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The Gradiflow (Life Therapeutics, Frenchs Forest, Australia) system is a novel electrophoretic technique that uses the dual characteristics of size and charge to separate target macromolecules from complex biological solutions. The system has the potential to selectively remove a range of moieties from blood and plasma in an in vivo system such as hemodialysis or, alternatively, in an in vitro setting such as a blood bank. In this study, the safety of a scaled down Gradiflow prototype device with a membrane surface area of 16 cm2 was investigated using an ex vivo ovine model. Physiologic, hematologic, and biochemical parameters were assessed in 12 sheep: 6 animals treated using the Gradiflow and 6 controls. The effects of the Gradiflow procedure on both whole blood and plasma were analyzed. The Gradiflow procedure was well tolerated, and the application of an electrical potential or exposure to the Gradiflow membrane did not cause any significant changes in the parameters measured. Hemoglobin levels remained stable in all groups during the 4-hour experiment. An early neutropenia was observed in all groups, although this appeared to be more pronounced with exposure to a plasma filter; the presence of the Gradiflow component had no separate influence. One sheep in the plasma group experienced septic shock, caused by Staphylococcus contamination of the separation membrane. Overall, the results indicate that there were no gross physiologic, hematologic, or biochemical adverse reactions to the ex vivo Gradiflow procedure.

Background: We have previously shown that monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T-cell expressed and secreted (RANTES) are significantly increased in renal cortex in adriamycin nephropathy. In this study, we tested the effect of DNA vaccination encoding the C-C chemokines MCP-1 and RANTES in a rat model of adriamycin nephropathy., Methods: Both reverse transcription-polymerase chain reaction (RT-PCR) products of MCP-1 and RANTES used as constructs were cloned into a pTarget vector for naked DNA vaccination. Two hundred micrograms of DNA was injected into the tibialis anterior muscle four times at weekly intervals. One week after the last DNA vaccination, rats received adriamycin. All animals were sacrificed 4 weeks after adriamycin administration. Changes in renal function and histologic features were assessed. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used for autoantibody determination. Antibody specificity was assessed in in vitro transmigration assays., Results: Chemokine DNA vaccination significantly reduced proteinuria (P < 0.05) and ameliorated creatinine clearance (P < 0.05) at 2, 3, and 4 weeks after adriamycin administration. Morphometric analysis showed less glomerular sclerosis (P < 0.001) and interstitial infiltrates (P < 0.005) in chemokine DNA vaccination group compared with control groups. Anti-MCP-1 and RANTES autoantibodies were detected in higher concentrations in chemokine DNA vaccinated rats than in control rats (P < 0.001) and serum from vaccinated rats blocked T-cell transmigration to MCP-1 and RANTES., Conclusion: In this study, we have shown that naked DNA vaccination against MCP-1 and RANTES ameliorates the progression of renal disease in the rat adriamycin nephropathy model of chronic proteinuric renal disease. The protective mechanism may involve the production of autoantibodies against MCP-1 and RANTES.

Apolipoprotein E is a genetic risk factor for Alzheimer's disease, and the apoE protein is associated with beta-amyloid deposits in Alzheimer's disease brain. We examined signaling pathways stimulated by apoE in primary neurons in culture. ApoE and an apoE-derived peptide activated several intracellular kinases, including prominently extracellular signal-regulated kinase 1/2 (ERK1/2). ERK1/2 activation by apoE was blocked by an inhibitor of the low-density lipoprotein receptor family, the specific NMDA glutamate receptor antagonist MK 801 and other calcium channel blockers. Activation of apoE receptors also induced tyrosine phosphorylation of Dab1, an adaptor protein of apoE receptors, but experiments in Dab1 knockout neurons demonstrated that Dab1 was not necessary for ERK activation. In contrast, apoE treatment of primary neurons decreased activation of c-Jun N-terminal kinase, a kinase that interacts with another apoE receptor adaptor protein, c-Jun N-terminal kinase-interacting protein. This change also depended on interactions with the low-density lipoprotein receptor family but was independent of calcium channels. c-Jun N-terminal kinase deactivation by apoE was blocked by gamma-secretase inhibitors and pertussis toxin. These results demonstrate that apoE affects several signaling cascades in neurons: increased disabled phosphorylation, activation of the ERK1/2 pathway (dependent on calcium influx via the NMDA receptor) and inhibition of the c-Jun N-terminal kinase 1/2 pathway (dependent on gamma-secretase and G proteins).

Background: Exploration of the role of tubulointerstitial inflammation in experimental chronic renal disease (CRD) is an essential step to understanding and finding new treatments for human CRD. Adriamycin nephrosis (AN) is an experimental analogue of human focal glomerular sclerosis and tubulointerstitial inflammation., Methods: Using murine and rat AN, we have systematically investigated the pathogenic roles of chemokines, costimulatory molecules, and inflammatory cells, such as macrophages and effector and regulatory T lymphocytes. The profile of humoral and cellular mediators was studied in vitro and in vivo. The pathogenic significance of various factors was investigated by DNA vaccination, leukocyte reconstitution and depletion, retroviral transduction, and blockade with monoclonal antibodies., Results: Renal cortical and tubular cell CC-chemokines, including MCP-1, RANTES, and MIP-1alpha, were up-regulated via mediation of NFkappaB, and contributed to disease by attracting inflammatory cells into the interstitium. The role of these chemokines was confirmed by DNA vaccination. CD40-CD40L costimulation signals were involved in expansion and activation of the inflammatory infiltrate, whereas PD-1 signals were inhibitory, and CD28-B7 appeared to have a neutral effect. Macrophage and CD8+ T cells were shown to be effectors of injury, whereas CD4+CD25+ and gammadelta T cells acted as regulatory cells. FoxP3 transduction was able to convert naive T cells to CD4+CD25+ regulatory T cells., Conclusion: There is a broad range of humoral and cellular factors involved in the pathogenesis of experimental CRD, some of which are potential targets for treatment of human CRD.

This report summarizes the discussions of the International Society of Nephrology (ISN) 2004 Consensus Workshop on Prevention of Progression of Renal Disease, which was held in Hong Kong on June 29, 2004. Three key areas were discussed during the workshop: (1) screening for chronic kidney disease; (2) evaluation and estimating progression of chronic kidney disease; and (3) measures to prevent the progression of chronic kidney disease. Fifteen consensus statements were made in these three areas, as endorsed by the participants of the workshop. The ISN can make use of and take reference to these statements in formulating its policy for tackling chronic kidney disease, a disease with significant global impact.

