the winning candidate. The college helps make the votes of people who didn't win count. That is the beauty of it.
It also guarantees the majority party can't beat up on the other minority parties. A pure democracy is really just a larger club of people who simply have all the power, and the minority has none. That is how it was done in Greece and Rome, and we all saw how that turned out.
weinerdog43
Oh please.
Very solid rebuttal. I'd give it a 10/10, two thumbs up, and a supersized diet Pepsi. The internet will sing your praises for years to come. That comment alone has single-handedly changed the way we view Democracy and the election process. Have you ever considered running for President? I can see it now:
Moderator: "This question is to weinerdog43: How do you end world hunger?"
weinerdog43: "Oh please".
**Major announcement: weinerdog43 wins the election by a landslide victory the likes that we've NEVER seen before**
I, myself, can't believe how much prep time you spent in specially crafting that solid response! You, my friend, are a master debater!
I dunno how obligated I'd feel to make an honest effort to debate someone who reduced the fall of a five-century old empire to "they didn't have an electoral college" I'd be myself.
Obviously it takes more than one factor to run a democracy. A well crafted Constitution, and a public willing to recognize its authority helps; a balance of power between the executive, courts, and congress another, etc, etc.
Mahhn
I'd rather see elimination of the party/gang system and make people run on their own merits, than on the coat tails and riches of others that have their own agenda and expect (and get) rewards for financing the puppets.
Sorry, but the reasoning for the Electoral College is wrong, and wouldn't make sense if it were the reason.
Firstly, an electoral college doesn't prevent multiple parties from participating. That is due to the system of "first past the post". That is a separate thing from the Electoral College. The FPTP system is responsible for the "spoiler effect" that suppresses 3rd parties from participation. It makes it inevitable for a republic to coalesce into a 2 party (polar) system, which is what we have.
Secondly, you are confusing the Electoral College, which is ONLY FOR PRESIDENTIAL ELECTIONS… with LEGISLATIVE REPRESENTATION 0f Congress. The Electoral College has NOTHING do do with representatives in Congress…. and thus NOTHING to do with the irrational fear of "too many parties" being ineffective or not reaching quorum.
Also, having multiple parties in Congress that don't have a majority may indeed decrease the efficiency of the government to make quick decisions…. but we have that anyway. The filibuster and requirements for super majorities for many actions exist today, and create the same problem that you may want to prevent.
Government isn't effective as is… even with a single party being in power. Moreover, with 2 parties swapping nearly every cycle, even laws that manage to get passed, get undone when the pendulum swings back.
"The college helps make the votes of people who didn't win count"
What? NO! The "Winner take all" component of the Electoral College does the exact OPPOSITE. It ensures that the people who voted for the losing candidate in that state, get ZERO electoral votes. Even if 49.9999% of the counted votes were for the loser… they get ZERO… It is how someone can win the white house without winning the popular vote.
"It also guarantees the majority party can't beat up on the other minority parties"
No… that is exactly what is happening, and has happened throughout the history of the US and our Electoral College.
Greece and Rome didn't have pure democracies either. Nobody has even really tried it. Greece and Rome didn't have a democracy like us… but they were fraught with corruption because only the elite could be in government. No women, peasants, etc.
We could eliminate the First Past the Post component of elections, and adopt Approval Voting. This will finally break the D vs. R cycle of nonsense and allow 3rd parties to run without spoiling. We have nearly 330 million people in the US.. it is stupid to think that we can be represented with only 2 party platforms. But again, this has nothing to do with the Electoral College
We could eliminate the Winner Take All component of the Electoral College too. 2 states have already done this. This would actually count votes for candidates who lose the state. This means conservatives in California and liberals in Texas finally get thier votes counted,.. instead of thrown away by the State.
Only when large red and large blue states do this at the same time, could it be fair. Until then, only if enough states override with the Interstate Compact agreement of the National Popular Vote will be able to balance out the ridiculousness of the Electoral College. If enough states sign, it will be yet another kludge fix to our democracy. Essentially a few big states will be agreeing to throw away their winner's votes to offset the many other smaller states that have thrown away their loser's votes.
Come on now Canuck, Justin Trudeau is your idea of a good leader?
