Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction

Semaphorin-3A (Sema3a), a guidance protein secreted by podocytes, is essential for normal kidney patterning and glomerular filtration barrier development. Here, we report that podocyte-specific Sema3a gain-of-function in adult mice leads to proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Reversibility of the glomerular phenotype upon removal of the transgene induction provided proof-of-principle of the cause-and-effect relationship between podocyte Sema3a excess and glomerular disease. Mechanistically, excess Sema3a induces dysregulation of nephrin, matrix metalloproteinase 9, and αvβ3 integrin in vivo. Sema3a cell-autonomously disrupts podocyte shape. We identified a novel direct interaction between the Sema3a signaling receptor plexinA₁ and nephrin, linking extracellular Sema3a signals to the slit-diaphragm signaling complex. We conclude that Sema3a functions as an extracellular negative regulator of the structure and function of the glomerular filtration barrier in the adult kidney. Our findings demonstrate a crosstalk between Sema3a and nephrin signaling pathways that is functionally relevant both in vivo and in vitro.The glomerular filter is a size-selective barrier composed of three layers: fenestrated endothelium, glomerular basement membrane (GBM), and podocyte foot processes.¹ Disruption of any of these components of the glomerular filtration barrier causes loss of permselectivity, proteinuria, and glomerular disease.¹ Podocyte foot processes are linked by slit diaphragms, which are modified adherens junctions composed of extracellular domains of nephrin molecules associated to a multiprotein complex.^2,3 Gene mutations in slit-diaphragm proteins and their actin-associated proteins cause familial nephrotic syndrome.^4–7 The GBM is a complex of type IV collagen (α3, α4, and α5) and laminin 521 (α5β2γ1) chains, perlecan, syndecan, entactin, and agrin. Imbalance of collagen and laminin chain expression results in abnormalities of GBM ultrastructure and proteinuria.^8,9 Loss of glomerular endothelial fenestration due to inhibition of vascular endothelial growth factor (VEGF-A) signaling or to excess soluble Flt-1 causes proteinuria and preeclampsia.^10,11Semaphorin-3A (Sema3a) is a secreted guidance protein involved in axon pathfinding and in cardiovascular, lung, and kidney patterning.^12,13 In the normal kidney, Sema3a is expressed in podocytes and collecting ducts.⁷ Loss-of-function studies during kidney development showed that Sema3a inhibits endothelial cell migration into glomeruli and limits ureteric bud branching.^14,15 Sema3a gain-of-function during development resulted in glomerular hypoplasia, delayed podocyte differentiation, and absent slit diaphragms.¹⁵ Exposure of cultured podocytes to recombinant Sema3a induced down-regulation of podocin and decreased the interactions among nephrin, podocin, and CD2AP.¹⁶ Systemic administration of Sema3a to adult mice induced transient, reversible foot-process effacement and proteinuria similar to that induced by protamine sulfate.^17,18 We observed increased podocyte Sema3a protein and mRNA expression in mice with diabetic nephropathy.^13,19 Taken together, our previous studies suggested that excess Sema3a might disrupt the glomerular filtration barrier in the mature kidney, particularly in the setting of diabetes.The goal of the present study was to define whether excess podocyte Sema3a per se causes glomerular disease in adult mice, and to examine the mechanism involved. Here, we report that induction of podocyte-specific Sema3a overexpression in adult mice causes a proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Mechanistically, we show that excess Sema3a induces dysregulation of nephrin, MMP-9, and αvβ3 integrin in vivo, and we identify a novel interaction between the Sema3a signaling receptor plexinA₁ and nephrin that links Sema3a signals to the slit-diaphragm signaling complex. Collectively, these findings establish that Sema3a functions as an extracellular negative regulator of the integrity and function of the glomerular filtration barrier.     