The incidence of end-stage renal disease is increasing. Progression to end stage can be slowed if kidney damage is detected at an early stage. Prognosis and outcomes in patients with chronic kidney disease have been related to the quality of predialysis care and the timing of referral. Many patients with chronic kidney disease are referred to a nephrologist close to commencement of renal replacement therapy. This leads to suboptimal management of complications of chronic renal insufficiency, and increased morbidity and mortality of patients on renal replacement therapy. This article addresses the evidence that examines the view that patients need to be referred early in order to avoid complications of chronic renal insufficiency. Early referral can be achieved through improved communication between primary health care givers and nephrology services. A multidisciplinary approach has a significant impact on outcomes. In the face of rising incidence of chronic kidney disease, early referral of all patients is not possible. Therefore, identification of patients at risk for rapid deterioration of renal function is important in order to rationalize and reduce health care expenditure.

Over the next decade, the number of patients with end-stage renal disease (ESRD) treated by dialysis may double, and even developed nations will have difficulty in coping with this alarming increase. This review will outline the proven and unproven strategies that have the potential to retard the progression of chronic kidney disease (CKD). Recently, a number of randomised clinical trials have demonstrated the efficacy of several strategies to slow the progression of CKD. Proven strategies include adequate blood pressure control (with angiotensin blockade), and for diabetic nephropathy good glycaemic control. Other potentially beneficial strategies include smoking cessation, lipid control and aldosterone blockade. The early institution of these strategies has the potential to regress established CKD as well as improve the long-term cardiovascular outcomes of these patients. Proof of the efficacy in humans of promising experimental approaches, such as the administration of growth factors (e.g., recombinant bone morphogenetic protein-7), anti-fibrotic agents (e.g., pirfenidone) and novel anti-proteinuric drugs (e.g., pentosan polysulphate), is awaited. Finally, the primary prevention of CKD, at least in part, by the eradication of type 2 diabetes and obesity (through improvement of lifestyle factors), and adequate treatment of hypertension, have the potential to eliminate up to half of the most common causes of CKD (or ESRD) in developed countries.

In a previous study we found that an episode of acute subclinical nephrotoxicity with gentamicin (G) (but not that induced by another proximal tubular cell nephrotoxin: ferric nitrilotriacetate, FeNTA), paradoxically reduced the progression of renal function and injury in uninephrectomized rats with nephrotic glomerular disease due to Adriamycin nephropathy (AN). Here, we hypothesized that subclinical exposure to G reduces early renal cortical tubulointerstitial inflammation and NF-kappaB activation in AN. To test this hypothesis, male Wistar rats with established AN received either G (10, 40, or 80 mg/kg by daily s.c.i. for 3 days), FeNTA (1.25, 5, or 10 mg/kg by a single i.p.i.), or vehicle (n=8 per group), 13 to 15 days after disease induction. Although G and FeNTA caused acute tubular necrosis in a dose-dependant manner (day 17), only the highest doses (10 mg/kg and 80 mg/kg) produced an acute elevation in the serum creatinine. On day 33, chronic tubulointerstitial inflammation (tubular atrophy, interstitial ED-1+/CD8+ cell accumulation) and NF-kappaB activation were exacerbated only in the groups that caused functional nephrotoxicity. These data suggest that: 1) the protective effect of subclinical G nephrotoxicity in chronic AN does not involve early changes in interstitial inflammation or NF-kappaB activation; and 2) a single episode of G exposure must be accompanied by clinically apparent nephrotoxicity in order to accelerate progression in a nonuremic model of chronic glomerular disease.

Cellular hypoxia has been proposed as a major factor in the pathogenesis of chronic renal injury, yet to date there has been no direct evidence to support its importance. Therefore, we examined cortical hypoxia in an animal model of chronic renal injury (murine adriamycin nephrosis; AN) by assessing nuclear localization of the oxygen-dependent alpha-subunit of hypoxia-inducible factor-1 (HIF-1alpha) in animals 7, 14, and 28 days after adriamycin. Results were assessed in conjunction with quantitation of the cortical microvasculature (by CD34 immunostaining) and cortical expression of VEGF. Cortical apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. A dramatic and significant increase in nuclear localization of HIF-1alpha was seen 28 days after adriamycin in the context of severe glomerular and tubulointerstitial damage. Areas of nuclear HIF-1alpha staining did not colocalize with areas of cellular apoptosis. AN was also associated with a significant attenuation of the peritubular capillaries that was significant at 14 and 28 days after adriamycin. Cortical VEGF expression fell in a stepwise manner from day 7 until day 28 after adriamycin. In conclusion, these data are consistent with a significant increase in cellular hypoxia occurring in the advanced stages of murine AN. Increased cortical hypoxia was preceded by significant reductions in both the number of peritubular capillaries (i.e., oxygen supply) and the angiogenic cytokine VEGF. Apart from providing the first direct evidence for cellular hypoxia in a model of chronic renal disease, these results suggest that a primary dysregulation of angiogenesis may be the cause of increased hypoxia in this model.

A fundamental question in the formation of the nervous system is the extent to which a neurotransmitter contributes to the development of the neurons that synthesize and release it. A complementary question is whether neurotransmitter signaling contributes to the development of postsynaptic targets. Prior studies have suggested that adrenergic signaling may promote adrenergic neuronal proliferation or survival and may be critical for the postnatal development of the cerebellum. To test these possibilities genetically, we studied mice that are unable to synthesize norepinephrine and epinephrine (NE/E), the endogenous adrenergic receptor ligands, due to a disruption the gene for dopamine beta-hydroxylase. These mice develop postnatally in the absence of NE/E. Here we report that the adrenergic neurons of these mutant mice are present in normal numbers and locations and exhibit typical innervation patterns throughout the central nervous system (CNS), as assessed by immunostaining for tyrosine hydroxylase and the NE transporter. Furthermore, cerebellar cortical development (size, foliation, layering, cell number, and position), which proceeds to a large degree postnatally, is unaltered in the mutants. These results indicate that the fate and innervation pattern of the adrenergic neurons, as well as the development of the cerebellum, do not depend on postnatal signaling by NE/E. The results also suggest that when restoration of adrenergic signaling is performed in this mutant mouse model (by administering a synthetic precursor of NE), reversal of phenotypes is due to the synthesis and release of NE/E from adrenergic terminals that are distributed normally within the CNS.

