Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells

Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells. Following nephrotoxic injury, renal repair is dependent on tubular regeneration. In the case of myoglobinuric acute renal failure (ARF), persistence of myoglobin within tubular cells, or sublethal injury sustained at the height of exposure to it, might retard this process. To test this hypothesis, a human proximal tubular cell line (HK-2) was cultured for 24 hours in the absence or presence of clinically relevant myoglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myoglobin removal, lethal cell injury (vital dye uptake), lipid peroxidation, and DNA damage (alkaline unwinding assay) were assessed. The extent of cell proliferation was estimated over the next four days by a tetrazolium based (MTT) assay and by determining total intracellular LDH. Myoglobin's effects on protein and DNA synthesis were also assessed (35S-methionine and bromodeoxyuridine incorporation, respectively). Myoglobin induced dose-dependent lipid peroxidation (malondialdehyde generation) and cell death (up to 80% vital dye uptake with the 4 mg/ml challenge). Although 1 mg/ml myoglobin caused no cell death, it induced nearly complete growth arrest. This lasted for approximately three days following myoglobin removal from the media. Neither of two control proteins (albumin; lysozyme) nor a second nephrotoxin (gentamicin; 1 mg/ml) reproduced this effect. The 1 mg/ml myoglobin challenge caused an 80 to 90% depression in protein and DNA synthesis. It also induced significant DNA damage, as assessed by the alkaline unwinding assay (P < 0.01). Iron chelation therapy (deferoxamine) mitigated myoglobin-induced cell killing. However, its addition following myoglobin loading worsened HK-2 outgrowth by exerting a direct anti-proliferative effect. These results indicate that: (1) sublethal myoglobin toxicity can induce transient proximal tubular cell growth arrest, potentially slowing recovery from ARF; (2) this effect correlates with, and could result from, heme-induced DNA damage and a blockade in DNA/protein synthesis; and (3) deferoxamine can inhibit proximal tubular cell proliferation. This possibility needs to be considered in designing clinical trials with DFO for myohemoglobinuric ARF

Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport

Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport. The purpose of this study was to gain direct insights into mechanisms by which myoglobin induces proximal tubular cell death. To avoid confounding systemic and hemodynamic influences, an in vitro model of myoglobin cytotoxicity was employed. Human proximal tubular (HK-2) cells were incubated with 10 mg/ml myoglobin, and after 24 hours the lethal cell injury was assessed (vital dye uptake; LDH release). The roles played by heme oxygenase (HO), cytochrome p450, free iron, intracellular Ca2+, nitric oxide, H2O2, hydroxyl radical (·OH), and mitochondrial electron transport were assessed. HO inhibition (Sn protoporphyrin) conferred almost complete protection against myoglobin cytotoxicity (92% vs. 22% cell viability). This benefit was fully reproduced by iron chelation therapy (deferoxamine). Conversely, divergent cytochrome p450 inhibitors (cimetidine, aminobenzotriazole, troleandomycin) were without effect. Catalase induced dose dependent cytoprotection, virtually complete, at a 5000 U/ml dose. Conversely, ·OH scavengers (benzoate, DMTU, mannitol), xanthine oxidase inhibition (oxypurinol), superoxide dismutase, and manipulators of nitric oxide expression (L-NAME, L-arginine) were without effect. Intracellular (but not extracellular) calcium chelation (BAPTA-AM) caused ∼50% reductions in myoglobin-induced cell death. The ability of Ca2+ (plus iron) to drive H2O2 production (phenol red assay) suggests one potential mechanism. Blockade of site 2 (antimycin) and site 3 (azide), but not site 1 (rotenone), mitochondrial electron transport significantly reduced myoglobin cytotoxicity. Inhibition of Na,K-ATPase driven respiration (ouabain) produced a similar protective effect. We conclude that: (1) HO-generated iron release initiates myoglobin toxicity in HK-2 cells; (2) myoglobin, rather than cytochrome p450, appears to be the more likely source of toxic iron release; (3) H2O2 generation, perhaps facilitated by intracellular Ca2+/iron, appears to play a critical role; and (4) cellular respiration/terminal mitochondrial electron transport ultimately helps mediate myoglobin's cytotoxic effect. Formation of poorly characterized toxic iron/H2O2-based reactive intermediates at this site seems likely to be involved

Decreased expression of mitochondrial-derived H2O2 and hydroxyl radical in cytoresistant proximal tubules