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in?Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.The apical side of endothelial cells is coated with an endothelial surface layer (ESL) composed of a surface-anchored glycocalyx which is itself composed of negatively charged proteoglycans [proteins with glycosaminoglycan (GAG) side chains] and glycoproteins (glycosylated proteins) and a more loosely associated layer of adsorbed plasma proteins.¹ The endothelial glycocalyx is 200 nm to 2 μm thick (depending on the vascular bed and also on the visualization technique).²The glycocalyx mediates shear, attenuates leukocyte and platelet adhesion,² regulates systemic vascular permeability,^3–6 and allows free passage of solutes but limits passage of charged macromolecules.⁷ The ESL is damaged in reperfusion injury, inflammation and trauma, hypervolemia, atherosclerosis, and diabetes (summarized by Becker et al²). ESL thickness is reduced by hyperglycemic infusions in healthy subjects, correlating with endothelial dysfunction and an increase in vascular permeability,⁸ and in type 1 diabetes correlating with microalbuminuria,⁹ suggesting a direct link between endothelial ESL dysfunction and this dysfunction of the glomerular filtration barrier. In mice, infusions of the GAG-specific enzymes hyaluronidase, chondroitinase, and heparinase reduced the glomerular endothelial cell (GEnC) ESL thickness, resulting in reduced charge selectivity and increased macromolecular passage (proteinuria).¹⁰ In addition, proteinuria is accompanied with a loss of charge selectivity and a reduction in the core proteins decorin, fibromodulin, and versican in nonobese diabetic mice.¹¹ Salmon et al¹² demonstrated an age-related reduction in ESL in glomerular (and other) microvessels of Munich Wistar Frömter rats; this was accompanied by an increase in glomerular albumin permeability, which could be rescued by intravenous injections of lectin. Finally, using doxorubicin (Adriamycin) to induce proteinuria in mice, Jeansson et al¹³ demonstrated increased fractional clearance of larger molecules in cooled, isolated, perfused kidneys, as well as reduced charge in the glomerular filtration barrier; this was accompanied by reduced synthesis of some core proteins and GAGs in the isolated glomeruli and reduced thickness of the ESL. Taken together, these studies strongly suggest that the ESL also regulates permeability in the glomerular filtration barrier. The glomerular filtration barrier is a tightly regulated filter that restricts macromolecular protein passage while allowing filtration of water and small solutes. Dysfunction of this barrier results in proteinuria, a hallmark of kidney disease. The glomerular filtration barrier consists of a trilayer of GEnCs, a glomerular basement membrane and glomerular epithelial cells (podocytes) whose foot processes interdigitate around the glomerular microvessels. Each of these layers contributes to the regulation of glomerular macromolecular permeability.^1,14 The GEnC contribution is increasingly thought to be largely dependent on the ESL.^10,15,16We have previously studied the GEnC ESL in vitro in a human conditionally immortalized (ci) cell line and, using electron microscopy, revealed the presence of an ESL layer measuring 200 nm, which is consistent with recent sophisticated measurements of endothelial glycocalyx in vivo.¹⁷ We confirmed the presence of proteoglycan core proteins, as previously demonstrated on human GEnCs,¹⁸ and of heparan sulfate (HS), a sulfated GAG.¹⁹ Enzymatic removal of HS increased macromolecular protein passage across a monolayer by 40%, whereas electrical resistance (a measure of pathways open to water and small molecules) was not affected. Furthermore, we have shown that high-glucose conditions reduce GEnC ESL and lead to a corresponding increase in macromolecular protein passage across GEnC monolayers,²⁰ demonstrating that the GEnC glycocalyx is present in vitro and plays a functional role.Glomerular enzyme infusion studies by Jeansson et al¹⁰ implicated, in addition to removal of HS, removal of hyaluronic acid (HA) and chondroitin sulfate (CS) in increased fractional albumin clearance. HA and CS are also thought to play a role in systemic macromolecular permeability.²¹ In contrast to other GAGs, HA is not synthesized in the Golgi apparatus, but rather at the plasma membrane (by HA synthases 1 to 3), and it is not attached to core proteins (reviewed by Genasetti et al²²). Furthermore, HA is unbranched and unsulfated, and therefore it does not have a strong negative charge. CS is sulfated on assembly within the Golgi apparatus, where it becomes attached to one of its core proteins (eg, aggrecan or versican), forming a proteoglycan. In the present study, we aimed to determine the contribution of HA and CS to the GEnC glycocalyx through in vitro studies using unique human ciGEnCs and selective enzymes.Vascular endothelial growth factor A (VEGFA), originally called vascular permeability factor, is a powerful angiogenic growth factor that has profound effects on vascular endothelial behavior (as summarized by Tammela et al²³), including GEnC maintenance,²⁴ repair,²⁵ and permeability.²⁶ VEGFA is highly expressed by podocytes.²⁷ Another member of the VEGF family of proteins, VEGFC, is a lymphangiogenic growth factor that can stimulate similar pathways to those of VEGFA in both lymphatic and vascular endothelial cells.²⁸ VEGFC also is expressed by podocytes.²⁹ Podocyte–endothelial signaling through VEGF is vital for GEnC maintenance and permeability regulation. It has been postulated that the effects of VEGFA on microvessel permeability are due in part to partial degradation of the glycocalyx.³⁰ In the present study, we aimed to determine whether VEGFs can modify the GEnC glycocalyx. Our central hypothesis was that CS and HA contribute to GEnC barrier maintenance and that they are modified by VEGFs.     