Background: Tubule cells can produce a variety of cytokines, extracellular matrix (ECM) components, and adhesion molecules in vitro and in vivo. It is generally assumed that stimulated tubule cells are proinflammatory and at least partially responsible for interstitial inflammation. However, the overall effect of tubular cells on interstitial cells is unknown. In this study, pro- and anti-inflammatory cytokine production and net effects on macrophages of tubule cells activated by lipopolysaccharide (LPS) were examined., Methods: Tubule cells stimulated with LPS expressed tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-12, monocyte chemoattractant protein-1 (MCP-1), IL-10, and transforming growth factor-beta (TGF-beta). Conditioned media were collected from confluent monolayers of rat tubule cells stimulated, or not, by LPS for 4 and 18 hours, respectively. Macrophages were cultured with conditioned media and/or LPS (0.5 microg/mL) for 18 hours., Results: TNF-alpha and IL-lbeta mRNA of macrophages stimulated by LPS increased more than fivefold when cultured with control conditioned media from unstimulated tubule cells. Surprisingly, TNF-alpha and IL-lbeta levels of macrophages stimulated by LPS were not increased when cultured with conditioned media from activated tubule cells. Neutralizing antibodies to IL-10 and TGF-beta were used to define the inhibitory component(s) in conditioned medium. Anti-IL-10, but not anti-TGF-beta, abolished partially the inhibitory effects of conditioned media on macrophages., Conclusion: Tubule cells produce both pro- and anti-inflammatory cytokines and the net effect, partially explained by IL-10, of tubule cells activated with LPS is to inhibit activity of macrophages. Thus, the net effect of activated tubule cells on interstitial pathology may in certain circumstances, be anti- rather than pro-inflammatory.

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF (adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary "baited"-global cluster analysis of gene expression profiles. Primary cluster analysis focused on mRNAs encoding matrix proteins and modulators of matrix turnover as classified by Onto-Compare and Gene Ontology and identified both molecules and pathways already implicated in the pathogenesis of TIF (e.g. transforming growth factor beta1-CTGF-fibronectin-1 pathway) and novel TIF-associated genes (e.g. SPARC and Matrilin-2). Specific gene expression patterns identified by primary extracellular matrix-focused cluster analysis were then used as bioinformatic bait in secondary global clustering, with which to search the renal transcriptome for novel modulators of TIF. Among the genes clustering with ECM proteins in the latter analysis were endoglin, clusterin, and gelsolin. In several notable cases (e.g. claudin-1 and meprin-1beta) the pattern of gene expression identified in adriamycin nephropathy in vivo was replicated during transdifferentiation of renal tubule epithelial cells to a fibroblast-like phenotype in vitro on exposure to transforming growth factor-beta and epidermal growth factor suggesting a role in fibrogenesis. The further exploration of these complex gene networks should shed light on the core molecular pathways that underpin TIF in renal disease.

Objectives: The primary objective of the IDEAL study is to determine whether the timing of dialysis initiation has an effect on survival in subjects with end-stage renal disease (ESRD). The secondary objectives are to determine the impact of "early start" versus "late start" dialysis on nutritional and cardiac morbidity, quality of life, and economic cost., Design: Prospective multicenter randomized controlled trial. Patients are randomized to commence dialysis at a glomerular filtration rate (by Cockcroft-Gault) of either 10-14 mL/minute/1.73 m2 ("early start") or 5-7 mL/min/1.73 m2 ("late start"), with stratification for dialysis modality (hemodialysis vs peritoneal dialysis), study center, and the presence or not of diabetes mellitus., Setting: Dialysis units throughout Australia and New Zealand., Patients: Patients with ESRD commencing chronic dialysis therapy., Outcome Measures: Three years from randomization, all-cause mortality, morbidity, and economic impact; structural and functional cardiac status, nutritional state, and quality of life will be assessed., Results: To date, 388 patients of a minimum 800 patients have been entered and randomized into the study. Current recruitment rates suggest sufficient patients will be enrolled by December 2004 and follow-up completed by December 2007., Conclusions: The IDEAL study will provide evidence for the optimal time to commence dialysis.

Background: Interaction between CD40 and CD40 ligand (CD40L) is involved in both cognate and innate immune responses. Blockade of CD40-CD40L interactions reduces severity of renal injury in murine lupus nephritis and membranous nephropathy. We hypothesized that CD40-CD40L could contribute to renal injury in models that are not antibody-dependent, and that anti-CD40L could diminish inflammation and fibrosis in murine adriamycin nephropathy., Methods: Male BALB/c mice were divided into three groups (N = 6 per group): (1). saline-treated, age-matched control; (2). adriamycin only; and (3). MR1 + adriamycin. In group 3, mice were treated with intraperitoneal injections of anti-CD40L antibody (clone MR1, 0.4 mg per mouse) after the onset of proteinuria at days 5, 7, 9, and 11 after adriamycin treatment. Animal subgroups were compared at 14 and 42 days after induction of adriamycin nephropathy. Functional and pathologic markers of disease severity, cellular components of interstitial inflammation, and the degree of CD40 expression were assessed. Relative cortical RNA expression of the chemokine monocyte-chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) was also compared between animal groups., Results: CD40 was weakly expressed in tubules of normal mice but was expressed in tubules, interstitium, and glomeruli of mice with adriamycin nephropathy in a time-dependent manner. MR1 treatment resulted in a significant attenuation of the severity of adriamycin nephropathy at day 42 [e.g., glomerular sclerosis (%), group 3, 20.1 +/- 4.7 vs. group 2, 30.2 +/- 7.2, P < 0.001]. CD40L blockade significantly reduced tubulointerstitial injury as well [tubular diameter microm), group 3, 42.5 +/- 6.9 vs. group 2, 66.3 +/- 13.7, P < 0.001; and group 1, 37.3 +/- 5.7, P < 0.01; tubular cell height microm), group 3, 16.3 +/- 1.7 vs. group 2, 11 +/- 1.8, P < 0.01; and group 1, 18.2 +/- 1.9, P < 0.01; interstitial volume (%), group 3, 13.9 +/- 5.1 vs. group 2, 26.2 +/- 4.9, P < 0.001; and group 1, 1.3 +/- 0.7, P < 0.001; proteinuria (mg/24 hours), group 3, 1.8 +/- 0.6 vs. group 2, 4.3 +/- 0.8, P < 0.001; and group 1, 0.7 +/- 0.2, P < 0.05; and creatinine clearance microL/min), group 3, 75 +/- 4 vs. group 2, 35 +/- 2, P < 0.001; and group 1, 82 +/- 4, P < 0.01] were also improved by MR1. MR1 treatment also resulted in a significant reduction in the number of cortical macrophages at both 14 and 42 days after adriamycin (P < 0.01). Cortical expression of MCP-1 and RANTES was significantly reduced by MR1 treatment at 42 days after adriamycin (P < 0.01 and P < 0.05, respectively)., Conclusion: Blockade of CD40-CD40L interaction protects against renal structural and functional injury in this murine model of chronic proteinuric renal disease.

