Steven Harwood

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.

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.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF‐κB (kidney), expression of iNOS and COX‐2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF‐κB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF‐κB to DNA and the degradation of IκBα, IKBβ, and IκBε. Selective inhibition of iNOS activity with L‐NIL reduced the circulatory failure and liver injury, while selective inhibition of COX‐2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF‐κB and thus prevention of the expression of NFκB‐dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX‐2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.—McDonald, M. C., Mota‐Filipe, H., Paul, A., Cuzzocrea, S., Abdelrahman, M., Harwood, S., Plevin, R., Chatterjee, P. K., Yaqoob, M. M., Thiemermann, C. Calpain inhibitor I reduces the activation of nuclear factor‐κB and organ injury/dysfunction in hemorrhagic shock. FASEB J. 15, 171–186 (2001)

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.

Ischemic Conditioning Protects the Uremic Heart in a Rodent Model of Myocardial Infarction

Background— Outcomes after acute myocardial infarction in patients with chronic kidney disease are extremely poor. Ischemic conditioning techniques are among the most powerful cytoprotective strategies discovered to date. However, experimental data suggest that comorbidity may attenuate the protective effects of ischemic conditioning. Methods and Results— We conducted investigations into the effects of chronic uremia on myocardial infarct size and the protective effects of ischemic preconditioning (IPC), remote ischemic preconditioning, and ischemic postconditioning in 2 rodent models of chronic uremia. In addition, a limited investigation into the signaling mechanisms involved in cardioprotection after IPC was performed in both uremic and nonuremic animals. Myocardial infarct size was increased in uremic animals, but all 3 conditioning strategies (IPC, remote IPC, ischemic postconditioning) proved highly efficacious in reducing myocardial infarct size (relative reduction, 86%, 39%, and 65% [P<0.005, P<0.05, and P<0.05], respectively). Moreover, some protocols (IPC and ischemic postconditioning) appeared to be more effective in uremic than in sham (nonuremic) animals. Analysis of the signaling mechanisms revealed that components of both the reperfusion injury salvage kinase and survivor activating factor enhancement pathways were similarly upregulated in both uremic and nonuremic animals after an IPC stimulus. Conclusion— Conditioning strategies may present the best opportunity to improve outcomes for patients with chronic kidney disease after an acute coronary syndrome.

Ergocalciferol and microcirculatory function in chronic kidney disease and concomitant vitamin d deficiency: an exploratory, double blind, randomised controlled trial.

Background and Objectives Vitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function. Study Design We conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3–4 and concomitant vitamin D deficiency (<16 ng/dl) who received oral ergocalciferol (50,000 IU weekly for one month followed by 50,000 IU monthly) or placebo over 6 months. The primary outcome was change in microcirculatory function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Secondary endpoints were tissue advanced glycation end products, sublingual functional capillary density and flow index as well as macrovascular parameters. Parallel in vitro experiments were conducted to determine the effect of ergocalciferol on cultured human endothelial cells. Results Twenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner. Conclusions Ergocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase. Trial Registration Clinical trials.gov NCT00882401

Ouabain-induced cell signaling.

When endogenous ouabain was first isolated from human plasma in 1991, many expressed doubts that such a compound could be endogenous to humans because of its unusual structure, its unknown synthesis, and its unidentified site of origin. Furthermore, the relevance of human ouabain was questioned because of its apparently low (< or =nmol/L) circulating concentration. Since then, much progress has been made on the origin and synthesis of endogenous ouabain, but perhaps the most significant finding is that nanomolar concentrations of ouabain can induce numerous signal transduction events in both primary and immortalized cultures of cells. Here we analyze the effects of low ouabain-induced signals including cell proliferation, calcium mobilization, cell cytotoxicity, apoptosis, mitogen-activated protein kinase (MAPK) activation and other signaling pathways. Furthermore, we consider how these low dose ouabain-induced events might enable a putative role for human endogenous ouabain to be assigned.

Inosine monophosphate dehydrogenase polymorphisms and renal allograft outcome.

Background Interindividual variation in inosine monophosphate dehydrogenase (IMPDH) enzyme activity and adverse effects caused by mycophenolate mofetil (MMF) inhibition may be genetically determined, and if so, transplant recipients should receive personalized dosing regimens of MMF, which would maximize efficacy and minimize toxicity. Some studies have demonstrated a relationship between the single nucleotide polymorphism and the risk of acute rejection with IMPDH I variants rs2278293 and rs2278294 and IMPDH II variant rs11706052, whereas others have failed to exhibit an effect. The aim of this work was to investigate the influence of these polymorphisms on acute rejection rates, graft survival and function, and MMF doses in a large cohort of patients. Methods A random sample of 1040 recipients from the Collaborative Transplant Study DNA bank was genotyped for the variants IMPDH I rs2278293 and rs2278294 and IMPDH II rs11706052. Results The presence of the T (rs2278293) and G alleles (rs2278294) in the IMPDH I variants and carriage of the G allele (rs11706052) in the IMPDH II variant did not increase the risk of rejection or affect graft function by 1 year after transplantation. There was no association with MMF dose tolerated at 1 year. Furthermore, these polymorphisms did not impact graft or patient survival at 5 years. Conclusion This study represents the largest cohort of patients with the longest follow-up to date and does not support previous evidence for an association between these IMPDH variants and renal allograft rejection and graft survival.

Plasma OLC is elevated in mild experimental uremia but is not associated with hypertension

BACKGROUND Little is known about the renal handling of endogenous ouabain-like compound (OLC). The aim of this study was to determine the normal renal clearance of OLC and the effect of mild experimental uremia on plasma OLC and its clearance. METHODS Male Wistar rats were studied 8 weeks after subtotal (5/6th) nephrectomy (n = 8) and compared with a control sham-operated group (n = 8). RESULTS Plasma creatinine and OLC were higher in uremic animals compared with controls (creatinine 76+/-5.6 micromol/L v 45+/-9.6 micromol/L, respectively, P < .00005; OLC 195+/-62 pmol/L v 121+/-62 pmol/L, P < .02). Creatinine clearance and OLC clearance were lower in uremic animals compared with controls (creatinine 1.06+/-0.12 mL/min v 1.58+/-0.32 mL/min, respectively, P < .002; OLC 23.6+/-10.4 microL/min v 33.2+/-11.4 microL/min, P < .05). There were no significant differences (all P > .05) between the uremic and control groups in the fractional clearance of OLC (uremic 2.3%+/-1.0% v control 2.2%+/-1.0%), OLC excretion rate (uremic 6.2+/-2.4 pmol/24 h v control 5.0+/-1.1 pmol/24 h) or in the mean systolic blood pressure (BP) (uremic 132+/-13 mm Hg v control 126+/-3 mm Hg). The amount of OLC excreted per unit of functioning nephron mass was 78% higher in uremic animals than in controls. The rate of tubular absorption varied linearly with filtered load, did not differ between groups, and showed no evidence of saturation. CONCLUSIONS The kidneys are an important excretion route for plasma OLC and moderate but significant increases may occur without inducing hypertension in the short term. The low fractional clearance of OLC is most likely due to tubular absorption and/or catabolism.