Treating Posttransplant Anemia With Erythropoietin Improves Quality of Life but Does Not Affect Progression of Chronic Kidney Disease.

OBJECTIVES: Posttransplant anemia affects 30% to 45% of kidney transplant recipients and is associated with increased morbidity. However, there is lack of evidence about safe hemoglobin levels after erythropoietin treatment. Studies are needed to better understand the potential benefits and risks, as well as to define safe target hemoglobin ranges in these patients. MATERIALS AND METHODS: In this single-center exploratory, open-label randomized controlled trial, kidney trans-plant recipients with anemia 3 months posttransplant were either treated with epoetin beta to a hemoglobin target level of 11.5 to 13.5 g/dL (n = 28) or given no treatment (n = 27). Treatment effects on graft function and health quality of life were assessed. RESULTS: After 2 years, hemoglobin concentrations were significantly higher in the epoetin beta treatment group than in the no treatment group (12.3 ± 0.18 vs 9.99 ± 0.22 g/dL; P < .0001). Estimated glomerular filtration rate, calculated by Modified Diet in Renal Disease 7, declined by 1.7 mL/min (interquartile range, -6 to 4.24) in the epoetin treatment group and by 4.16 mL/min (interquartile range, -12.42 to 2.78) in the no treatment group (P = .32). Rate of progression, determined by estimated glomerular filtration rate slope, was not significantly different between groups (-0.09 ± 0.1 vs -0.12 ± 0.15 mL/min for treated vs not treated; P = .78). Moreover, we observed no significant differences in proteinuria and blood pressure. Treated patients had greater improvements in the vitality and mental health domains of the Medical Outcomes Short Form Health Survey quality of life scores. CONCLUSIONS: Treatment of anemia in kidney transplant recipients to a hemoglobin level of 11.5 to 13.5 g/dL with erythropoietin improves some quality of life scores. The treatment was safe and not associated with adverse outcomes. There were no changes in rate of decline of graft function.

Ergocalciferol improves endothelial vasodilatory and vasoconstrictor function in an in vivo model of mild uraemia.

Endothelial dysfunction and vitamin D deficiency are prevalent in patients with cardiovascular disease (CVD) and chronic kidney disease (CKD). Both are risk factors for cardiovascular events in patients with CKD. No studies have investigated the effect of nutritional forms of vitamin D on endothelial function in earlier stages of CKD, when vascular endothelium may be more amenable to this therapy. We studied the effect of ergocalciferol in a pre-clinical model of mild uraemia. Male Wistar rats underwent either a 5/6th nephrectomy or sham surgery. Four weeks after the final stage of the surgery, these two groups were randomly allocated to placebo or an oral dose of 1000 iu of ergocalcfierol at day 7 and 2 pre sacrifice. Vascular responses to acetylcholine, Spermine NONOate and phenylephrine were determined in aortic rings. Blood pressure, calcium, phosphate and parathyroid hormone were measured in all groups. Ergocalciferol significantly improved the endothelium-dependent responses to acetylcholine and overcame the blunting of the contractile response to phenylephrine seen in uraemic animals. Ergocalciferol improved the contractile response to potassium chloride in uraemic, but not sham animals. All effects occurred independently of changes to calcium, phosphate, parathyroid hormone and systolic blood pressure. There were no differences in endothelium-independent relaxation to Spermine NONOate. In summary, in a model of mild uraemia, ergocalciferol improved vasodilator and vasoconstrictor tone independently of blood pressure and bone mineral parameters suggesting a direct effect of ergocalciferol on the endothelium.

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling.

BACKGROUND: During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. METHODS: We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. RESULTS: Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-ß1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. CONCLUSIONS: Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

Monomeric eNAMPT in the development of experimental diabetes in mice: a potential target for type 2 diabetes treatment.

