Alkylating histone deacetylase inhibitors may have therapeutic value in experimental myeloperoxidase-ANCA vasculitis.

Current therapies for treating antineutrophil cytoplasm autoantibody (ANCA)-associated vasculitis include cyclophosphamide and corticosteroids. Unfortunately, these agents are associated with severe adverse effects, despite inducing remission in most patients. Histone deacetylase inhibitors are effective in rodent models of inflammation and act synergistically with many pharmacological agents, including alkylating agents like cyclophosphamide. EDO-S101 is an alkylating fusion histone deacetylase inhibitor molecule combining the DNA alkylating effect of Bendamustine with a pan-histone deacetylase inhibitor, Vorinostat. Here we studied the effects of EDO-S101 in two established rodent models of ANCA-associated vasculitis: a passive mouse model of anti-myeloperoxidase IgG-induced glomerulonephritis and an active rat model of myeloperoxidase-ANCA microscopic polyangiitis. Although pretreatment with EDO-S101 reduced circulating leukocytes, it did not prevent the development of passive IgG-induced glomerulonephritis in mice. On the other hand, treatment in rats significantly reduced glomerulonephritis and lung hemorrhage. EDO-S101 also significantly depleted rat B and T cells, and induced DNA damage and apoptosis in proliferating human B cells, suggesting a selective effect on the adaptive immune response. Thus, EDO-S101 may have a role in treatment of ANCA-associated vasculitis, operating primarily through its effects on the adaptive immune response to the autoantigen myeloperoxidase.

Role of the Immune System in Diabetic Kidney Disease.

PURPOSE OF REVIEW: The purpose of this review is to examine the proposed role of immune modulation in the development and progression of diabetic kidney disease (DKD). RECENT FINDINGS: Diabetic kidney disease has not historically been considered an immune-mediated disease; however, increasing evidence is emerging in support of an immune role in its pathophysiology. Both systemic and local renal inflammation have been associated with DKD. Infiltration of immune cells, predominantly macrophages, into the kidney has been reported in a number of both experimental and clinical studies. In addition, increased levels of circulating pro-inflammatory cytokines have been linked to disease progression. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease. Although no current therapies for DKD are directly based on immune modulation many of the therapies in clinical use have anti-inflammatory effects along with their primary actions. Macrophages emerge as the most likely beneficial immune cell target and compounds which reduce macrophage infiltration to the kidney have shown potential in both animal models and clinical trials.

Urinary Soluble CD163 in Active Renal Vasculitis.

A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3 ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV.

IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-ß1-induced fibrotic responses.

TGF-ß1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-ß1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localisation of IHG-1 is pivotal in the amplification of TGF-ß1 signalling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-ß1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (Δmts-IHG-1) repressed TGF-ß1 fibrotic signalling in renal epithelial cells. In cells expressing Δmts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-ß1. Δmts-IHG-1 modulation of TGF-ß1 signalling was associated with increased Smad7 protein expression. Δmts-IHG-1 modulated TGF-ß1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-ß1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-ß1 signal transduction and that IHG-1 is a key player in this modulation.

IHG-1 amplifies TGF-ß1 signalling and mitochondrial biogenesis and is increased in diabetic kidney disease.

PURPOSE OF REVIEW: This review focuses on the role of the mitochondrial protein induced in high glucose 1 (IHG-1) in kidney fibrosis. RECENT FINDINGS: Diabetic nephropathy is the most common cause of end-stage renal disease. Transforming growth factor-ß1 (TGF-ß1) is a pivotal mediator of fibrosis and diabetic nephropathy. IHG-1 was identified in a screen for genes differentially expressed in renal cells exposed to high glucose. Here we review the biology of this novel functionally uncharacterized gene transcript. Data from human biopsy material and experimental models indicate increased expression of IHG-1 is a critical component of fibrogenesis as it amplifies TGF-ß1 signalling. IHG-1 is expressed in mitochondria, stabilizes PGC-1α protein and increases mitochondrial biogenesis. Recently the crystal structure of IHG-1 has been determined revealing structural homology with canonical 5'→ 3' DNA polymerases and adenylyl/guanylyl cyclases, whereas the closely related yeast homologue has been shown to function as a tRNA(HIS) guanyltransferase. SUMMARY: IHG-1 is a transcript up-regulated in renal cells exposed to high glucose, in animal models of renal fibrosis and in human diabetic nephropathy. IHG-1 encodes a mitochondrial protein that amplifies fibrotic responses to TGF-ß1 and promotes mitochondrial biogenesis. Investigation of the functional significance of the highly conserved domains of IHG-1 may lead to new therapeutic strategies.

Connective tissue growth factor antagonizes transforming growth factor-ß1/Smad signalling in renal mesangial cells.

