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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., Graphical abstract fx1, Highlights • Endotoxins (LPS) increase cellular oxidative stress during sepsis. • During its translocation, HMGB1 gets oxidized in the cytoplasm. • Thioredoxin and glutathione keep HMGB1 in a reduced redox state during sepsis. • HMGB1 oxidation enhances its stimulation of inflammatory cyto-/chemokine release.

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.

Significance: A common link between all forms of acute and chronic kidney injuries, regardless of species, is enhanced generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during injury/disease progression. While low levels of ROS and RNS are required for prosurvival signaling, cell proliferation and growth, and vasoreactivity regulation, an imbalance of ROS and RNS generation and elimination leads to inflammation, cell death, tissue damage, and disease/injury progression. Recent Advances: Many aspects of renal oxidative stress still require investigation, including clarification of the mechanisms which prompt ROS/RNS generation and subsequent renal damage. However, we currently have a basic understanding of the major features of oxidative stress pathology and its link to kidney injury/disease, which this review summarizes. Critical Issues: The review summarizes the critical sources of oxidative stress in the kidney during injury/disease, including generation of ROS and RNS from mitochondria, NADPH oxidase, and inducible nitric oxide synthase. The review next summarizes the renal antioxidant systems that protect against oxidative stress, including superoxide dismutase and catalase, the glutathione and thioredoxin systems, and others. Next, we describe how oxidative stress affects kidney function and promotes damage in every nephron segment, including the renal vessels, glomeruli, and tubules. Future Directions: Despite the limited success associated with the application of antioxidants for treatment of kidney injury/disease thus far, preventing the generation and accumulation of ROS and RNS provides an ideal target for potential therapeutic treatments. The review discusses the shortcomings of antioxidant treatments previously used and the potential promise of new ones. Antioxid. Redox Signal. 25, 119–146.

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.

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.

We sought to characterize a minor renal cryoinjury that allows investigation into renal damage processes and subsequent endogenous repair mechanisms. To achieve this, we induced a small cryoinjury to mice, in which the transient superficial application of a liquid nitrogen-cooled cryoprobe to the exposed kidney induces a localized lesion that did not impair renal function. The resulting cryoinjury was examined by immunohistochemistry and Laser-Doppler flowmetry. Within hours of cryoinjury induction, tubular and vascular necrotic damage was observed, while blood flow in the directly injured area was reduced by 65%. The injured area demonstrated a peak in tubular and perivascular cell proliferation at 4 days postinjury, while apoptosis and fibrosis peaked at day 7. Infiltration of macrophages into the injury was first observed at day 4, and peaked at day 7. Vascular density in the direct injured area was lowest at day 7. As compared to the direct injured area, the (peripheral) penumbral region surrounding the directly injured area demonstrated enhanced cellular proliferation (2.5-6-fold greater), vascular density (1.6-2.9 fold greater) and blood perfusion (twofold greater). After 4 weeks, the area of damage was reduced by 73%, fibrosis decreased by 50% and blood flow in the direct injured area was reestablished by 63% with almost complete perfusion restoration in the injury's penumbral region. In conclusion, kidney cryoinjury provides a flexible facile model for the study of renal damage and associated endogenous repair processes.