AIMS/HYPOTHESIS: Serum extracellular nicotinamide phosphoribosyltransferase (eNAMPT) concentrations are elevated in type 2 diabetes. However, the relationship between abnormally elevated serum eNAMPT and type 2 diabetes pathophysiology is unclear. eNAMPT circulates in functionally and structurally distinct monomeric and dimeric forms. Dimeric eNAMPT promotes NAD biosynthesis. The role of eNAMPT-monomer is unclear but it may have NAD-independent proinflammatory effects. However, studies of eNAMPT in type 2 diabetes have not distinguished between monomeric and dimeric forms. Since type 2 diabetes is characterised by chronic inflammation, we hypothesised a selective NAD-independent role for eNAMPT-monomer in type 2 diabetes. METHODS: Two mouse models were used to examine the role of eNAMPT-monomer in type 2 diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10 weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14 days. RESULTS: Serum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3 weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion and the presence of systemic and tissue inflammation, without changes in NAD levels. CONCLUSIONS/INTERPRETATION: We demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes.

Myocardial bioenergetic abnormalities in experimental uremia.

PURPOSE: Cardiac bioenergetics are known to be abnormal in experimental uremia as exemplified by a reduced phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio. However, the progression of these bioenergetic changes during the development of uremia still requires further study and was therefore investigated at baseline, 4 weeks and 8 weeks after partial nephrectomy (PNx). METHODS: A two-stage PNx uremia model in male Wistar rats was used to explore in vivo cardiac and skeletal muscles' bioenergetic changes over time. High-energy phosphate nucleotides were determined by phosphorus-31 nuclear magnetic resonance ((31)P-NMR) and capillary zone electrophoresis. RESULTS: (31)P-NMR spectroscopy revealed lower PCr/ATP ratios in PNx hearts compared to sham (SH)-operated animals 4 weeks after PNx (median values given ± SD, 0.64±0.16 PNx, 1.13±0.31 SH, P<0.02). However, 8 weeks after PNx, the same ratio was more comparable between the two groups (0.84±0.15 PNx, 1.04±0.44 SH, P= not significant), suggestive of an adaptive mechanism. When 8-week hearts were prestressed with dobutamine, the PCr/ATP ratio was again lower in the PNx group (1.08±0.36 PNx, 1.55±0.38 SH, P<0.02), indicating a reduced energy reserve during the progression of uremic heart disease. (31)P-NMR data were confirmed by capillary zone electrophoresis, and the changes in myocardial bioenergetics were replicated in the skeletal muscle. CONCLUSION: This study provides evidence of the changes that occur in myocardial energetics in experimental uremia and highlights how skeletal muscle bioenergetics mirror those found in the cardiac tissue and so might potentially serve as a practical surrogate tissue during clinical cardiac NMR investigations.

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.

Aging and uremia: Is there cellular and molecular crossover?

Many observers have noted that the morphological changes that occur in chronic kidney disease (CKD) patients resemble those seen in the geriatric population, with strikingly similar morbidity and mortality profiles and rates of frailty in the two groups, and shared characteristics at a pathophysiological level especially in respect to the changes seen in their vascular and immune systems. However, whilst much has been documented about the shared physical characteristics of aging and uremia, the molecular and cellular similarities between the two have received less attention. In order to bridge this perceived gap we have reviewed published research concerning the common molecular processes seen in aging subjects and CKD patients, with specific attention to altered proteostasis, mitochondrial dysfunction, post-translational protein modification, and senescence and telomere attrition. We have also sought to illustrate how the cell death and survival pathways apoptosis, necroptosis and autophagy are closely interrelated, and how an understanding of these overlapping pathways is helpful in order to appreciate the shared molecular basis behind the pathophysiology of aging and uremia. This analysis revealed many common molecular characteristics and showed similar patterns of cellular dysfunction. We conclude that the accelerated aging seen in patients with CKD is underpinned at the molecular level, and that a greater understanding of these molecular processes might eventually lead to new much needed therapeutic strategies of benefit to patients with renal disease.

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.

Elevated hepatic 11ß-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia.

Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11ßHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11ßHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11ßHSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11ßHSD1(-/-) mice and rats treated with a specific 11ßHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11ßHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11ßHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.

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.

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.

A possible explanation for anemia in patients treated with mycophenolic acid.