The critical involvement of TGF-ß1 (transforming growth factor-ß1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-ß1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-ß1 and its physiological significance. CTGF was determined to bind directly to the TßRIII (TGF-ß type III receptor) and antagonize TGF-ß1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-ß1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-ß1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-ß1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF. Knockdown of TßRIII restored TGF-ß1-mediated Smad signalling and cell contractility, suggesting that TßRIII is key for CTGF-mediated regulation of TGF-ß1. Comparison of gene expression profiles from CTGF/TGF-ß1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-ß1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

IHG-1 promotes mitochondrial biogenesis by stabilizing PGC-1α.

Increased expression of Induced-by-High-Glucose 1 (IHG-1) associates with tubulointerstitial fibrosis in diabetic nephropathy. IHG-1 amplifies TGF-ß1 signaling, but the functions of this highly-conserved protein are not well understood. IHG-1 contains a putative mitochondrial-localization domain, and here we report that IHG-1 is specifically localized to mitochondria. IHG-1 overexpression increased mitochondrial mass and stabilized peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Conversely, inhibition of IHG-1 expression decreased mitochondrial mass, downregulated mitochondrial proteins, and PGC-1α-regulated transcription factors, including nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM), and reduced activity of the TFAM promoter. In the unilateral ureteral obstruction model, we observed higher PGC-1α protein expression and IHG-1 levels with fibrosis. In a gene-expression database, we noted that renal biopsies of human diabetic nephropathy demonstrated higher expression of genes encoding key mitochondrial proteins, including cytochrome c and manganese superoxide dismutase, compared with control biopsies. In summary, these data suggest that IHG-1 increases mitochondrial biogenesis by promoting PGC-1α-dependent processes, potentially contributing to the pathogenesis of renal fibrosis.

von Willebrand Factor Antigen, von Willebrand Factor Propeptide, and ADAMTS13 in Carotid Stenosis and Their Relationship with Cerebral Microemboli.

BACKGROUND: The relationship between von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio, ADAMTS13 activity, and microembolic signal (MES) status in carotid stenosis is unknown. METHODS: This prospective, multicenter study simultaneously assessed plasma VWF:Ag levels, VWFpp levels and ADAMTS13 activity, and their relationship with MES in asymptomatic versus symptomatic moderate-to-severe (≥50-99%) carotid stenosis patients. One-hour transcranial Doppler ultrasound of the middle cerebral arteries classified patients as MES+ve or MES-ve. RESULTS: Data from 34 asymptomatic patients were compared with 43 symptomatic patients in the "early phase" (≤4 weeks) and 37 patients in the "late phase" (≥3 months) after transient ischemic attack (TIA)/ischemic stroke. VWF:Ag levels were higher (p = 0.049) and VWFpp/VWF:Ag ratios lower (p = 0.006) in early symptomatic than in asymptomatic patients overall, and in early symptomatic versus asymptomatic MES-ve subgroups (p ≤0.02). There were no intergroup differences in VWFpp expression or ADAMTS13 activity (p ≥0.05). VWF:Ag levels and ADAMTS13 activity decreased (p ≤ 0.048) and VWFpp/VWF:Ag ratios increased (p = 0.03) in symptomatic patients followed up from the early to late phases after TIA/stroke. Although there were no differences in the proportions of symptomatic and asymptomatic patients with blood group O, a combined analysis of early symptomatic and asymptomatic patients revealed lower median VWF:Ag levels in patients with blood group O versus those without blood group O (9.59 vs. 12.32 µg/mL, p = 0.035). DISCUSSION: VWF:Ag expression, a marker of endothelial ± platelet activation, is enhanced in recently symptomatic versus asymptomatic carotid stenosis patients, including in MES-ve patients, and decreases with ADAMTS13 activity over time following atherosclerotic TIA/ischemic stroke.

Pro-inflammatory Stimulation of Monocytes by ANCA Is Linked to Changes in Cellular Metabolism.

Clinical and experimental data suggest that pathogenesis in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is driven by ANCA-mediated activation of neutrophils and monocytes. While the role of neutrophils has been extensively investigated, the function of monocytes remains relatively understudied. We have previously demonstrated that stimulation of monocytes with anti-myeloperoxidase (MPO), but not anti-proteinase-3 (PR3), antibodies results in production of the pro-inflammatory cytokine IL-1ß. Changes in cellular metabolism, particularly a switch to glycolysis, have recently been linked to activation of immune cells and production of IL-1ß. Therefore, we investigated the metabolic profile of monocytes following ANCA stimulation. We found a significant increase in glucose uptake in anti-MPO stimulated monocytes. Interestingly, both anti-MPO and anti-PR3 stimulation resulted in an immediate increase in glycolysis, measured by Seahorse extracellular flux analysis. However, this increase in glycolysis was sustained (for up to 4 h) in anti-MPO- but not anti-PR3-treated cells. In addition, only anti-MPO-treated cells exhibited increased oxidative phosphorylation, a metabolic response that correlated with IL-1ß production. These data indicate that monocyte metabolism is altered by ANCA, with divergent responses to anti-MPO and anti-PR3 antibodies. These metabolic changes may underlie pathologic immune activation in ANCA associated vasculitis, as well as potentially contributing to the differing clinical phenotype between PR3- and MPO-ANCA positive patients. These metabolic pathways may therefore be potential targets for therapeutic intervention.