This supposes that Russian meddling is as simplistic as the Russians (or any foreign power, really), interfering in order to get us to take some specific position- which you suppose we are too smart to fall for.
It doesn't at all account for the idea that the meddling is as often to sow confusion, enhance division, and cause general chaos as it is to elicit support for some specific agenda.
Also I'm honestly not sure that "intelligence" alone is the relevant measure for deciding the success or failure of these efforts. You might be confusing the American Electorate for one that has a long and storied history of decisions made on purely cerebral, philosophical grounds, and never an aggregate that vacillates wildly between emotional extremes… often coming to decisions based on our fears and "feels".
Caffeineguru
I'm sure Americans are too smart to be influenced to buy a Coke after seeing a Coke commercial, yet Coca-Cola spent $4B on advertising in 2017 (investopedia).
The American advertising industry is worth hundreds of billions of dollars, which is clear evidence that Americans are too smart to be swayed by mere advertising… /s
Best comment Ive seen rebutting the above. Google wouldnt exist if marketing didnt work.
Not a very good example, as Coke has lost market share for years and years.
Robert Adjemian
I'd like to believe that people aren't swayed by Russian fake news, certainly I'm not, but see the PBS documentary on Facebook: https://www.youtube.com/watch?v=T48KFiHwexM&t=6s
Maybe people simply want to believe what they read. However it works, riots have started and people killed – all due to fake postings on Facebook. Check out the program if you haven't seen it already.
There is no doubt that the Arab spring was derailed by hatred speech, and fake news. However, when you have a society founded on hatred, it isn't surprising to see the best laid plans of mice and men go bad. I'd like to think the American public condemnation of hate speech and similar human proclivities are one of our best advantages. I've seen Facebook groups and individual members correcting each other when fake news is attempted to pass as the truth. As I see it the members themselves are the best regulators of Facebook(or other social media) information. Maybe someday soon they will have an AI to add more balance, and police fake accounts.
What fantasy land are you living in? The US has no hate speech laws, is home to the Proud Boys and the KKK, among others, and hate speech is cheered on as much as it is condemned.
That comes across as islamophobic, and definitely revisionist.
What would they say about our society. Literally built on slavery.
"Current campaign finance law prohibits corporate contributions to campaigns." LOL. When begin with such a ludicrous statement, the entire article reads as ,"blah, blah, blah." American democracy is doomed. Find a soft landing place. Canada is nice.
jdgalt
How far does this in-kind contribution rule go? I'd love it if deliberately biased sites such as the legacy media plus Facebook and Twitter were made to conform to limits on their huge in-kind contributions to Democrats.
How far does this in-kind contribution rule go? I'd love it if deliberately biased media such as Fox News (Hannity) and Rush Limbaugh plus sites like Breitbart and Infowars were made to conform to limits on their huge in-kind contributions to Republicans.
But there is this pesky little "First Amendment" issue that prevents contribution rules limiting blatant criticism and promotion by talking heads and pundits even when they are lying.
I've seen plenty of deliberately biased news on all the cable news networks – I see no difference at all. The only one I see that stands out is the BBC – which is amazing considering it is run by the UK government. I try to check out the BBC to get a true balance when I can.
repubes treason dreams
repubes want russian hacking because that's the only way they can win the presidency again. they already won the presidency and put trump in office with the help of russian hacking. and repubes want it again.
LMAO!! You are too funny!
red ospray
Seems to me that neither side has the peoples best interest at heart. Only power. Right now I have an issue with the inadvertent/unintended Demos helping the Russians achieve their goals of distracting the US. So who is profiting?
InfoSec Pro
"Under Republicans Man Exploits Man.
Under Democrats It's Just the Opposite."
How are Democrats distracting the US away from Russian involvement? They've opened every investigation they could.
Will these campaign-focused security firms actually need to prove they are in the business of providing security?
Do the requisite 'non-political' entities need to prove they are not in turn being operated by the same campaign office the 'security firm' is being so helpful with?
Marco Belmonte
Anyone else think it is hilarious that Area 1 reserves helping organizations which don't exceed $1,337 in profits? That was "leet!" 😉
Yes, I found that hilarious.