BACKGROUND: Clinical studies suggest that the immunosuppressant mycophenolate mofetil is associated with anemia. However, the mechanism for this is not known. Here, we studied the effect of mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil, on erythropoiesis in vitro. METHODS: Both UT-7 cells and primary murine bone marrow cells were studied. Cells were initially treated with erythropoietin and MPA and proliferation and caspase-3 assays were performed. The effect of guanosine-5'-triphosphate, guanosine, and caspase inhibitors was also investigated. RESULTS: MPA was found to decrease the proliferation of UT-7 cells and erythropoiesis in murine bone marrow cells. This inhibition was associated with an increase in caspase-3 activity in the UT-7 cells. Inhibition was reversed in UT-7 cells and in murine bone marrow by guanosine, but not by caspase inhibitors. The apoptosis induced by MPA was also reversed by guanosine. UT-7 cells treated with MPA showed a decreased inosine-5'-monophosphate dehydrogenase activity. CONCLUSION: These results suggest that MPA inhibits inosine-5'-monophosphate dehydrogenase activity in erythroid cells and that this is a likely mechanism of action of anemia in MPA-treated patients.

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.

The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts.

The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp31 may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp31 gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp31 gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp31 knockout in the AcMNPV genome. The pp31 knockout was subsequently rescued by reinserting the pp31 gene into the polyhedrin locus of the same virus genome. We found that pp31 was not essential for viral replication although the absence of pp31 resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp31 showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp31 gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes.

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.

Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications.

Cellular responses to high glucose are numerous and varied but ultimately result in functional changes and, often, cell death. High glucose induces oxidative and nitrosative stress in many cell types causing the generation of species such as superoxide, nitric oxide and peroxynitrite and their derivatives. The role of these species in high glucose-mediated apoptotic cell death is relevant to the complications of diabetes such as neuropathy, nephropathy and cardiovascular disease. High glucose causes activation of several proteins involved in apoptotic cell death, including members of the caspase and Bcl-2 families. These events and the relationship between high glucose-induced oxidative stress and apoptosis are discussed here with reference to additional regulators of apoptosis such as the mitogen-activated protein kinases (MAPKs) and cell-cycle regulators.

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.

Albumin stimulates cell growth, l-arginine transport, and metabolism to polyamines in human proximal tubular cells.

BACKGROUND: Pure albumin stimulates proximal tubular epithelial cell (PTEC) proliferation, and may have a role in homeostasis in health, as well as in disrupted PTEC turnover in proteinuric nephropathies. We investigated a role for arginine and its metabolites, the polyamines, in this process, given the ability of polyamines to trigger proliferation in other mammalian cells. METHODS: [(3)H]-L-arginine uptake was examined after incubation with 20 mg/mL recombinant human serum albumin (rHSA) in HK-2 PTEC monolayers. Nitric oxide synthase (NOS) and arginase activity was measured; NOS, arginase, and ornithine decarboxylase (ODC) expression was identified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Polyamine synthesis and intracellular amino acid concentrations were compared using high-performance liquid chromatography, and cell growth measured by [(3)H]-thymidine incorporation. RESULTS: In HK-2 PTEC exposed to 20 mg/mL rHSA for 24 hours, cell proliferation as determined by [(3)H]-thymidine incorporation was increased. In parallel, L-arginine transport capacity was increased in a dose- and time-dependent manner. This effect was specific to rHSA, and was not seen with transferrin or immunoglobulin G. The intracellular concentration of L-arginine remained unchanged, although L-ornithine was increased with rHSA incubation. rHSA up-regulated type II arginase mRNA, and increased arginase activity, although no difference in nitric oxide synthase expression or activity was seen. ODC mRNA was increased, as were intracellular polyamine concentrations. alpha-Difluoromethylornithine (DFMO), an ODC inhibitor, reduced intracellular polyamine concentrations and rHSA-induced cell proliferation to control levels. CONCLUSION: The arginine-ornithine-polyamine pathway appears enhanced in PTEC incubated with rHSA and is involved in cellular proliferation; this may offer novel approaches to understanding progressive proteinuric nephropathies.

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.