Decreased expression of mitochondrial-derived H2O2 and hydroxyl radical in cytoresistant proximal tubules. Increased production of reactive oxygen metabolites (ROM) can contribute to the initiation phase of nephrotoxic and ischemic acute renal failure (ARF). However, whether altered ROM expression also exists during the maintenance phase of ARF has not been adequately assessed. Since diverse forms of tubular injury can initiate a “cytoresistant state,” this study tested whether a down-regulation of ROM expression might develop in the aftermath of acute tubular damage, potentially limiting renal susceptibility to further attack. To test this hypothesis, rats were subjected to either mild myohemoglobinuria (glycerol injection) or bilateral ureteral obstruction and 24 hours later, cytoresistant proximal tubular segments (PTS) were isolated to assess ROM expression. PTS from sham operated rats were used to establish normal values. Both sets of cytoresistant PTS manifested ∼ 75% reductions in H2O2 levels, as assessed by the phenol red/horseradish peroxidase technique (P < 0.01 to 0.001). A 40% reduction in hydroxyl radical (˙OH) levels was also observed (salicylate trap method), thereby substantiating decreased oxidant stress in cytoresistant PTS. Catalase, glutathione peroxidase, and free iron levels were comparable in control and cytoresistant PTS, suggesting that decreased H2O2 production (such as by mitochondria) was the cause of the decreased oxidant stress. To test this latter hypothesis, H2O2 expression by control and cytoresistant PTS was assessed in the presence of respiratory chain inhibitors. Although site 1 and site 3 inhibition markedly suppressed H2O2 production in control PTS, they had no impact on H2O2 production in cytoresistant PTS, implying that production at these sites was already maximally suppressed. Correlates of the decreased mitochondrial H2O2 production were improvements in cell energetics (increased ATP/ADP ratios with Na ionophore treatment) and ∼ 40 to 90% increases in PTS/renal cortical glutathione content. We conclude that: (1) proximal tubule H2O2/˙OH expression can be down-regulated during the maintenance phase of ARF; (2) this seemingly reflects a decrease in mitochondrial ROM generation; and (3) the associated improvements in glutathione content and/or cellular energetics could conceivably contribute to a post-injury cytoresistant state

Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury

Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury.BackgroundCholesterol accumulates in renal cortical proximal tubules in response to diverse forms of injury or physiologic stress. However, the fate of triglycerides after acute renal insults is poorly defined. This study sought new insights into this issue.MethodsCD-1 mice were subjected to three diverse models of renal stress: (1) endotoxemia [Escherichia coli lipopolysaccharide (LPS), injection]; (2) ischemia/reperfusion (I/R); or (3) glycerol-induced rhabdomyolysis. Renal cortical, or isolated proximal tubule, triglyceride levels were measured ∼18 hours later. To gain mechanistic insights, triglyceride levels were determined in (1) proximal tubules following exogenous phospholipase A2 (PLA2) treatment; (2) cultured HK-2 cells after mitochondrial blockade (antimycin A) ± serum; or (3) HK-2 cells following “septic” (post-LPS) serum, or exogenous fatty acid (oleate) addition.ResultsEach form of in vivo injury evoked three-to fourfold triglyceride increases in renal cortex and/or proximal tubules. PLA2 treatment of proximal tubules evoked acute, dose-dependent, triglyceride formation. HK-2 cell triglyceride levels rose with antimycin A. With serum present, antimycin A induced an exaggerated triglyceride loading state (vs. serum alone or antimycin A alone). “Septic” serum stimulated HK-2 triglyceride formation (compared to control serum). Oleate addition caused striking HK-2 cell triglyceride accumulation. Following oleate washout, HK-2 cells were sensitized to adenosine triphosphate (ATP) depletion or oxidant attack.ConclusionDiverse forms of renal injury induce dramatic triglyceride loading in proximal tubules/renal cortex, suggesting that this is a component of a cell stress response. PLA2 activity, increased triglyceride/triglyceride substrate (e.g., fatty acid) uptake, and possible systemic cytokine (e.g., from LPS) stimulation, may each contribute to this result. Finally, in addition to being a marker of prior cell injury, accumulation of triglyceride (or of its constituent fatty acids) may predispose tubules to superimposed ATP depletion or oxidant attack

Cholesterol ester accumulation: An immediate consequence of acute in vivo ischemic renal injury

