May M. Rabadi

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 EPs 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.

Endothelial Sirtuin 1 Deficiency Perpetrates Nephrosclerosis through Downregulation of Matrix Metalloproteinase-14: Relevance to Fibrosis of Vascular Senescence

Sirtuin 1 (SIRT1) depletion in vascular endothelial cells mediates endothelial dysfunction and premature senescence in diverse cardiovascular and renal diseases. However, the molecular mechanisms underlying these pathologic effects remain unclear. Here, we examined the phenotype of a mouse model of vascular senescence created by genetically ablating exon 4 of Sirt1 in endothelial cells (Sirt1(endo-/-)). Under basal conditions, Sirt1(endo-/-) mice showed impaired endothelium-dependent vasorelaxation and angiogenesis, and fibrosis occurred spontaneously at low levels at an early age. In contrast, induction of nephrotoxic stress (acute and chronic folic acid-induced nephropathy) in Sirt1(endo-/-) mice resulted in robust acute renal functional deterioration followed by an exaggerated fibrotic response compared with control animals. Additional studies identified matrix metalloproteinase-14 (MMP-14) as a target of SIRT1. In the kidneys of Sirt1(endo-/-) mice, impaired angiogenesis, reduced matrilytic activity, and retention of the profibrotic cleavage substrates tissue transglutaminase and endoglin accompanied MMP-14 suppression. Furthermore, restoration of MMP-14 expression in SIRT1-depeleted mice improved angiogenic and matrilytic functions of the endothelium, prevented renal dysfunction, and attenuated nephrosclerosis. Our findings establish a novel mechanistic molecular link between endothelial SIRT1 depletion, downregulation of MMP-14, and the development of nephrosclerosis.

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.

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.

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 pro-inflammatory 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.

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.

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

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.

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-1β, 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.

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 alarmins 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 HMGB1s pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state.

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.