Icodextrin is a starch-derived, high molecular weight glucose polymer, which has been shown to promote sustained ultrafiltration equivalent to that achieved with hypertonic (3.86%/4.25%) glucose exchanges during prolonged intraperitoneal dwells (up to 16 h). Patients with impaired ultrafiltration, particularly in the settings of acute peritonitis, high transporter status and diabetes mellitus, appear to derive the greatest benefit from icodextrin with respect to augmentation of dialytic fluid removal, amelioration of symptomatic fluid retention and possible prolongation of technique survival. Glycaemic control is also improved by substituting icodextrin for hypertonic glucose exchanges in diabetic patients. Preliminary in vitro and ex vivo studies suggest that icodextrin demonstrates greater peritoneal membrane biocompatibility than glucose-based dialysates, but these findings need to be confirmed by long-term clinical studies. This paper reviews the available clinical evidence pertaining to the safety and efficacy of icodextrin and makes recommendations for its use in peritonal dialysis.

Context: Patients with peritoneal carcinomatosis have a dismal prognosis despite systemic chemotherapy or palliative surgery. A novel strategy of complete tumor debulking with intraoperative hyperthermia with chemotherapy has been proposed to provide prolonged survival., Objective: To retrospectively analyze the preliminary experience with this technique at Baylor University Medical Center., Methods: All patients underwent attempted tumor debulking followed by intraperitoneal hyperthermia with 40 mg mitomycin-C over 2 hours., Results: Patient diagnoses included nonmucinous colorectal carcinomatosis (n = 9), diffuse peritoneal adenomucinosis (n = 1), peritoneal mucinous carcinomatosis (n = 2), and gastric carcinomatosis (n = 3). Tumors in most patients (13/15) were resected to < or = 5 mm, and those in 10 of 15 were resected to no gross disease. Complications included ileus (n = 9), bowel leak (n = 2), infection (n = 1), and fistula (n = 1). One patient died of progressive gastric cancer at 1 month. Within a median follow-up of 4 months, 8 patients had no tumor by radiologic or tumor marker analysis., Conclusion: Intraoperative hyperthermia with chemotherapy is a viable treatment for patients with isolated peritoneal carcinomatosis from colorectal or gastric origin.

Objective: To examine the effects of patient age on the growth and transfer of blastocyst-stage embryos., Design: Retrospective clinical study., Setting: Private assisted reproductive technologies center., Patient(s): Three hundred patients between the ages of 18 and 45 undergoing in vitro fertilization., Intervention(s): Bipronucleate oocytes were grown for up to 144 hours and subsequently transferred when at least one embryo attained the expanded blastocyst stage., Main Outcome Measure(s): Oocytes retrieved and fertilized, blastocyst formation rates, implantation rates, and pregnancy rates per retrieval and transfer., Result(s): The rate of cycle cancellation before oocyte retrieval increased significantly with age, and the average number of oocytes per retrieval declined significantly with age. Fertilization rates were unrelated to patient age. The proportion of cycles with expanded blastocysts declined significantly with age. Pregnancy rates per stimulation declined with age, but pregnancy rates per transfer were approximately 50% across the entire age range studied., Conclusion(s): The decline in female fertility with age appears to be the result of reduced numbers of oocytes and the inability of fertilized oocytes to develop to the blastocyst stage. Implantation and pregnancy rates appear to be unaffected by age when blastocysts do form.

Background: Since the Fas/Fas Ligand (FasL) interaction is recognized as a major pathway of apoptosis in immune cells, we hypothesized that selective expression of FasL by tubular cells (TC) may promote the resolution of interstitial inflammation by inducing apoptosis of infiltrating immune cells. In this study, the effect of FasL transfection of rat TC on apoptosis of leukocytes was examined., Methods: Rat tubule cells (NRK52E) were transfected with plasmids constructed using human and rat FasL (hFasL and rFasL). The propensity of activated, transfected TC to undergo apoptosis was examined. Similarly, the effects of FasL transfection on apoptosis of Jurkat cells and activated leukocytes were assessed directly following co-culture for 12 hours and in a cell insert system intended to assess the effects of soluble FasL. Fas and FasL expression was assessed by flow cytometry and apoptosis was examined using Annexin V staining and the TUNEL method., Results: Expression of FasL in TC was increased after FasL transfection. Transfected TC showed no detectable increase in apoptosis following lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activation. Jurkat cell apoptosis was increased ninefold and eightfold after co-culture with TC transfected with hFasL and rFasL, respectively (67.0 +/- 12.1% and 60.1 +/- 8.8% vs. 6.7 +/- 1.8% with un-transfected TC, P < 0.01). Similarly, apoptosis of activated leukocytes was increased fourfold by co-culture (26.8 +/- 4.9% vs. 6.7 +/- 2.0% with untransfected TC, P < 0.01). Leukocyte apoptosis also was increased in an insert culture system (18.2 +/- 4.4% vs. 5.8 +/- 2.3% with un- transfected TC, P < 0.01). No increase of TC apoptosis was detected in any of the co-culture experiments., Conclusion: Enhanced expression of FasL by TC is capable of inducing apoptosis of activated leukocytes, without evidence for increased susceptibility to apoptosis of the transfected cells themselves. This suggests a potential role for this approach in the limitation and resolution of renal tubulointerstitial inflammation.

