David A. Allen

Erythropoietin Protects the Kidney against the Injury and Dysfunction Caused by Ischemia-Reperfusion

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.

High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases

Diabetic nephropathy is the leading cause of end‐stage renal disease in the Western world. Poor glycemic control contributes to the development of diabetic nephropathy, but the mechanisms underlying high glucose‐induced tissue injury are not fully understood. In the present study, the effect of high glucose on a proximal tubular epithelial cell (PTEC) line was investigated. Reactive oxygen species (ROS) were detected using the fluorescent probes dichlorofluorescein diacetate, dihydrorhodamine 123, and 2,3‐diaminonapthalene. Peroxynitrite (ONOO−) generation and nitrite concentrations were increased after 24 h of high glucose treatment (P<0.05). LLC‐PK1 cells exposed to high d‐glucose (25 mM) for up to 48 h had increased DNA fragmentation (P<0.01), caspase‐3 activity (P<0.001), and annexin‐V staining (P<0.05) as well as decreased expression of XIAP when compared with controls (5 mM d‐glucose). The ONOO− scavenger ebselen reduced DNA fragmentation and caspase‐3 activity as well as the high glucose‐induced nitrite production and DCF fluorescence. High glucose‐induced DNA fragmentation was completely prevented by an inhibitor of caspase‐3 (P<0.01) and a pan‐caspase inhibitor (P<0.001). Caspase inhibition did not affect ROS generation. This study, in a PTEC line, demonstrates that high glucose causes the generation of ONOO−, leading to caspase‐mediated apoptosis. Ebselen and a caspase‐3 inhibitor provided significant protection against high glucose‐mediated apoptosis, implicating ONOO− as a proapoptotic ROS in early diabetic nephropathy.

Gsk-3β inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat

Objective:Serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in regulation of many cell functions, but its role in regulation of inflammatory response is unknown. Here we investigate the effects of GSK-3β inhibition on organ injury/dysfunction caused by lipopolysaccharide or coadministration of lipopolysaccharide and peptidoglycan in the rat. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Ninety-nine anesthetized male Wistar rats. Interventions:Study 1: Rats received either intravenous Escherichia coli lipopolysaccharide (6 mg/kg) or vehicle (1 mL/kg; saline). Study 2: Rats received either intravenous E. coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle. The potent and selective GSK-3β inhibitors TDZD-8 (1 mg/kg intravenously), SB216763 (0.6 mg/kg intravenously), and SB415286 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 mins before lipopolysaccharide or lipopolysaccharide and peptidoglycan. Measurements and Main Results:Endotoxemia resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), aspartate aminotransferase, alanine aminotransferase (markers for hepatocellular injury), lipase (indicator of pancreatic injury), and creatine kinase (indicator of neuromuscular injury). Coadministration of lipopolysaccharide and peptidoglycan resulted in hepatocellular injury and renal dysfunction. All GSK-3β inhibitors attenuated the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan. GSK-3β inhibition reduced the Ser536 phosphorylation of nuclear factor-&kgr;B subunit p65 and the messenger RNA expression of nuclear factor-&kgr;B-dependent proinflammatory mediators but had no effect on the nuclear factor-&kgr;B/DNA binding activity in the lung. GSK-3β inhibition reduced the increase in nuclear factor-&kgr;B p65 activity caused by interleukin-1 in human embryonic kidney cells in vitro. Conclusions:The potent and selective GSK-3β inhibitors TDZD-8, SB216763, and SB415286 reduced the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan in the rat. We propose that GSK-3β inhibition may be useful in the therapy of the organ injury/dysfunction associated with sepsis, shock, and other diseases associated with local or systemic inflammation.

Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis:

Calpain and caspase are families of cysteine proteases that have important roles in the initiation, regulation and execution of cell death. The function of both groups of proteases in the progression of apoptotic and necrotic pathways is presented here in the context of a concise overview of regulated cell death. Many of the morphological differences between apoptotic and necrotic processes are thought to be as a consequence of the action of cysteine proteases. Recent studies suggest that caspase and calpain cascades are tightly interrelated and an appreciation of how these proteases cross-talk should enable a greater understanding of how the boundaries between apoptotic and necrotic cell death have become blurred. Furthermore, an assessment of the contribution that caspase and calpain make to human physiology and pathology is provided, with a description of how these proteases can be detected and quantified. Lastly, an evaluation is made of how caspase and calpain activation might be exploited diagnostically.

Dexamethasone Ameliorates Renal Ischemia-Reperfusion Injury

In the setting of renal ischemia-reperfusion injury (IRI), the effect and mechanism of action of glucocorticoids are not well understood. In rat renal IRI, a single dose of dexamethasone administered before ischemia, or at the onset of reperfusion, ameliorated biochemical and histologic acute kidney injury after 24 h. Dexamethasone upregulated Bcl-xL, downregulated ischemia-induced Bax, inhibited caspase-9 and caspase-3 activation, and reduced apoptosis and necrosis of proximal tubular cells. In addition, dexamethasone decreased the number of infiltrating neutrophils and ICAM-1. We observed the protective effect of dexamethasone in neutrophil-depleted mice, suggesting a neutrophil-independent mechanism. In vitro, dexamethasone protected human kidney proximal tubular (HK-2) cells during serum starvation and IRI-induced apoptosis, but inhibition of MEK 1/2 abolished its anti-apoptotic effects in these conditions. Dexamethasone stimulated rapid and transient phosphorylation of ERK 1/2, which required the presence of the glucocorticoid receptor and was independent of transcriptional activity. In summary, in the setting of renal ischemia-reperfusion injury, dexamethasone directly protects against kidney injury by a receptor-dependent, nongenomic mechanism.

Insulin reduces the multiple organ injury and dysfunction caused by coadministration of lipopolysaccharide and peptidoglycan independently of blood glucose: Role of glycogen synthase kinase-3β inhibition*

Objective:Insulin reduces morbidity and mortality among critically ill patients, but the molecular mechanisms of its effect remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3, which may play an important role in systemic inflammation and shock. Here we investigate the role of blood glucose and glycogen synthase kinase-3&bgr; inhibition in the protective effect of insulin on the organ injury/dysfunction associated with excessive systemic inflammation. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Eighty-five anesthetized Wistar rats. Interventions:Rats received Escherichia coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle intravenously. Insulin (1.4 units/kg intravenously) was administered in the absence or presence of continuous glucose administration (4.5 mg/kg/hr intravenously) either prophylactically or therapeutically. The potent and selective glycogen synthase kinase-3&bgr; inhibitor TDZD-8 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered either prophylactically or therapeutically. Measurements and Main Results:Coadministration of lipopolysaccharide and peptidoglycan resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), alanine aminotransferase, and aspartate aminotransferase (indicators of liver injury) at 6 hrs. Insulin or TDZD-8 similarly attenuated the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan when given either prophylactically or therapeutically. Continuous glucose administration had no effect on blood glucose levels or organ injury/dysfunction at 6 hrs. Treatment with insulin or TDZD-8 reduced the plasma levels of the proinflammatory cytokine interleukin-1&bgr;. In vitro, insulin or TDZD-8 caused similar reductions in the nuclear factor-&kgr;B p65 activity and similar increases in the phosphorylation of Ser9 of glycogen synthase kinase-3&bgr;. Conclusions:Therapy with insulin or the potent and selective glycogen synthase kinase-3&bgr; inhibitor TDZD-8 reduced the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan in the rat. We propose that the inhibitory effect of insulin on the activity of glycogen synthase kinase-3&bgr; contributes to the protective effect of insulin against the organ injury/dysfunction caused by excessive systemic inflammation independently of any effects on blood glucose.