That's not how I read it.
"Area 1 .. offers .. services to certain clients .. capped at $1,337."
That figure is surely what Area 1 charges the client, and has nothing to do with the client's profitability.
If in doubt ask the company for clarification.
Actually, you are right. It's still funny 🙂
some guy
While I didn't take it to mean what you suggested, I'm certain the number was chosen deliberately. Made me lol.
JimV
Transactional focus in both regulatory control and "policy" formulation is a fundamental component of this neofascist administration.
ChrisSuperPogi
From a cybersecurity perspective: This is an important development as it addresses one (of the three) major channels of threats (the other two are: threats to the voting machines and the social engineering attacks / fake news that influence our voters).
By allowing campaigns to accept cybersecurity services that they can afford helps them address threats without being suspected of accepting in-kind contributions.
Remember, DNC hack and Hillary Clinton's phishing events are " "a dress rehearsal for the big show in 2020." If we don't address these now, we are bound to repeat our mistakes.
My $0.02
I appreciate that BK tried presenting the article in a non-partisan manner and I want to respond in kind.
But I can't.
Wyden annoys me. The FEC annoys me. So what follows is a big rant.
You're welcome to ignore.
Rant/
Wyden's bill is a $10,000,000 bribe to each state to vote Democrat. There is no other reason for the federal government to be handing money over to state offices of elections BEFORE changes are implemented. It's not a reimbursement for making improvements, it's a bribe.
This bill would force states to hire more election workers to handle the mandated paper ballots. Civil servants inevitably vote for the party of big government, as it is a cycle of self-preservation.
McConnell is right. The Wyden bill is a Democrat job protection measure.
Now, that doesn't mean Wyden is entirely wrong. It is a great idea for election records to be secure from blackmail, tampering, and exploitation. Voting needs to be secure.
But what he's wrong about is trying to mandate these things at the federal level. Election security is not a national issue. It's a state issue. States run their elections as their citizens demand.
If Wyden really wanted to improve election security, he'd offer reimbursement to state election offices that voluntarily improve their systems. But as long as it's mandated and a bribe, I wouldn't stand with him.
All votes, except for president, are for state issues and state representation in federal and state government. The only job of the federal government in elections is to oversee the electoral college tabulation. Everything else is a state responsibility.
The FEC would do well to stop issuing letters. As a federal agency, they don't have any business sticking their nose into the affairs of private companies or political campaigns, save for presidential campaigns. And even for those, they have no business interpreting the will of Congress and acting as part of the judicial branch.
Stop it, FEC. Stop it.
Wyden should instead be focused on reeling in the power of social media, marketing, and search companies from influencing politics by censorship, data mining, and targeting advertising.
He should look into amending section 230 of the Communication Decency Act to eliminate the loophole that allows Facebook, Instagram, and Twitter to silence Americans and spout anti-American propaganda. These companies are the ones who gave "the Russians", ISIS, and others a platform to try influencing the last two big elections. (Unsuccessfully, I might add).
He should make it impossible for Google to shift voters to or from political candidates by manipulation of search algorithms. Currently, Google has the ability to shift 20 million votes, just by changing how search results are ordered for different users.
(Pranksters can manipulate results, too. Google "pranksters santorum").
And Wyden should be looking to keep Chinese spy technology out of our country's 5G rollout, by codification of the Entities List, so that whoever wins in 2020 cannot undo the current ban on Huawei infrastructure tech.
But he won't. Because in 2016, he took in twice the average money among senators. He's bought and paid for by special interests. He really is a partisan hack.
/rant
I should add: thank you, Brian, for allowing the rant to stay up, unedited.
PrivateCitizen
The party of Big Government is the Republican party. They only *talk* about a smaller government. The Republican party grows government every time they are in power and only fight to shrink it when a Democrat is in the White House. There's a good reason why too, because government jobs are *jobs*. Normally, while they are in power, they want full employment and for people to associate the good economy with their policies. They hide the facts by still talking tough on Big Government, and then expand the spending quietly (as in not talking about the cost or the growth of government from their policies). The DHS is one big example, but there's plenty of others too.