Background/aims: Quercetin, a naturally occurring dietary flavonoid, is a potent in vitro inhibitor of the transcription factors, nuclear factor (NF)-kappa B and activator protein (AP)-1. Here we investigated the efficacy of quercetin to suppress renal cortical NF-kappa B/AP-1 activation and tubulointerstitial injury, in vivo, in a nephrotic rat model of chronic glomerular disease., Methods: Adult male Wistar rats with (n = 27) or without (n = 15) Adriamycin nephropathy (AN) were stratified into six groups (according to proteinuria and endogenous creatinine clearance, measured on day 10) and pair-fed a semipurified diet supplemented with or without quercetin (0.5, 2%) from day 14 to 36., Results: Quercetin-fed rats had minimal weight gain during the study (p < 0.05). In AN, proteinuria, the decline in endogenous creatinine clearance, hypercholesterolaemia and cortical tubulointerstitial injury were not reduced by quercetin. Quercetin had no effect on renal cortical NF-kappa B activation, but unexpectedly exacerbated the induction of AP-1 in AN (by 67%, p < 0.05). In normal rats, quercetin increased proteinuria (by 75%, p < 0.05), renal cortical AP-1 activation (by 198%, p = 0.06), and malondialdehyde production (by 110%, p < 0.05)., Conclusion: These data suggest that supplementation of the diet with quercetin is not an effective therapeutic strategy alone to reduce NF-kappa B/ AP-1 activation in chronic glomerular diseases, and paradoxically could be associated with adverse renal effects., (Copyright 2002 S. Karger AG, Basel)

Objective: To investigate the predictive value of quantitative measurements of blastocyst morphology on subsequent implantation rates after transfer., Design: Prospective observational study., Setting: Private assisted reproductive technology center., Patient(s): One hundred seventy-four IVF patients receiving transfers of expanded blastocyst-stage embryos on day 5 (n = 112) or day 6 (n = 62) after oocyte retrieval., Intervention(s): None., Main Outcome Measure(s): Blastocyst diameter, number of trophectoderm cells, inner cell mass (ICM) size, ICM shape, and implantation and pregnancy rates., Result(s): Blastocyst diameter and trophectoderm cell numbers were unrelated to implantation rates. Day 5 expanded blastocysts with ICMs of >4,500 microm(2) implanted at a higher rate than did those with smaller ICMs (55% vs. 31%). Day 5 expanded blastocysts with slightly oval ICMs implanted at a higher rate (58%) compared with those with either rounder ICMs (7%) or more elongated ICMs (33%). Implantation rates were highest (71%) for embryos with both optimal ICM size and shape. Pregnancy rates were higher for day 5 transfers of optimally shaped ICMs compared with day 5 transfers of optimally sized ICMs., Conclusion(s): Quantitative measurements of the inner cell mass are highly indicative of blastocyst implantation potential. Blastocysts with relatively large and/or slightly oval ICMs are more likely to implant than other blastocysts.

The persistence of NF-kappaB independent inflammatory signals in the cortical tubulointerstitium may explain the incomplete suppression of interstitial monocyte accumulation by the antioxidant NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), in nephrotic rats with established Adriamycin nephropathy (AN). Because PDTC is known to have anti-proteinuric effects, in this study we investigated whether earlier commencement, during the pre-nephrotic phase of AN, would be more effective in reducing interstitial monocyte accumulation. Male Wistar rats with AN received either vehicle or PDTC (50 mg/kg bd i.p.i.) from d7 until d30 (n = 8 per group). On d30, PDTC reduced renal cortical lipid peroxidation (43%), wet kidney weight and tubulointerstitial injury in AN, but did not decrease proteinuria. Accordingly, inhibition of interstitial ED-1 accumulation remained incomplete (52%). Interestingly, the early administration of PDTC in AN, induced polyuria and renal cortical NF-kappaB DNA-binding activity was reduced by only 35%. These results suggest that: (i) the combination of an anti-proteinuric agent with PDTC may be required to completely suppress interstitial monocyte cell accumulation in AN and, (ii) the timing and duration of PDTC therapy are an important determinant of its efficacy to reduce NF-kappaB activation, in vivo.

Objective: To compare the outcome of second and third cycles of in vitro fertilization with blastocyst transfer to the outcome of first attempts at IVF with blastocyst transfer., Design: Retrospective study., Setting: Private ART center., Patient(s): Three hundred and four patients undergoing treatment with in vitro fertilization with blastocyst transfer, 87 of which underwent at least one cycle of re-treatment after failing to achieve pregnancy in their first cycle., Intervention(s): Bipronucleate oocytes were grown for up to 144 hours and subsequently transferred when at least one embryo attained the expanded blastocyst stage., Main Outcome Measure(s): Pregnancy and implantation rates., Result(s): Pregnancy rates per retrieval were significantly higher for patients undergoing their first cycle of in vitro fertilization with blastocyst transfer (36%) compared to those undergoing their second (19%) or their third (9%) cycles of treatment. Implantation rates per embryo were also higher for first cycles of in vitro fertilization with blastocyst transfer (30%) compared to second (18%) or third cycles (8%)., Conclusion(s): Pregnancy and implantation rates decline dramatically in repeated cycles of in vitro fertilization with blastocyst transfer following one or more unsuccessful cycles of in vitro fertilization with blastocyst transfer.

Adriamycin nephropathy (AN) is a widely used nonimmune-mediated rat model of proteinuric chronic glomerular disease and is usually induced by a single intravenous injection of doxorubicin hydrochloride (DX) into the tail vein. However, this route can be associated with local skin necrosis and variability in disease induction and poses an occupational hazard to the investigator. Here we describe a simple technique of administering DX (1.7 mg) to ketamine:xylazine-sedated adult male Wistar rats (mean +/- 1 standard deviation, 238 +/- 8 g; n = 28) by using a single substernal intracardiac injection. The procedure was associated with minimal morbidity and mortality (1 death related to anesthesia). By day 21, severe nephrotic syndrome with effacement of glomerular epithelial cell foot processes and diffuse cortical tubulointerstitial injury was induced in all animals. Therefore, intracardiac injection of DX is a safe and consistent method of inducing AN in the rat and provides an alternative to the tail-vein route.

