Peptidyl arginine deiminase-4 exacerbates ischemic AKI by finding NEMO.

Peptidyl arginine deiminase-4 (PAD4) catalyzes the conversion of peptidylarginine residues to peptidylcitrulline. We have previously shown that kidney ischemia-reperfusion (I/R) injury increases renal proximal tubular PAD4 expression and activity. Furthermore, kidney PAD4 plays a critical role in ischemic acute kidney injury (AKI) by promoting renal tubular inflammation, neutrophil infiltration, and NF-κB activation. However, the mechanisms of PAD4-mediated renal tubular inflammation and NF-κB activation after I/R remain unclear. Here, we show that recombinant PAD4 preferentially citrullinates recombinant IKKγ [also called NF-κB essential modulator (NEMO)] over recombinant IKKα or IKKß. Consistent with this finding, PAD4 citrullinated renal proximal tubular cell IKKγ and promoted NF-κB activation via IκBα phosphorylation in vitro. NEMO inhibition with a selective NEMO-binding peptide attenuated PAD4-mediated proinflammatory cytokine mRNA induction in HK-2 cells. Moreover, NEMO inhibition did not affect proximal tubular cell survival, proliferation, or apoptosis, unlike global NF-κB inhibition. In vivo, NEMO-binding peptide treatment protected against ischemic AKI. Finally, NEMO-binding peptide attenuated recombinant PAD4-mediated exacerbation of ischemic AKI, renal tubular inflammation, and apoptosis. Taken together, our results show that PAD4 exacerbates ischemic AKI and inflammation by promoting renal tubular NF-κB activity and inflammation via NEMO citrullination. Targeting NEMO activation may serve as a potential therapy for this devastating clinical problem.

Maternal malnourishment induced upregulation of fetuin-B blunts nephrogenesis in the low birth weight neonate.

Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.

Kidney dysfunction in the low-birth weight murine adult: implications of oxidative stress.

Maternal undernutrition (MUN) during pregnancy leads to low-birth weight (LBW) neonates that have a reduced kidney nephron endowment and higher morbidity as adults. Using a severe combined caloric and protein-restricted mouse model of MUN to generate LBW mice, we examined the progression of renal insufficiency in LBW adults. Through 6 mo of age, LBW males experienced greater albuminuria (ELISA analysis), a more rapid onset of glomerular hypertrophy, and a worse survival rate than LBW females. In contrast, both sexes experienced a comparable progressive decline in renal vascular density (immunofluorescence analysis), renal blood flow (Laser-Doppler flowmetry analysis), glomerular filtration rate (FITC-sinistrin clearance analysis), and a progressive increase in systemic blood pressure (measured via tail-cuff method). Isolated aortas from both LBW sexes demonstrated reduced vasodilation in response to ACh, indicative of reduced nitric oxide bioavailability and endothelial dysfunction. ELISA and immunofluorescence analysis revealed a significant increase of circulating reactive oxygen species and NADPH oxidase type 4 (NOX4) expression in both LBW sexes, although these increases were more pronounced in males. Although more effective in males, chronic tempol treatment did improve all observed pathologies in both sexes of LBW mice. Chronic NOX4 inhibition with GKT137831 was more effective than tempol in preventing pathologies in LBW males. In conclusion, despite some minor differences, LBW female and male adults have a reduced nephron endowment comparable with progressive renal and vascular dysfunction, which is associated with increased oxidative stress and subsequent endothelial dysfunction. Tempol treatment and/or NOX4 inhibition attenuates renal and vascular dysfunction in LBW adults.

HMGB1 redox during sepsis.

During sepsis, the alarmin HMGB1 is released from tissues and promotes systemic inflammation that results in multi-organ damage, with the kidney particularly susceptible to injury. The severity of inflammation and pro-damage signaling mediated by HMGB1 appears to be dependent on the alarmin's redox state. Therefore, we examined HMGB1 redox in kidney cells during sepsis. Using intravital microscopy, CellROX labeling of kidneys in live mice indicated increased ROS generation in the kidney perivascular endothelium and tubules during lipopolysaccharide (LPS)-induced sepsis. Subsequent CellROX and MitoSOX labeling of LPS-stressed endothelial and kidney proximal tubule cells demonstrated increased ROS generation in these cells as sepsis worsens. Consequently, HMGB1 oxidation increased in the cytoplasm of kidney cells during its translocation from the nucleus to the circulation, with the degree of oxidation dependent on the severity of sepsis, as measured in in vivo mouse samples using a thiol assay and mass spectrometry (LC-MS/MS). The greater the oxidation of HMGB1, the greater the ability of the alarmin to stimulate pro-inflammatory cyto-/chemokine release (measured by Luminex Multiplex) and alter mitochondrial ATP generation (Luminescent ATP Detection Assay). Administration of glutathione and thioredoxin inhibitors to cell cultures enhanced HMGB1 oxidation during sepsis in endothelial and proximal tubule cells, respectively. In conclusion, as sepsis worsens, ROS generation and HMGB1 oxidation increases in kidney cells, which enhances HMGB1's pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state.