α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. l-NAME salt–induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2–deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2–null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1–dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology.Alport syndrome is characterized by delayed-onset progressive glomerulonephritis associated with sensorineural hearing loss and retinal flecks.¹ The most common form (80%) is X-linked and caused by mutations in the type IV collagen COL4A5 gene.² The two autosomal forms of the disease account for the remaining 20% of Alport patients, and result from mutations in the COL4A3 and COL4A4 genes.³ The α3(IV), α4(IV), and α5(IV) proteins form a heterotrimer that is assembled into a subepithelial network in the glomerular basement membrane (GBM) that is physically and biochemically distinct from a subendothelial type IV collagen network comprising α1(IV) and α2(IV) heterotrimers.⁴ Mutations in any one of the three type IV collagen genes that cause Alport syndrome result in the absence of all three proteins in the GBM due to an obligatory association to form functional heterotrimers.⁵ Thus, the net result for all genetic forms of Alport syndrome is the absence of the α3(IV) α4(IV) α5(IV) subepithelial collagen network, resulting in a GBM type IV collagen network comprising only α1(IV) and α2(IV) heterotrimers.This change in basement membrane composition does not result in immediate pathology. The GBM appears to function adequately for the first few years of life and sometimes past the first decade.⁶ This delayed onset predicts a triggering mechanism for glomerular disease initiation and a theoretical window for therapeutic intervention that may arrest or significantly ameliorate Alport renal disease in its earliest stages. The activation of genes encoding GBM matrix molecules, matrix metalloproteinases (MMPs), and proinflammatory cytokines have all been linked to the progression of Alport glomerular disease. These, however, are events that occur after the onset of proteinuria, and therefore, downstream of disease initiation events.^7–11 Consistent with this notion, experiments aimed at blocking these pathways have offered only limited therapeutic benefit in mouse models for Alport syndrome.^8–10,12 One of the earliest events we have documented is the appearance of an irregular deposition of laminin 211 in the GBM of Alport mice,⁸ an observation confirmed in both Alport dogs and human patients with the disease.¹³ This laminin is normally found only in the mesangium of the glomerulus, and is not expressed in the GBM at any stage of embryonic development.¹⁴ Indeed, several other mesangial matrix proteins appear in the GBM of Alport mice, including laminin 111 and fibronectin.^15,16In the Alport glomerulus, the podocytes are exposed to GBM that has an embryonic type IV collagen composition.^17,18 This could result in altered cell signaling that may trigger the onset of the disease. It has been proposed that this type of mechanism may account for the reactivation of laminin 111 expression in podocytes,¹⁹ because laminin 111 is found in the GBM during development.¹⁴ Because the α1(IV)/α2(IV) collagen network contains significantly fewer interchain disulfide crosslinks,²⁰ and the Alport GBM is thinner than normal,²¹ the Alport GBM is likely to be more elastic, resulting in elevated biomechanical strain on the glomerular cells at their points of contact with the GBM. Consistently, glomeruli from Alport mice have been shown to have elevated deformability relative to wild-type glomeruli,²² and salt-induced hypertension has been shown to accelerate glomerular disease progression in Alport mice.²³In this work, we show that the cellular origin of GBM laminin 211 in Alport glomeruli is mesangial cell process invasion, and that deletion of laminin 211 in Alport mice ameliorates the mesangial process invasion of the glomerular capillary loops in Alport mice. Salt-mediated hypertension exacerbates this mesangial process invasion. A knockout mouse for the integrin α3β1 coreceptor CD151 also develops mesangial process invasion of the capillary loops with GBM deposition of laminin 211, demonstrating the same phenotype for a completely unrelated component of the capillary structural barrier. The CD151 knockout mouse model also shows accelerated glomerular disease progression in response to hypertension.