Objective: To compare implantation and pregnancy rates according to the day of embryo transfer (day 5 or 6 after oocyte retrieval) when transfer was postponed until expanded blastocysts developed., Design: Retrospective clinical study., Setting: Private ART center., Patient(s): One-hundred and eighty-three women undergoing blastocyst-stage embryo transfer following in vitro fertilization., Intervention(s): Bipronucleate oocytes were grown for up to 144 hours and subsequently transferred only when at least one embryo attained the expanded blastocyst stage., Main Outcome Measure(s): Implantation and pregnancy rates., Result(s): Blastocysts transferred on day 5 implanted at nearly twice the rate of blastocysts transferred on day 6 (36.3% vs. 19.0%). Pregnancy rates were also almost twice as high among the day 5 transfer patients (59.3% vs. 32.3%). In addition, more blastocysts developed (3.6 vs. 2.4), and more were transferred (2.7 vs. 2.3) to the day 5 transfer patients, although the proportion of expanded blastocysts among the blastocysts that were transferred was the same for the two groups (91.7% vs. 93.6%)., Conclusion(s): Embryos that develop to the expanded blastocyst stage and are transferred on day 5 after retrieval are approximately twice as likely to implant compared to those for which expansion and transfer are delayed until day 6.

Tubulointerstitial damage, in progressive chronic renal disease of all types, arises because of a complex interplay between factors in the tubular lumen, tubular epithelial cells, peritubular capillaries, resident and infiltrating interstitial cells and extracellular matrix. Particularly in proteinuric renal disease, tubular epithelial cells play a central role in orchestrating these events. In response to mediators arising systemically, in the tubular lumen or from other renal cells, tubular epithelial cells undergo a complex series of structural and functional changes and produce a bewildering number of soluble and fixed mediators, which in turn lead to interstitial inflammation and fibrosis. Knowledge of these interactions has increased exponentially over the past decade, and has defined a number of new targets for treatment. Both expansion and consolidation of this knowledge is needed to determine which of these targets holds the most promise for future treatment.

Background: CD4(+) T cells play an important role in various types of immunologic renal disease, including lupus nephritis, IgA nephropathy, and crescentic glomerulonephritis. CD4(+) T cells are also major infiltrating lymphocytes in chronic tubulointerstitial inflammation associated with nonimmunological renal diseases. We suspected that CD4(+) T cells might contribute to disease progression and loss of renal function in chronic proteinuric renal disease (CPRD). To investigate this possibility, the effect of monoclonal antibody against CD4(+) lymphocytes (anti-CD4) was studied in a murine model (adriamycin nephropathy) of CPRD., Methods: Adriamycin nephropathy was produced in male BALB/c mice by a single intravenous injection of adriamycin (11 mg/kg). Anti-CD4 was given by intraperitoneal injection following the development of proteinuria at days 5, 6, 7, 21, and 37 after adriamycin. After six weeks, renal function and histology were studied by histomorphometry, immunohistochemistry, and flow cytometry., Results: Flow cytometric analysis showed a marked decrease in the number of CD4(+) T cells in blood and spleen of the antibody-treated animals (N = 7, P < 0.01). Adriamycin plus CD4(+) depletion mice had significantly greater mesangial expansion, glomerular sclerosis, and interstitial expansion than the mice on adriamycin alone. Interstitial infiltration with macrophages and CD8(+) cells was significantly increased in adriamycin plus CD4(+) depletion mice. Creatinine clearance (17.5 +/- 0.54 vs. 29.2 +/- 0.89 microL/min, P < 0.001) was significantly worse in the adriamycin plus CD4(+) depletion mice than in adriamycin alone mice and correlated with histologic change in glomeruli and interstitium., Conclusions: Depletion of CD4(+) T cells promotes glomerular and interstitial injury in mice with established adriamycin nephropathy. These findings suggest that CD4(+) T cells have a protective role against the progression of adriamycin nephropathy.

Background: Many studies have shown that interstitial inflammation in human and experimental renal disease is characterized by T-cell infiltration, but published data on the involvement of inflammatory cell subsets in progressive tubulointerstitial lesions are often conflicting. A previous study suggested a role for cytotoxic T lymphocytes in the damaging effect of CD4(+) T-cell depletion in murine adriamycin (ADR) nephropathy, a model of focal segmental glomerulosclerosis (FSGS), and tubulointerstitial inflammation. The aim of this study was to investigate the role of CD8(+) cells in this model., Methods: Male BALB/c mice were treated with five intraperitoneal injections of anti-CD8 monoclonal antibody (mAb), beginning from five days after ADR treatment, when overt proteinuria was established. Seven mice in each of groups A (ADR + mAb), B (ADR only), and C (saline treated, age matched) were sacrificed at week 6. Changes in renal function and histopathological features were assessed. Tubulointerstitial inflammation and glomerular inflammation were examined immunohistochemically., Results: mAb treatment reduced CD8(+) cell levels to <2% of normal in spleen. Proteinuria in group A was no different from that in group B at week 6, but was markedly higher than in group C. Creatinine clearance was significantly ameliorated by anti-CD8 treatment (71.8 +/- 4.9 microL/min vs. 29.2 +/- 2.8 in group B and 81.9 +/- 3.7 in group C). Morphometric analysis showed less FSGS in group A compared with group B (6.5 +/- 1.9 vs. 13.0 +/- 2.8, P < 0.001), as well as less tubular atrophy (indicated by increased ratio of tubule cell height to tubular diameter, 0.25 +/- 0.24 in group A vs. 0.04 +/- 0.02 in group B, P < 0.05). CD8 depletion also reduced interstitial expansion (6.3 +/- 2.2% vs. 16.4 +/- 3.1 in group B, P < 0.001) and fibrosis (P < 0.01). Macrophage infiltration in tubulointerstitium was less in group A than in group B (P = 0.052). The number of interstitial CD4(+) cells appeared to increase after anti-CD8 treatment, but was not statistically different between groups A and B., Conclusion: Anti-CD8 treatment protects against renal functional and structural injury in this murine model of chronic proteinuric renal disease.