Low birth weight is associated with impaired murine kidney development and function.

BackgroundLow birth weight (LBW) neonates have impaired kidney development that leaves them susceptible to kidney disease and hypertension during adulthood. The study here identifies events that blunt nephrogenesis and kidney development in the murine LBW neonate.MethodsWe examined survival, kidney development, GFR, gene expression, and cyto-/chemokines in the LBW offspring of malnourished (caloric and protein-restricted) pregnant mice.ResultsMalnourished pregnant mothers gave birth to LBW neonates that had 40% reduced body weight and 54% decreased survival. Renal blood perfusion was reduced by 37%, whereas kidney volume and GFR were diminished in the LBW neonate. During gestation, the LBW neonatal kidney had 2.2-fold increased apoptosis, 76% decreased SIX2+ progenitor cells, downregulation of mesenchymal-to-epithelial signaling factors Wnt9b and Fgf8, 64% less renal vesicle formation, and 32% fewer nephrons than controls. At birth, increased plasma levels of IL-1ß, IL-6, IL-12(p70), and granulocyte-macrophage colony-stimulating factor in the LBW neonate reduced SIX2+ progenitor cells.ConclusionIncreased pro-inflammatory cytokines in the LBW neonate decrease SIX2+ stem cells in the developing kidney. Reduced renal stem cells (along with the decreased mesenchymal-to-epithelial signaling) blunt renal vesicle generation, nephron formation, and kidney development. Subsequently, the mouse LBW neonate has reduced glomeruli volume, renal perfusion, and GFR.

The Secretome of Hydrogel-Coembedded Endothelial Progenitor Cells and Mesenchymal Stem Cells Instructs Macrophage Polarization in Endotoxemia.

UNLABELLED: : We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid-based (HA)-hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC-MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA-hydrogel-embedded delivery of cells to LPS-induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC-MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned medium changed their polarization to anti-inflammatory M2. Incubation of coembedded EPCs-MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti-inflammatory interleukin (IL)-4 and IL-10. EPC-MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs-MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned. SIGNIFICANCE: This report provides insight into a new therapeutic approach for treatment of sepsis and provides a new and improved strategy using hydrogels for the delivery of stem cells to treat sepsis and, potentially, other injuries and/or diseases. The delivery of two different stem cell lines (endothelial progenitor cells and mesenchymal stem cells; delivered alone and together) embedded in a protective bioengineered scaffolding (hydrogel) offers many therapeutic benefits for the treatment of sepsis. This study shows how hydrogel-delivered stem cells elicit their effects and how hydrogel embedding enhances the therapeutic efficacy of delivered stem cells. Hydrogel-delivered stem cells influence the components of the overactive immune system during sepsis and work to counterbalance the release of many proinflammatory and prodamage substances from immune cells, thereby improving the associated vascular and kidney damage.

High-mobility group box 1 is a novel deacetylation target of Sirtuin1.

High-mobility group box 1 (HMGB1) undergoes acetylation, nuclear-to-cytoplasmic translocation, and release from stressed kidneys, unleashing a signaling cascade of events leading to systemic inflammation. Here, we tested whether the deacetylase activity of Sirtuin1 (SIRT1) participates in regulating nuclear retention of HMGB1 to ultimately modulate damage signaling initiated by HMGB1 secretion during stress. When immunoprecipitated acetylated HMGB1 was incubated with SIRT1, HMGB1 acetylation decreased by 57%. Proteomic analysis showed that SIRT1 deacetylates HMGB1 at four lysine residues (55, 88, 90, and 177) within the proinflammatory and nuclear localization signal domains of HMGB1. Genetic ablation or pharmacological inhibition of SIRT1 in endothelial cells increased HMGB1 acetylation and translocation. In vivo, deletion of SIRT1 reduced nuclear HMGB1 while increasing its acetylation and release into circulation during basal and ischemic conditions, causing increased renal damage. Conversely, resveratrol pretreatment led to decreased HMGB1 acetylation, its nuclear retention, decreased systemic release, and reduced tubular damage. Thus, a vicious cycle is set into motion in which the inflammation-induced repression of SIRT1 disables deacetylation of HMGB1, facilitates its nuclear-to-cytoplasmic translocation, and systemic release, thereby maintaining inflammation.