²⁴ We show that biomechanical stretching of cultured mesangial cells induces promigratory cytokines transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), both known to be induced in Alport glomeruli.^7,12 Inhibitor studies indicate that mesangial cell migration is mediated by integrin α1β1 signaling through the Rho GTPases RAC1 and CDC42. Consistently, integrin α1 deletion in Alport mice was previously shown to ameliorate glomerular disease progression and slow the accumulation of laminin 211 in Alport GBM.⁸ Here, we show that mesangial process invasion of the capillary loops is ameliorated in integrin α1–null Alport mice. These data define a role for biomechanical strain-mediated induction of mesangial cell process invasion as a key aspect of Alport glomerular disease initiation, and set the stage for defining novel therapeutic targets aimed at blocking this process.     

Glypican-5 Increases Susceptibility to Nephrotic Damage in Diabetic Kidney

Type 2 diabetes mellitus is a leading health issue worldwide. Among cases of diabetes mellitus nephropathy (DN), the major complication of type 2 diabetes mellitus, the nephrotic phenotype is often intractable to clinical intervention and demonstrates the rapid decline of renal function to end-stage renal disease. We recently identified the gene for glypican-5 (GPC5), a cell-surface heparan sulfate proteoglycan, as conferring susceptibility for acquired nephrotic syndrome and additionally identified an association through a genome-wide association study between a variant in GPC5 and DN of type 2 diabetes mellitus. In vivo and in vitro data showed a progressive increase of GPC5 in type 2 DN along with severity; the excess was derived from glomerular mesangial cells. In this study, diabetic kidney showed that accumulation of fibroblast growth factor (Fgf)2 strikingly induced progressive proteinuria that was avoided in Gpc5 knockdown mice. The efficacy of Gpc5 inhibition was exerted through expression of the Fgf receptors 3 and 4 provoked in the diabetic kidney attributively. Extraglomerular Fgf2 was pathogenic in DN, and the deterrence of Gpc5 effectively inhibited the glomerular accumulation of Fgf2, the subsequent increase of mesangial extracellular matrix, and the podocytes'' small GTPase activity. These findings elucidate the pivotal role of GPC5, identified as a susceptible gene in the genome-wide association study, in hyperglycemia-induced glomerulopathy.Among diabetes mellitus nephropathy (DN) cases, intractable dysfunction rapidly propagating to end-stage renal disease often accompanies the nephrotic proteinuria phenotype. Our recent study implicated glypican-5 (GPC5) as a susceptible gene for acquired nephrotic syndrome and demonstrated its functional significance by using podocyte-specific knockout mice; a genome-wide association study of DN satisfying the criterion of nephrotic syndrome also clarified GPC5 as the candidate gene [odds ratio 1.45 (95% CI, 1.18–1.79)].¹ Glypicans share 14 conserved and unique cysteine residues and covalently link to the cell membrane via a glycosylphosphatidylinositol anchor.² Glypican is a heparan sulfate proteoglycan, which facilitates selective protein–protein interactions establishing transient cell-signal platforms in concert with lipid rafts; glypican plays a critical role in regulating the signaling of Wnt; hedgehogs; bone morphogenetic protein; and, especially, of fibroblast growth factor (FGF)2.³FGF2 is a highly conserved 18-kDa cationic protein released from the cytosolic storage site through plasma membrane disruptions.^4,5 In the kidney, the release of FGF2 is probable from mesangial cells⁶ and podocytes.⁷ It stimulates in vitro proliferation of mesangial cells,⁸ endothelial cells,⁹ and podocytes.⁷ An excess of FGF2 clearly injures podocytes in vivo and induces proteinuria.^10,11 Based on the information from the genome-wide association study that GPC5 is a susceptible gene, this study was undertaken to clarify the functional role of GPC5 in DN and related pathways.     

Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity: Implications for the Protective Effect of Low-Dose Lithium in Podocytopathy

Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion–associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy.Glomerular visceral epithelial cells or podocytes are a core structural constituent of the glomerular filtration barrier, with elaborate interdigitating foot processes that envelop the capillaries of the glomeruli in the kidney, control glomerular permselectivity, and prevent protein in the bloodstream from leaking into the urine.^1–4 Converging evidence suggests that the podocytic filter barrier is not static but a highly dynamic structure that is regulated via the motility of podocyte foot processes.^5–7 The molecular basis of foot process motility lies in the constant dynamics of the molecular machinery that sustains the foot process architecture.^5–7 Actin is the principal component of the cytoskeletal machinery of foot processes and forms a subcortical network of branched filaments as well as bundled filaments that run longitudinally through the processes with contractility.⁸ Actin exists in foot processes in a state of dynamic equilibrium between assembly and disassembly, which is important for maintaining the homeostasis of the glomerular filtration barrier. In response to various pathogenic mediators, including oxidative stress, circulating permeability factors, and nephrotoxins such as doxorubicin (Adriamycin), the parallel actin bundles depolymerize, resulting in foot process effacement, a pathologic hallmark of podocyte injury and dysfunction.^9–13Focal adhesions (FAs), by which cells are anchored to the extracellular matrix, are a crucial determinant of actin cytoskeleton integrity and cell motility.^14,15 Molecules from FA structures connect the extracellular matrix to bundles of actin filaments, enabling the growing actin network to push the plasma membrane and the contractile stress fibers to pull the cell body, corresponding to protrusive and retractive activities.^14,16 Dynamic turnover of FAs is indispensable for constant motility and reorganization of cell edges that manifest as boundary curvature waves.¹⁷ A cycle of cellular boundary motion commences with the formation of nascent adhesions, which initiate actin assembly and, thus, allow the growing actin network to push the cell protrusion forward. The nascent adhesion or focal complex, a precursor of the FA, is smaller in size, with weaker adhesive force and rapid turnover.^18,19 Subsequently, nascent adhesions will either disassemble rapidly or mature to be FAs. The FAs usually contain multiple structural and regulatory molecules, among which paxillin acts as a pivotal adaptor protein to provide docking sites for cytoskeletons and to recruit FA regulators that control actin dynamics and FA stability.²⁰ The rate of FA turnover determines cell motility and governs the podocyte foot process dynamics. Consistently, targeted manipulation of FA turnover in podocytes by enhancing or intercepting the activity of FA regulatory molecules incurred foot process effacement and podocyte dysfunction.^21–23Glycogen synthase kinase 3β (GSK3β), a well-conserved and ubiquitously expressed serine/threonine protein kinase, plays a key role in the regulation of cytoskeleton organization and cellular motility.^24,25 Indeed, inhibition of GSK3β has been found to reduce cell motility in multiple cells, including vascular smooth muscle cells,²⁶ glioma cells,²⁷ gastric cancer cells,²⁸ and renal tubular epithelial cells.²⁹ In the kidney, GSK3β has lately been implicated in acute kidney injury and repair.³⁰ However, its role in podocyte injury and foot process cytoskeletal disarrangement remains unknown. This study examined the role of GSK3β in a model of hypermotility-associated podocytopathy induced by doxorubicin injury in vivo and in vitro. The potential intervention effect of GSK3β blockade by a single low dose of lithium, a selective GSK3β inhibitor and US Food and Drug Administration–approved mood stabilizer, on podocyte motility and dysfunction was accordingly delineated.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Podocyte Injury–Driven Lipid Peroxidation Accelerates the Infiltration of Glomerular Foam Cells in Focal Segmental Glomerulosclerosis

