Jeffrey B. Hodgin

Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues, and Expose Immunostimulatory Molecules in Systemic Lupus Erythematosus

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.

A Noninvasive Computerized Tail-Cuff System for Measuring Blood Pressure in Mice

We have validated a noninvasive computerized tail-cuff system for measuring blood pressure in mice. The system was designed to perform all functions automatically, including a programmable routine of cuff inflation and deflation, analysis and assignment of pulse rate and blood pressure, and recording of data electronically. To evaluate this system over a range of blood pressures, we gave groups of mice enalapril or NG-nitro-L-arginine methyl ester in their drinking water. For each of these groups, an equal number of control mice were given nothing in their drinking water. Tail-cuff blood pressures were recorded as the means of blood pressures determined on at least 3 days after at least 7 days of training. Tail-cuff enalapril and control group means were measured both 3 and 4 months after enalapril (or no drug) was begun; the group means at 3 months were not significantly different from the group means at 4 months. These results demonstrate that the system gives reproducible results. After the tail-cuff measurements were completed, intra-arterial blood pressures were attempted in all mice under unrestrained, unanesthetized conditions, and individual mouse (n = 22) blood pressures with the use of the two methods were compared. The blood pressures from individual mice by tail-cuff and intra-arterial methods were highly correlated (r = .86, P < .01). The means for the four mouse groups were also highly correlated (r = .98, P < .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptor α is a major mediator of 17β-estradiol’s atheroprotective effects on lesion size in Apoe–/– mice

The inhibitory effects of estrogen (17β-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERα and ERβ). To investigate the role of ERα in the atheroprotective effect of 17β-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERα (ααee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized ααee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with ααee mice without E2, demonstrating that ERα is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the ααee females, although not to the same degree as in AAee females, suggesting the existence of ERα-independent atheroprotective effects of E2.

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Objectives An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs—knockout of NOX2—accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. Methods NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice. Results Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease. Conclusions PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.

Identification of Cross-Species Shared Transcriptional Networks of Diabetic Nephropathy in Human and Mouse Glomeruli

Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human–mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS−/− db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human–mouse networks were discovered. The human–mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed and cofilin was de-phosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiologic steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

A Molecular Profile of Focal Segmental Glomerulosclerosis from Formalin-Fixed, Paraffin-Embedded Tissue

Focal segmental glomerulosclerosis (FSGS) is a common form of idiopathic nephrotic syndrome defined by the characteristic lesions of focal glomerular sclerosis and foot process effacement; however, its etiology and pathogenesis are unknown. We used mRNA isolated from laser-captured glomeruli from archived formalin-fixed, paraffin-embedded renal biopsies, until recently considered an unsuitable source of mRNA for microarray analysis, to investigate the glomerular gene expression profiles of patients with primary classic FSGS, collapsing FSGS (COLL), minimal change disease (MCD), and normal controls (Normal). Amplified mRNA was hybridized to an Affymetrix Human X3P array. Unsupervised (unbiased) hierarchical clustering revealed two distinct clusters delineating FSGS and COLL from Normal and MCD. Class comparison analysis of FSGS + COLL combined versus Normal + MCD revealed 316 significantly differentially regulated genes (134 up-regulated, 182 down-regulated). Among the differentially regulated genes were those known to be part of the slit diaphragm junctional complex and those previously described in the dysregulated podocyte phenotype. Analysis based on Gene Ontology categories revealed overrepresented biological processes of development, differentiation and morphogenesis, cell motility and migration, cytoskeleton organization, and signal transduction. Transcription factors associated with developmental processes were heavily overrepresented, indicating the importance of reactivation of developmental programs in the pathogenesis of FSGS. Our findings reveal novel insights into the molecular pathogenesis of glomerular injury and structural degeneration in FSGS.

The Atheroprotective Effect of Dietary Soy Isoflavones in Apolipoprotein E−/− Mice Requires the Presence of Estrogen Receptor-α

Objective—Although the mechanisms by which dietary soy inhibits atherosclerosis are unclear, one line of evidence implicates an important role for its phytoestrogenic isoflavones. We sought to determine whether soy isoflavones exert atheroprotective effects through estrogen receptor–dependent processes and, if so, which estrogen receptor subtype (ie, &agr; or &bgr;) is involved. Methods and Results—We compared the effects of diets rich in soy protein that were either isoflavone depleted (0.04 mg/g protein isolate) or isoflavone-replete, or Soy(+IF) (1.72 mg/g protein isolate) in apolipoprotein E–deficient (ee) mice that had been crossed with estrogen receptor-&agr;– and -&bgr;–deficient mice to produce double-knockout &agr;&agr;ee and &bgr;&bgr;ee mice and (estrogen receptor) wild-type controls (AA ee and BB ee). Both male and ovariectomized female mice were studied (n=10 to 17 per treatment group; total n=201). After 16 weeks, atherosclerosis was assessed by quantifying the aortic content of esterified cholesterol. Atherosclerosis was reduced 20% to 27% (P <0.05) by Soy(+IF) in &bgr;&bgr;ee, BB ee, and AA ee mice but was unaffected in &agr;&agr;ee mice. The inhibitory effect of Soy(+IF) was unrelated to sex, total plasma cholesterol, VLDL, LDL, and HDL cholesterol. Conclusions—The results indicate a necessary role for estrogen receptor-&agr;–dependent processes in mediating the atheroprotective effects of dietary soy isoflavones.

Defining cell-type specificity at the transcriptional level in human disease

Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.