Messengers without borders: mediators of systemic inflammatory response in AKI.

The list of signals sent by an injured organ to systemic circulation, so-called danger signals, is growing to include multiple metabolites and secreted moieties, thus revealing a highly complex and integrated network of interlinked systemic proinflammatory and proregenerative messages. Emerging new data indicate that, apart from the well established local inflammatory response to AKI, danger signaling unleashes a cascade of precisely timed, interdependent, and intensity-gradated mediators responsible for development of the systemic inflammatory response. This fledgling realization of the importance of the systemic inflammatory response to the localized injury and inflammation is at the core of this brief overview. It has a potential to explain the additive effects of concomitant diseases or preexisting chronic conditions that can prime the systemic inflammatory response and exacerbate it out of proportion to the actual degree of acute kidney damage. Although therapies for ameliorating AKI per se remain limited, a potentially powerful strategy that could reap significant benefits in the future is to modulate the intensity of danger signals and consequently the systemic inflammatory response, while preserving its intrinsic proregenerative stimuli.

HMGB1 in renal ischemic injury.

Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H(2)O(2) (oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.

Interaction between uric acid and HMGB1 translocation and release from endothelial cells.

We aimed to investigate the potential relationship between alarmins [acting via Toll-like receptor-4 (TLR4)], uric acid (UA), and high-mobility group box-1 protein (HMGB1) during acute kidney injury. UA, which is significantly increased in the circulation following renal ischemia-reperfusion injury (IRI), was used both in vitro and in vivo as an early response-signaling molecule to determine its ability to induce the secretion of HMGB1 from endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with UA resulted in increased HMGB1 mRNA expression, acetylation of nuclear HMGB1, and its subsequent nuclear-cytoplasmic translocation and release into the circulation, as determined by Western blotting and immunofluorescence. Treatment of HUVEC with UA and a calcium mobilization inhibitor (TMB-8) or a MEK/Erk pathway inhibitor (U0126) prevented translocation of HMGB1 from the nucleus, resulting in reduced cytoplasmic and circulating levels of HMGB1. Once released, HMGB1 in autocrine fashion promoted further HMGB1 release while also stimulating NF-κB activity and increased angiopoietin-2 expression and protein release. Transfection of HUVEC with TLR4 small interfering (si) RNA reduced HMGB1 levels during UA and HMGB1 treatment. In summary, UA after IRI mediates the acetylation and release of HMGB1 from endothelial cells by mechanisms that involve calcium mobilization, the MEK/Erk pathway, and activation of TLR4. Once released, HMGB1 promotes its own further cellular release while acting as an autocrine and paracrine to activate both proinflammatory and proreparative mediators.

Hydrogel-embedded endothelial progenitor cells evade LPS and mitigate endotoxemia.

Sepsis and its complications are associated with poor clinical outcomes. The circulatory system is a well-known target of lipopolysaccharide (LPS). Recently, several clinical studies documented mobilization of endothelial progenitor cells (EPCs) during endotoxemia, with the probability of patients' survival correlating with the rise in circulating EPCs. This fact combined with endotoxemia-induced vascular injury led us to hypothesize that the developing functional EPC incompetence could impede vascular repair and that adoptive transfer of EPCs could improve hemodynamics in endotoxemia. We used LPS injection to model endotoxemia. EPCs isolated from endotoxemic mice exhibited impaired clonogenic potential and LPS exerted Toll-like receptor 4-mediated cytotoxic effects toward EPCs, which was mitigated by embedding them in hyaluronic acid (HA) hydrogels. Therefore, intact EPCs were either delivered intravenously or embedded within pronectin-coated HA hydrogels. Adoptive transfer of EPCs in LPS-injected mice improved control of blood pressure and reduced hepatocellular and renal dysfunction. Specifically, EPC treatment was associated with the restoration of renal microcirculation and improved renal function. EPC therapy was most efficient when cells were delivered embedded in HA hydrogel. These findings establish major therapeutic benefits of adoptive transfer of EPCs, especially when embedded in HA hydrogels, in mice with LPS-induced endotoxemia, and they argue that hemodynamic and renal abnormalities of endotoxemia are in significant part due to developing incompetence of endogenous EPCs.