Dysregulated Monocyte and Neutrophil Functional Phenotype in Infants With Neonatal Encephalopathy Requiring Therapeutic Hypothermia.

Neonatal encephalopathy (NE) is a significant cause of morbidity and mortality. Persistent inflammation and activation of leukocytes mediate brain injury in NE. The standard of care for NE, therapeutic hypothermia (TH), does not improve outcomes in nearly half of moderate to severe cases, resulting in the need for new adjuvant therapies, and immunomodulation holds promise. Our objective was to explore systemic leukocyte phenotype in infants with NE and healthy controls in response to lipopolysaccharide (LPS). Twenty-four infants with NE (NE II-20; NE III = 4) requiring TH and 17 term neonatal controls were enrolled, and blood samples were analyzed between days 1 and 4 of life at a mean (SD) timepoint of 2.1 (± 0.81) days of postnatal life at the time of the routine phlebotomy. Leukocyte cell surface expression levels of Toll-like receptor 4, NADPH oxidase (NOX2), CD11b, mitochondrial mass, and mitochondrial superoxide production were measured by flow cytometry. Gene expression of TRIF (TIR domain-containing adapter-inducing interferon-ß), MyD88 and IRAK4 was measured by reverse transcription-polymerase chain reaction. Infants with NE had significantly lower expression of neutrophil CD11b and NOX2 with LPS stimulation compared to healthy term controls. Mitochondrial mass in neutrophils and monocytes was significantly increased in NE infants with LPS compared to controls, potentially indicating a dysregulated metabolism. Infants with NE had significantly lower IRAK4 at baseline than controls. NE infants display a dysregulated inflammatory response compared to healthy infants, with LPS hyporesponsiveness to CD11b and NOX2 and decreased IRAK4 gene expression. This dysregulated immune profile may indicate an adaptable response to limit hyperinflammation.

Diabetes mellitus and apoptosis: inflammatory cells.

Since the early observation that similarities between thyroiditis and insulitis existed, the important role played by inflammation in the development of diabetes has been appreciated. More recently, experiments have shown that inflammation also plays a prominent role in the development of target organ damage arising as complications, with both elements of the innate and the adaptive immune system being involved, and that cytokines contributing to local tissue damage may arise from both infiltrating and resident cells. This review will discuss the experimental evidence that shows that inflammatory cell-mediated apoptosis contributes to target organ damage, from beta cell destruction to both micro- and macro-vascular disease complications, and also how alterations in leukocyte turnover affects immune function.

Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells.

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.

Bcr-Abl regulates osteopontin transcription via Ras, PI-3K, aPKC, Raf-1, and MEK.

Chronic myeloid leukemia (CML) is caused by the constitutively active Bcr-Abl tyrosine kinase. This fusion protein is generated by the Philadelphia translocation t(9;22). CML is a progressive condition that invariably advances from a drug-sensitive to a drug-resistant, aggressive, acute leukemia. The mechanisms responsible for this progression are largely unknown; however, in many cases, progression is accompanied by an increase in Bcr-Abl expression. Osteopontin (OPN) expression has been shown to be involved in the progression and increased aggression and invasiveness of many solid tumors. Here, we demonstrate that OPN expression is induced in a model of leukemia, and we describe the identification of specific signaling pathways required for the induction of OPN expression by p210 Bcr-Abl. We have determined that high levels of Bcr-Abl activate a signaling cascade involving the sequential activation of Ras, phosphatidylinositol-3 kinase, atypical protein kinase C, Raf-1, and mitogen-activated protein kinase kinase, leading to the ultimate expression of OPN. Our results suggest that these molecules represent a single pathway and also that there is no redundancy in this pathway, as inhibition of any individual component results in a block in the induction of OPN. The data presented here define for the first time the ability of Bcr-Abl to stimulate the expression of OPN and also identify the signaling pathway involved. This may not only prove important in understanding the mechanisms of progression of CML but also highlights a pathway that may prove significant in many other cases of oncogenesis, where OPN expression is implicated.