To clarify "normally", there's nothing normal about this Presidency. Trump is inconsistent and incoherent most of the time, and as a result, even when the Republicans were in full control of both houses of Congress, they couldn't figure out how to enact his policies.
The one thing I agree with is that we have a corruption issue in the US. It isn't just a one sided issue. There's corruption on both sides, and that is something we shouldn't accept.
Any word if they are going to provide discounted personal protection to Seth Rich types?

redpajama_set_name: RedPajamaCommonCrawl


text:
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Home Defense Robo-Raven may one day fly for Soldiers
Robo-Raven may one day fly for Soldiers
Army photograph by Todd Lopez
John W. Gerdes III, mechanical engineer at the Vehicle Technology Directorate, prepares to fly Robo-Raven at Aberdeen Proving Ground's Spesutie Island Robotics Research Facility on Chesapeake Bay, Md., Nov. 3, 2015.
In the future, it's possible that some unmanned aerial vehicles, or UAVs, might sport wings that flap like a bird or a butterfly.
The Army Research Lab, or ARL, is testing that concept at the Spesutie Island Robotics Research Facility on Chesapeake Bay.
John W. Gerdes III, mechanical engineer at the Vehicle Technology Directorate, has been testing such a UAV, known as Robo-Raven. He designed the vehicle in collaboration with the University of Maryland.
During an open house Nov. 3, Gerdes took Robo-Raven for three test flights. He held it aloft in his hand, sort of like a falconer might do. With the other hand, he switched on the transmitter – the sort found in hobby shops for drones and toy vehicles.
The wings started flapping immediately as soon as he threw it aloft. Up and away it went, flapping around in a light breeze more like a butterfly than a bird. A gust blew it backward, but Robo-Raven made course corrections on its own so that Gerdes continued to maintain nearly full control of its flight.
After a minute or two, a curious raptor, possibly a hawk, circled Robo-Raven from above. At this point, Gerdes decided to land his mechanical bird. He raised his arm, and Robo-Raven obediently landed on his outstretched hand.
Raptors, if given the chance, will destroy Robo-Raven, he said. Once in the past, he said he flew his bird up to about 300 feet and a falcon dive bombed it, destroying its gossamer wings.
Once the falcon disappeared, Gerdes launched a second flight. This time, a flock of seagulls circled it. Gerdes noted that non-birds of prey will come over to investigate, but will not attack Robo-Raven – at least not yet.
Unfortunately, Gerdes' landing didn't go as smoothly as the first and it crashed into the grass nearby. Fortunately, his half-pound bird sustained no damage. Observers wore hard hats and goggles, just to be safe.
Had his Robo-Raven been destroyed in a crash or by a raptor, Gerdes had two backups, each of which looked similar, but were slightly different in shape and size for testing.
The third flight went well and landed back in Gerdes' hand.
"Ultimately, what we're trying to go after is a vehicle which, at least in part, duplicates some of the great things animals can do," he said. "The benefit of the flapping air vehicle is obvious when you look at nature."
Flying creatures are so good at what they do because of how well in harmony all their subsystems work, he said. "Everything is perfectly matched to itself so they are able to perform at the highest possible level, given their size and weight. We're curious to discover the tricks they're using."
The challenge is to engineer a system, which can duplicate that behavior, he added.
Gerdes provided another illustration.
A quad-copter is great for flying at slow speeds, hovering, maneuvering and avoiding obstacles, he said. On the other hand, an airplane is great at flying high, far and fast.
"When you take either of those platforms out of their intended mission space they do very poorly," he said. "Animals, on the other hand, can do all these things quite well." The reason why is they can reconfigure their wings and orient themselves to take advantage of the airflow. "All these different details we're trying to learn about here."
Animals that fly are able to do so because of an enormous number of muscles and nerves that control their flexible wing and body, he said. The slightest alteration of a wing can send a bird soaring hundreds of feet into the air on an updraft.
"It's extraordinarily difficult to unravel all of the pieces of that problem," where there are flexible wings, sensors and controls distributed everywhere, he said. "It's just too difficult to engineer at this point. But, we can approach that solution at least."
On the other hand, Robo-Raven has some advantages over real birds.