Intracapillary foam cell infiltration with podocyte alterations is a characteristic pathology of focal segmental glomerulosclerosis (FSGS). We investigated the possible role of podocyte injury in glomerular macrophage and foam cell infiltration in a podocyte-selective injury model (NEP25 mice) and hypercholesterolemic model [low-density lipoprotein receptor deficiency (LDLR^−/−) mice] with doxorubicin–induced nephropathy. Acute podocyte selective injury alone failed to induce glomerular macrophages in the NEP25 mice. However, in the doxorubicin-treated hypercholesterolemic LDLR^−/− mice, glomerular macrophages/foam cells significantly increased and were accompanied by lipid deposition and the formation and ingestion of oxidized phospholipids (oxPLs). Glomerular macrophages significantly correlated with the amount of glomerular oxPL. The NEP25/LDLR^−/− mice exhibited severe hypercholesterolemia, glomerular lipid deposition, and renal dysfunction. Imaging mass spectrometry revealed that a major component of oxidized low-density lipoprotein, lysophosphatidylcholine 16:0 and 18:0, was present only in the glomeruli of NEP25/LDLR^−/− mice. Lysophosphatidylcholine 16:0 stimulated mesangial cells and macrophages, and lysophosphatidylcholine 18:0 stimulated glomerular endothelial cells to express adhesion molecules and chemokines, promoting macrophage adhesion and migration in vitro. In human FSGS, glomerular macrophage–derived foam cells contained oxPLs accompanied by the expression of chemokines in the tuft. In conclusion, glomerular lipid modification represents a novel pathology by podocyte injury, promoting FSGS. Podocyte injury–driven lysophosphatidylcholine de novo accelerated glomerular macrophage–derived foam cell infiltration via lysophosphatidylcholine–mediated expression of adhesion molecules and chemokines in glomerular resident cells.Focal segmental glomerulosclerosis (FSGS) is a progressive kidney disease caused by podocyte injury.^1–4 The pathology of FSGS includes a variety of glomerular features. The Columbia classification system divides FSGS into the following five variants: not otherwise specified, perihilar variant, cellular variant, tip variant, and collapsing variant.^1,5,6 The classification is useful for predicting the prognosis in FSGS.^1,5–7Glomerulosclerosis is defined by its pathological characteristics, including capillary collapse accompanied by deposition of extracellular matrices and/or hyaline materials. This classic feature of sclerosis is usually observed in not otherwise specified and perihilar variants, whereas it is not observed in the definition of other variants. To diagnose FSGS in the absence of classic sclerosis, the infiltration of foam cells in the glomerular segment is a good marker, particularly in cellular variants.^1,7 Although glomerular foam cells can be observed in any variant of FSGS and are thought to be involved in disease progression, the mechanism underlying foam cell infiltration in FSGS remains largely unknown.Studies have shown that glomerular foam cells were associated with extremely high levels of serum lipids in patients with familial hypercholesterolemia and lecithin-cholesterol acyltransferase deficiency.^8,9 Glomerular foam cells were also observed in experimental models of hypercholesterolemia,^10–15 suggesting that high levels of serum lipids may be a plausible basis for glomerular foam cell infiltration. Some studies reported that glomerular foam cells were associated with nephrotic syndrome in humans, including membranous glomerulonephritis, diabetic nephropathy, and IgA glomerulonephritis.^16–18 However, in these cases, the glomerular foam cells were not always associated with hyperlipidemia. A previous study demonstrated that glomerular foam cells were not correlated with serum cholesterol levels in FSGS patients.⁷ Likewise, glomerular foam cells were absent in minimal change disease, despite the presence of similar levels of serum lipids observed in FSGS.