Nitric oxide (NO) levels are increased after exposure of cultured proximal tubule cells (PTC) to non-haem iron, potentially contributing to PTC injury in disease states associated with increased iron exposure, including proteinuric renal disease. The mechanisms underlying this observed increase were investigated. After 3 h exposure to 400 microM nitrilotriacetate (NTA)-Fe, inducible nitric oxide synthase (iNOS) mRNA expression was significantly increased, with a corresponding increase in iNOS protein after 12 h. The nuclear binding activity of NFkappaB with 400 microM NTA-Fe was increased, and pyrrolidine dithiocarbamate (PDTC), an antioxidant inhibitor of NFkappaB, prevented both activation of NFkappaB and NO production in response to NTA-Fe. Inhibition of protein tyrosine kinase reduced iNOS mRNA, iNOS protein levels and NO production in response to NTA-Fe. The effect of tyrosine kinase inhibition on NFkappaB activation was variable, with herbimycin but not genistein having an inhibitory effect. Activation of either protein kinase A or C increased iNOS mRNA and protein levels, and NO production in response to NTA-Fe, whereas only the protein kinase C activator phorbol dibutyrate (PDBu) had a stimulatory effect on NFkappaB activation. The protein kinase A activator forskolin did not alter iron-induced activation of NFkappaB. These data suggest that the observed increase in NO production by PTC in response to iron is due to increased transcription of iNOS. The transcriptional regulation of this response is complex and involves NFkappaB, protein tyrosine kinase and the protein kinases A and C., (Copyright 2001 S. Karger AG, Basel)

Proximal tubule cells (PTC) in chronic renal disease produce chemokines which cause renal interstitial inflammation, and also transport more Na+ than normal. To investigate whether these two events might be related, monocyte chemoattractant protein-1 (MCP-1) production was examined in rat PTC in primary culture. Amiloride reduced, while ouabain increased levels of MCP-1 mRNA and protein. Amiloride reduced MCP-1 in cells stimulated with ouabain, lipopolysaccharide (LPS) or albumin. Intracellular Na+ rose with ouabain, but not LPS or albumin. Effects of amiloride, ouabain, LPS and albumin were abrogated by sodium-free but not chloride-free culture medium, and were not explained by changes in intracellular pH. Intracellular Ca2+ rose with ouabain, LPS or albumin and sodium-free medium. BAPTA-AM reduced intracellular Ca2+ and MCP-1 mRNA levels in unstimulated cells, and cells stimulated with ouabain, LPS or albumin. Thus, amiloride and ouabain may alter tubular cell MCP-1 by changing intracellular Na+, with secondary changes in intracellular Ca2+, whereas stimulation by LPS and albumin may involve Ca2+ directly., (Copyright 2001 S. Karger AG, Basel)

Background/aim: Inhibition of nuclear factor kappaB with the antioxidant pyrrolidine dithiocarbamate (PDTC) reduced tubulointerstitial injury in Adriamycin nephropathy (AN), whereas N-acetylcysteine (NAC) was ineffective. Here we hypothesize that PDTC reduces the renal cortical expression of nuclear factor kappaB dependent cytokines in AN., Methods: Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride (7.5 mg/kg). NAC (150 mg/kg twice daily i.p.), PDTC (50 mg/kg twice daily i.p.), or vehicle were commenced on day 14 and continued until day 30., Results: On day 30, mRNAs of selected cytokines were increased in AN (TNF-alpha 3.4-fold, MCP-1 5.1-fold, IL-10 2.7-fold, TGF-beta1 3.5-fold, all p < 0.05) as determined by RT-PCR. PDTC reduced IL-10 and TGF-beta1 mRNAs (p < 0.05), whereas the upregulation of MCP-1 and TNF-alpha mRNAs was not affected. In contrast, NAC increased TNF-alpha and IL-10 mRNAs (p < 0.05). Nuclear protein levels of activator protein-1 were increased in AN (4.4-fold, p < 0.01) and not significantly altered by PDTC (3.0-fold, p = 0.13) or NAC (5. 2-fold, p = 0.18)., Conclusions: The protective effects of PDTC in AN are not associated with a local reduction in TNF-alpha and MCP-1 gene expression. The latter may be due to continued transactivation by activator protein-1. These data also suggest that IL-10 and TGF-beta1 mRNA expressions are PDTC dependent and have a role in mediating tubulointerstitial injury.

ABSTRACT The incidence of strawberry flower infection by Botrytis cinerea was monitored in unsprayed field plots in three successive years together with meteorological data and numbers of conidia in the air. There were large differences in conidia numbers and weather conditions in the 3 years. Three sets of models were derived to relate inoculum and weather conditions to the incidence of flower infection; by inoculum only, by weather variables only, and by both inoculum and weather variables. All the models fitted the observed incidence satisfactorily. High inoculum led to more infection. Models using weather variables only gave more accurate predictions than models using inoculum only. Models using both weather variables and inoculum gave the best predictions, but the improvement over the models based on weather variables only was small. The relationship between incidence of flower infection and inoculum and weather variables was generally consistent between years. Of the weather variables examined, daytime vapor pressure deficit and nighttime temperature had the greatest effect in determining daily incidence of flower infection. Infection was favored by low day vapor pressure deficit and high night temperature. The accuracy and consistency of the weather-based models suggest they could be explored to assist in management of gray mold.

Background: As an experimental analogue of human focal glomerular sclerosis (FGS), adriamycin (ADR)-induced nephropathy is well-characterized in rats. Previously, this model has not been fully established in mice. The extension of this model to the mouse would be useful in the application of genetic and monoclonal antibody technology to characterize mechanisms of progressive renal disease. Herein, we describe a stable and reproducible murine model of chronic proteinuria induced by ADR., Methods: Male BALB/c mice were intravenously injected with a single dose of ADR (10 to 11 mg/kg). Seven mice were sacrificed at weeks 1, 2, 4, and 6. Renal function, quantitative morphometric analysis, and electron microscopic studies were performed. Peripheral CD4+ and CD8+ T cells were evaluated using flow cytometric analysis of splenocytes. The leukocytic inflammatory pattern was analyzed by immunohistochemical examination., Results: Overt proteinuria was observed from day 5 and remained significantly elevated throughout the study period. A focal increase in reabsorption droplets in tubular cells appeared at weeks 1 and 2, and numerous intraluminal casts were present after two weeks. Glomerular vacuolation and mild FGS appeared at week 4. At week 6, extensive focal and even global glomerular sclerosis, associated with moderate interstitial expansion and severe inflammation, were observed. A prominent macrophage infiltration appeared within both interstitium and glomeruli at week 2, followed by accumulation of both CD4+ and CD8+ T cells in interstitium but not glomeruli. There were almost no B lymphocytes seen at any time., Conclusion: This model should be useful in unraveling the pathogenesis of glomerular and interstitial inflammation and fibrosis in chronic proteinuric renal disease.