For instance, Robo-Raven doesn't need to eat or reproduce. "Animals make sacrifices that are different that don't make sense from a robotics standpoint, so it gives me hope that one day we can do better than an animal," he said.
Robo-Raven's design is a terrible approach from an efficiency standpoint, Gerdes said.
For one thing, it has two motors that independently control each wing. There are commercial flappy-bird designs that work more efficiently using just one motor to control both wings. For example, "you wouldn't drive a car with a different motor on each wheel. You'd be carrying unnecessary extra weight," he said.
But from a scientific standpoint, this approach lets one learn a lot more about the platform and explore interesting spaces of the design, which wouldn't be possible with a traditional flapping wing single-motor design, he said. With a single motor, one can only speed the wings up and slow them down, which doesn't do much to inform learning of the system.
So, the goal is not to build the best flapping wing possible. "It's our goal to build the most amount of knowledge about flapping-wing air vehicles as possible."
Robo-Raven's wings were designed with the help of ARL's 3-D printer. Gerdes programmed a commercial-off-the-shelf Arduino micro-controller, which controls the flapping motion.
A hobby shop transmitter and receiver is used to send and receive commands to Robo-Raven. It can fly for 10 to 15 minutes before the motors overheat.
Inside Gerdes' lab is a breadboard with wires connected to sensors. In about a month, he said he hopes to have a more robust Robo-Raven that can carry this full suite of sensors, which will measure altitude, air speed, wing position, flapping speed, power draw, battery charge, acceleration, roll and so on.
These sensors will provide a huge body of data, he said, to begin to get at the "diabolically hard problem to solve" of flight similar to birds.
Down the road, he said ARL might come up with an innovative flexible material that would boost the chance of bird-like flight. He said the various labs across the Army collaborate very well on projects like this and others are aware of what he's doing.
Ultimately in the future, Gerdes said he wants Soldiers to have a single platform that's safer, quieter, stealthier, more versatile, that can do a wide range of missions.
"It's an exciting project, full of possibilities, but unfortunately right now, it's not the finished product we're after," he said. That will be years from now. At that point, the U.S. Army Aviation and Missile Research, Development and Engineering Center might work on it.
Click here to watch on YouTube – Ed.
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Business hours: 6.30am to 10pm, daily.
Renaissance KL restaurant brings in guest chef from Middle East to tempt diners.
INDULGE yourself with delicacies from the Middle East at Vogue Cafe, Renaissance Kuala Lumpur Hotel.
Guest chef Youssef Issa has brought with him some authentic Arabian flavours all the way from Dubai and patrons can enjoy his selection of dishes until Aug 29.
Youssef, who uses traditional recipes and original ingredients, said patrons were guaranteed to taste the best of Arabian cuisine.
Chef Youssef Issa with the Pomegranate Walnut Salad.
Patrons will also be spoilt for choice as there is a wide spread of cold mezze — dishes served as appetisers that range from dips to salad.
At Vogue Cafe, patrons can expect to get a taste of flavoursome salads such as taboulleh (a fusion of parsley, tomato, onion, bulgur wheat, lemon juice and olive oil), fattoush (crisp romaine lettuce, cucumber, tomato, radish, fresh mint mixed with pomegranate sauce) and baba ghanoush (roasted eggplant, tomato, parsley, lemon juice and olive oil).
Tabouleh is a famous Middle East salad and tasted a bit sour, perhaps from the tomato and lemon juice.
Fattoush is a part of the mezze available at the buffet spread.
Apart from the salad, patrons can dip pita bread with signature spreads from the Middle East such as hummus (chickpeas, tahina, fresh lemon juice and olive oil) andmuhammara (bread crumb, walnuts, tahini and pomegranates molasses).
A small portion of rice is also available among the mezze, such as the Stuffed Vine Leaves and Mujaddara.
The rolls of vine leaves are stuffed with Egyptian rice, tomato, parsley and onion, which are then cooked with lemon juice and olive oil.
Meanwhile, the mujaddara is a cooked lentil dish with fragrant rice, caramelised onion and olive oil.
At a recent media preview, we tried the Arabic Menu Day 3, from the five day rotating menu.
Continuing to tantalise the tastebuds of patrons, the main course offers a flavourful choice of rice, lamb or shrimp.