Cholesterol ester accumulation: An immediate consequence of acute in vivo ischemic renal injury.BackgroundCholesterol is a major constituent of plasma membranes, and recent evidence indicates that it is up-regulated during the maintenance phase of acute renal failure (ARF). However, cholesterol's fate and that of the cholesterol ester (CE) cycle [shuttling between free cholesterol (FC) and CEs] during the induction phase of ARF have not been well defined. The present studies sought to provide initial insights into these issues.MethodsFC and CE were measured in mouse renal cortex after in vivo ischemia (15 and 45 minutes)/reperfusion (0 to 120 minutes) and glycerol-induced myoglobinuria (1 to 2 hours). FC/CE were also measured in (1) cultured human proximal tubule (HK-2) cells three hours after ATP depletion and in (2) isolated mouse proximal tubule segments (PTSs) subjected to plasma membrane damage (with cholesterol oxidase, sphingomyelinase, phospholipase A2, or cytoskeletal disruption with cytochalasin B). The impact of cholesterol synthesis inhibition (with mevastatin) and FC traffic blockade (with progesterone) on injury-evoked FC/CE changes was also assessed.ResultsIn vivo ischemia caused approximately threefold to fourfold CE elevations, but not FC elevations, that persisted for at least two hours of reperfusion. Conversely, myoglobinuria had no effect. Isolated CE increments were observed in ATP-depleted HK-2 cells. Neither mevastatin nor progesterone blocked this CE accumulation. Plasma membrane injury induced with sphingomyelinase or cholesterol oxidase, but not with phospholipase A2 or cytochalasin B, increased tubule CE content. High CE levels, induced with cholesterol oxidase, partially blocked hypoxic PTS attack.ConclusionsIn vivo ischemia/reperfusion acutely increases renal cortical CE, but not FC, content, indicating perturbed CE/FC cycling. The available data suggest that this could stem from specific types of plasma membrane damage, which then increase FC flux via aberrant pathways to the endoplasmic reticulum, where CE formation occurs. That CE levels are known to inversely correlate with both renal and nonrenal cell injury suggests the potential relevance of these observations to the induction phase of ischemic ARF

HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney

HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney. Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, α3β1 integrin, fibronectin; negative for factor VHI-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 ± iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application

Growth and development alter susceptibility to acute renal injury.

Many of the studies of acute renal injury have been conducted in young mice usually during their rapid growth phase; yet, the impact of age or growth stage on the degree of injury is unknown. To address this issue, we studied three forms of injury (endotoxemic-, glycerol-, and maleate-induced) in mice ranging in age from adolescence (3 weeks) to maturity (16 weeks). The severity of injury within each model significantly correlated with weight and age. We also noticed a progressive age-dependent reduction in renal cholesterol content, a potential injury modifier. As the animals grew and aged they also exhibited stepwise decrements in the mRNAs of HMG CoA reductase and the low density lipoprotein receptor, two key cholesterol homeostatic genes. This was paralleled by decreased amounts of RNA polymerase II and the transcription factor SREBP1/2 at the reductase and lipoprotein receptor gene loci as measured by chromatin immunoprecipitation. Our study shows that the early phase of mouse growth can profoundly alter renal susceptibility to diverse forms of experimental acute renal injury

A function-based typology for Earth’s ecosystems

As the United Nations develops a post-2020 global biodiversity framework for the Convention on Biological Diversity, attention is focusing on how new goals and targets for ecosystem conservation might serve its vision of ‘living in harmony with nature’(1,2). Advancing dual imperatives to conserve biodiversity and sustain ecosystem services requires reliable and resilient generalizations and predictions about ecosystem responses to environmental change and management(3). Ecosystems vary in their biota(4), service provision(5) and relative exposure to risks(6), yet there is no globally consistent classification of ecosystems that reflects functional responses to change and management. This hampers progress on developing conservation targets and sustainability goals. Here we present the International Union for Conservation of Nature (IUCN) Global Ecosystem Typology, a conceptually robust, scalable, spatially explicit approach for generalizations and predictions about functions, biota, risks and management remedies across the entire biosphere. The outcome of a major cross-disciplinary collaboration, this novel framework places all of Earth’s ecosystems into a unifying theoretical context to guide the transformation of ecosystem policy and management from global to local scales. This new information infrastructure will support knowledge transfer for ecosystem-specific management and restoration, globally standardized ecosystem risk assessments, natural capital accounting and progress on the post-2020 global biodiversity framework

Strategies for Creating Engaging Videos for your Course

This workshop will focus on strategies for planning and creating videos for your online and hybrid courses. Learn how to engage your students and use Kaltura to record and deliver videos to your students