¹⁹ These findings suggest that additional factors other than serum lipid levels may account for the formation of glomerular foam cells in FSGS.The characteristic histology of glomerular foam cell formation in FSGS is its segmental and intracapillary localization associated with overlaying podocyte abnormalities. Because podocyte injury represents the common basis of FSGS, there may be a causal relationship between podocyte injury and foam cell accumulation in particular variants of FSGS.Most glomerular foam cells are derived from CD68-positive macrophages,^20,21 which transform into foam cells by ingesting lipids within the glomerulus in situ. As demonstrated in the mechanism of atherosclerosis, it seems that the development of glomerular foam cell infiltration in FSGS is because of the interaction and catenation of several factors, including the extent and duration of hyperlipidemia, lipid characteristics, and the response of glomerular resident cells.^22–24The present study assessed the stepwise development of glomerular foam cell infiltration using transgenic murine models of FSGS, with podocyte-specific injury and hypercholesterolemia caused by low-density lipoprotein receptor (LDLR) deficiency. We performed imaging mass spectrometry (IMS) analysis to determine the biochemical characteristics of glomerular lipid peroxidation and conducted an in vitro study to assess changes in the molecular profiles of mesangial cells and endothelial cells in response to podocyte injury–driven lipid modifications. Our results suggest that podocyte injury promotes hypercholesterolemia-based lipid deposition and specific peroxidation, which activate a molecular network within a glomerular microenvironment that induces macrophage recruitment and foam cell formation in FSGS.     

Astrocytic Dynamin-Like Protein 1 Regulates Neuronal Protection against Excitotoxicity in Parkinson Disease

Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca²⁺ in response to extracellular glutamate, resulting from compromised intracellular Ca²⁺ buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca²⁺-coupled glutamate uptake.Parkinson disease (PD) is a neurodegenerative disorder clinically characterized by both motor and nonmotor symptoms.^1–3 The loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) is the primary cause of the motor deficits,⁴ whereas nonmotor symptoms are the result of dysfunction in multiple brain regions.^2,3 Until recently, most investigations have focused on the neuronal pathogenesis. However, accumulating evidence shows that astrocytes, which have multiple neuroprotective roles,^5,6 contribute to neuronal loss in PD.⁷Decreased respiratory chain activity⁸ and increased oxidative damage in the SNpc^9,10 contribute to neuronal demise in PD. Among proteins that regulate mitochondrial functions,¹¹ those that regulate mitochondrial fission [dynamin-like protein 1 (Dlp1; official name DNM1L), fission 1], and fusion [(mitofusins 1 and 2 and optic atrophy 1)] are particularly interesting. Models of PD show dramatic effects in mitochondrial morphological features, which can be rescued by altering expression of these proteins.^12,13 Our previous study profiled the mitochondrial fraction of the SNpc in healthy control and PD patients and identified specific alterations in expression of Dlp1, but not other mitochondrial fission and fusion proteins in PD brains,¹⁴ suggesting Dlp1 plays a role in PD pathogenesis. However, the cellular origin and consequences of decreased Dlp1 expression remain to be fully elucidated.A recent investigation found that Dlp1 potentially interacts with the glutamate transporter 1 (GLT-1; official name SLC1A2),¹⁵ which is specifically expressed by astrocytes,¹⁶ implicating this cell type in Dlp1-mediated pathogenic effects. In theory, disrupting astrocyte regulation of extracellular glutamate [also a function of the glutamate aspartate transporter (GLAST; official name SLC1A3¹⁷)] could result in excessive extracellular glutamate. This excess glutamate could prolong the opening of neuronal N-methyl-d-aspartate (NMDA) receptors,¹⁸ resulting in excessive Ca²⁺ entry and, ultimately, neuronal death. Indeed, this phenomenon of excitotoxicity has been implicated in PD and animal models of PD,^19,20 as well as in other neurodegenerative diseases.To test the hypothesis that decreased astrocytic Dlp1 expression contributes to neurodegeneration in PD, we measured astrocytic and neuronal Dlp1 expression, in the SNpc and cortex, in both PD and healthy control patients and explored the molecular mechanisms related to astrocytic dysfunction resulting from decreased Dlp1 expression.