Myeloid Engraftment in Humanized Mice: Impact of Granulocyte-Colony Stimulating Factor Treatment and Transgenic Mouse Strain.

Poor myeloid engraftment remains a barrier to experimental use of humanized mice. Focusing primarily on peripheral blood cells, we compared the engraftment profile of NOD-scid-IL2Rγc(-/-) (NSG) mice with that of NSG mice transgenic for human membrane stem cell factor (hu-mSCF mice), NSG mice transgenic for human interleukin (IL)-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and stem cell factor (SGM3 mice). hu-mSCF and SGM3 mice showed enhanced engraftment of human leukocytes compared to NSG mice, and this was reflected in the number of human neutrophils and monocytes present in these strains. Importantly, discrete classical, intermediate, and nonclassical monocyte populations were identifiable in the blood of NSG and hu-mSCF mice, while the nonclassical population was absent in the blood of SGM3 mice. Granulocyte-colony stimulating factor (GCSF) treatment increased the number of blood monocytes in NSG and hu-mSCF mice, and neutrophils in NSG and SGM3 mice; however, this effect appeared to be at least partially dependent on the stem cell donor used to engraft the mice. Furthermore, GCSF treatment resulted in a preferential expansion of nonclassical monocytes in both NSG and hu-mSCF mice. Human tubulointerstitial CD11c(+) cells were present in the kidneys of hu-mSCF mice, while monocytes and neutrophils were identified in the liver of all strains. Bone marrow-derived macrophages prepared from NSG mice were most effective at phagocytosing polystyrene beads. In conclusion, hu-mSCF mice provide the best environment for the generation of human myeloid cells, with GCSF treatment further enhancing peripheral blood human monocyte cell numbers in this strain.

Changes in urinary metabolomic profile during relapsing renal vasculitis.

Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.

Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1ß secretion in response to anti-MPO antibodies.

ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1ß, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1ß in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

IHG-1 increases mitochondrial fusion and bioenergetic function.

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-ß1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species-induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion-mitofusins (Mfns) 1 and 2-and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1-mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-ß, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.

Diabetic kidney disease and immune modulation.

Immune modulation is now known to contribute to the development of glomerulosclerosis, tubulointerstitial fibrosis and end-stage renal disease in a large number of kidney diseases. Similarly, diabetic nephropathy is increasingly considered an inflammatory disease, with immune modulation being involved in both the development and progression of the disease. Infiltration of immune cells including macrophages, T cells, B cells and mast cells into the kidney has been reported. A number of pro-inflammatory cytokines and chemokines also play a major role in pathogenesis of diabetic nephropathy. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease.

Deletion of Gremlin1 increases cell proliferation and migration responses in mouse embryonic fibroblasts.

Gremlin1 (Grem1) is an antagonist of bone morphogenetic proteins (BMPs) that plays a critical role in embryonic and postnatal development. Grem1 has been implicated as both a promoter and an inhibitor of cell proliferation driven by BMP-4 and other mitogens in a diverse range of cell types. Recent data showed that Grem1 can trigger angiogenesis via vascular endothelial growth factor receptor (VEGFR2) binding, highlighting that the precise modalities of Grem1 signalling require further elucidation. In an attempt to enhance our understanding of the role of Grem1 in cell proliferation, mouse embryonic fibroblasts lacking grem1 (grem1⁻/⁻) were generated. Grem1⁻/⁻ cells showed elevated levels of proliferation in vitro compared to wild-type and grem1⁺/⁻, with accelerated scratch wound repair but no obvious changes in cell cycle profile. Modest increases in BMP-4-stimulated Smad1/5/8 phosphorylation were detected in grem1⁻/⁻ cells, with concomitant modest changes in Smad-dependent gene expression. Surprisingly, levels of ERK phosphorylation were reduced in grem1⁻/⁻ cells compared to wild-type. These data suggest Grem1 is an inhibitor of embryonic fibroblast proliferation in vitro. Furthermore, the signalling pathways causing increased cell proliferation in the absence of Grem1 may involve other pathways distinct from canonical Smad and non-canonical ERK signalling.

Insulin receptor substrate 2 and FoxO3a signalling are involved in E-cadherin expression and transforming growth factor-ß1-induced repression in kidney epithelial cells.

Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3-kinase and mitogen-activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)-ß1 is the primary cytokine shown to induce epithelial-mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF-ß1-induced epithelial-mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF-ß1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)-mediated targeting of IRS2 increased E-cadherin expression, although it did not alter TGF-ß1-mediated E-cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24 h greatly reduced FoxO3a phosphorylation on Ser253 but over-expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF-ß1-mediated E-cadherin repression at 24 h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E-cadherin.