What stood out among these delights was the lamb.
There are several lamb dishes available, which are flavourful and not overwhelming.
The Vegetable Tagine is a slow-cooked lamb dish with olives and saffron, while lamb cooked in yoghurt sauce, shallots, pine seeds and coriander results in the Lamb Umo. The Mix Grill of shish taouk, lamb kebab and kebab hallabi are also available to complete your dining experience.
The dinner buffet, from 6.30pm to 10pm, is priced at RM108++ per person and children between six to 12 years old dine at half price.


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Home / New Music and Video / Reviews / Record Review: White Hinterland
Record Review: White Hinterland
By Daryl Lazaro on February 1, 2011
White Hinterland
"Celestial pop companion"
Portland duo White Hinterland is back with Eidolon, the ancient Greek word for a ghostly reincarnation. The songs on this album are just that: reincarnations of past compositions, into which Casey Dienel and Shawn Creeden have breathed new life.
It opens with a remarkable cover of a '90s R&B hit, Monica's "Don't Take It Personal (Just One of Dem Days)." Supported by steady drums and a mellow keyboard, Dienel's sultry voice sounds distant, wandering in and out of the foreground like an apparition inside an empty house. Both eerie and sensuous, she treats the song with a fresh indie-pop sensibility without sacrificing the soulfulness of the original.
The rest of the EP is essentially a companion piece to the band's well-received LP Kairos: a vaporous reworking of Kairos' closer "Magnolias" and two remixes of the popular single "Icarus." The first "Icarus" remix, featured on Project Runway, revolves around the original version's catchy intro motif; the second, remixed by Connecticut DJ GhostWaves, features harsher, dance club beats. Though Eidolon might be considered a minor work in White Hinterland's acclaimed catalog, its four fine tracks guarantee that the duo's celestial pop aesthetic remains on track for their next release. (Dead Oceans)
Recorded and mixed at Studio 1907, Portland, OR // Produced by Casey Dienel // Engineered by Shawn Creeden
www.myspace.com/whitehinterland
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text:
From left to right: Wilbur (played by HSU alumnus Catlin Hatfield), Homer Zuckerman (played by HSU student Isaiah Alexander) and Fern (played by HSU communications and theater arts major Isabella Ceja). Photo by Heather Karns.
Charlotte's Web wins hearts
By Tyrone Mcdonald on Thursday, March 8, 2018
Chances are you know about Charlotte's Web, a bestselling children's novel. Now that we are grown up, we can return to the days of being read to and tap into the inspiration of animals, language and the importance of community.
Through the immaculate performance of Charlotte's Web by Humboldt State students and the HSU theater department, this children's story flows with laughter, touches you with sadness and sweeps you up and out the door in waves of joy. The cast hopes to bring happiness to families and give people the opportunity to take a break from the chaos of life.
Director Derek Lane, HSU theater arts lecturer, used the play adaptation of Charlotte's Web by Joseph Robinette and added his own composition from copyright-free music and lyrics.
Charlotte is played by Camille Borrowdale, an HSU environmental science major.
"Charlotte's story is important," Borrowdale said. "It's about sticking together and being there for one another in our scary world."
During intermission, the lobby of the HSU Van Duzer Theatre was transformed into a carnival midway from the county fair. There were games and chances for children to win prizes.
HSU theater major Roman Sanchez is the production manager of Charlotte's Web and an activist for getting arts into education.
"This might be some children's first play," Sanchez said. "We want to inspire them for a lifetime of the arts."
Sanchez believes the domino effect can change the world.
"The togetherness of rehearsing with the cast and crew was really fun," HSU zoology student and cast member Rachael Robinson said.
Veronica Brooks is an HSU double major in theater and anthropology, as well as the costume designer for Charlotte's Web.
"Come and see Charlotte's Web," Brooks said. "It is happy and rewarding. It will knock your socks off!" .
Charlotte's Web continues tonight and tomorrow in the Van Duzer Theatre at 7:30 p.m., as well as a matinee show on Sunday at 10 a.m. You can find more information at centerarts.humboldt.edu.
Published in Life & Arts and Reviews
Tyrone Mcdonald
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