Background: Carbamylation is an irreversible process of non-enzymatic modification of proteins by the breakdown products of urea. For haemoglobin (Hb), the extent of carbamylation is a marker of urea exposure and has been proposed as an indicator of the control of uraemia by dialysis, analogous to the use of Hb glycosylation in diabetic patients., Methods: We performed a cross-sectional study of haemodialysis (HD) and peritoneal dialysis (PD) patients in order to study potential determinants of carbamylated Hb (CarbHb) and to investigate the relationship between CarbHb and established measures of dialysis dose/adequacy by multivariate analysis., Results: In 80 HD patients, CarbHb was independently predicted by post-dialysis urea (r=0.40, P:<0.01), serum albumin (r=0.24, P:<0.05) and serum bicarbonate (r=-0.40, P:<0. 05). No correlation was found between CarbHb and measures of dialysis dose/adequacy (Kt/V, urea reduction ratio, weekly dialysis duration, and normalized protein catabolic rate (nPCR)). In 42 PD patients, serum urea was the only significant independent predictor of CarbHb (r=-0.51, P:=0.001). No relationship was found between CarbHb and Kt/V, corrected creatinine clearance (CrCl) or nPCR in PD patients., Conclusions: Serum urea is the most consistent independent predictor of CarbHb in dialysis patients. This association in combination with the lack of a relationship with conventional measures of dialysis dose and a positive relationship with serum albumin suggest that a single measurement of CarbHb is unlikely to be a useful indicator of the adequacy of dialysis.

Background: Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in response to LPS is incomplete., Methods: Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-kappaB (NF-kappaB) and Western blot for the NF-kappaB inhibitory protein IkappaB. To prove the role for NF-kappaB, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5'-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E)., Results: LPS increased NF-kappaB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IkappaBalpha decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IkappaBbeta levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5'-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-kappaB binding sites, located in the enhancer region of the 5'-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 and Sp1 sites, located in the proximal promoter region of MCP-1, reduced basal luciferase activity in unstimulated cells, but had no effect on LPS-stimulated luciferase activity., Conclusions: These studies prove that NF-kappaB is critical for LPS-induced MCP-1 transcription, and AP-1 and Sp1 are essential for basal expression of MCP-1 in rat tubule cells. The species-specific nature of transcriptional regulation of MCP-1 has important implications for the delineation of treatment to prevent endotoxin-mediated renal injury.

Objective: To determine the predictive value of 72-hour blastomere cell number on blastocyst development and to compare success rates of subsequent transfer based on the degree of blastocyst development., Design: Retrospective clinical study., Setting: Private assisted reproductive technology center., Patient(s): Ninety-three women aged 32.0 +/- 5.1 years undergoing oocyte retrieval for IVF., Intervention(s): Bipronucleate oocytes obtained from IVF were grown for up to 168 hours after fertilization and subsequently transferred at the blastocyst stage., Main Outcome Measure(s): Percentages of embryos developing to blastocyst from 72-hour embryos by blastomere cell number and subsequent implantation and pregnancy rates of transferred blastocysts., Result(s): Rates of blastocyst formation and expansion increased as cell numbers at 72 hours increased. Implantation rates were 43% for embryos transferred to women receiving only expanded blastocysts and 17% for embryos transferred to women receiving one or more less developed blastocysts. Pregnancy rates were higher for women receiving only expanded blastocysts than for women receiving one or more less developed blastocysts, although the difference was not significant., Conclusion(s): More developed 72-hour embryos are more likely to become blastocysts and expand. Implantation rates are greater for the transfer of expanded rather than unexpanded blastocysts.

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.

We recently reported that inhibition of the transcription factor nuclear factor-kappaB (NFkappaB) with pyrrolidinedithiocarbamate (PDTC) reduced interstitial monocyte infiltration in rats with proteinuric tubulointerstitial disease, whereas N-acetylcysteine (NAC) was not effective. Here we investigate the effects of antioxidants (PDTC, NAC, and quercetin) on NFkappaB activation and cytokine transcription in primary cultured rat proximal tubular epithelial cells (PTC) stimulated with lipopolysaccharide. Antioxidant-mediated inhibition of NFkappaB activation (PDTC, 20-100 microM; NAC, 100 mM; and quercetin, 50 microM) diminished the induction of both pro- [interleukin (IL)-1beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-2] and anti-inflammatory (IL-10, transforming growth factor-beta1) cytokine transcription in PTC (RT-PCR analysis). PDTC and quercetin did not affect PTC viability, but NAC (100 mM) caused a threefold increase in lactate dehydrogenase leakage (P < 0.001). We conclude that NAC is unable to suppress NFkappaB activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFkappaB is correlated with the nonselective reduction of cytokine transcription in activated tubular cells. These data might explain the protective effects of PDTC-mediated NFkappaB inhibition in tubulointerstitial disease in vivo.

Background: Protein-induced chemokine expression in proximal tubular cells is mediated by the transcription factor nuclear factor-kappa B (NF-kappaB). We hypothesized that in vivo inhibition of renal NF-kappaB activation would reduce interstitial monocyte infiltration in a rat model of nonimmune proteinuric tubulointerstitial inflammation., Methods: Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride [adriamycin (ADR), 7.5 mg/kg] and were studied 7, 14, 21, and 28 days later. In a second study, inhibitors of NF-kappaB [N-acetylcysteine (NAC; 150 mg/kg, b.i.d., i.p.), pyrrolidine dithiocarbamate (PDTC, 50 mg/kg, b. i.d., i.p.)] or vehicle were commenced on day 14 after the onset of proteinuria and were continued until day 30., Results: Rats injected with ADR had increased proteinuria (UpV, day 28, 474 +/- 57; control, 18 +/- 2 mg/day; P < 0.01) and cortical tubulointerstitial injury [tubule cell atrophy, interstitial volume, and monocyte/macrophage (ED-1) infiltration]. Electrophoretic mobility shift assay of nuclear extracts from whole cortex of ADR rats demonstrated that NF-kappaB activation (p50/65, p50/c-Rel) increased from day 7 (4.7 +/- 0.2 fold-increase above control; P < 0.01) was maximal at day 28 (6.2 +/- 0.7; P < 0.01) and correlated with UpV (r = 0.63; P < 0.05) and interstitial ED-1 infiltration (r = 0.67; P < 0.01). Chronic treatment of ADR rats with PDTC suppressed NF-kappaB activation (by 73%; P < 0.05) without any effect on UpV. NF-kappaB inhibition with PDTC was accompanied by a reduction in tubule cell atrophy (59%; P < 0.01), interstitial volume (49%; P < 0.05) and ED-1 infiltration (48%; P < 0.01), and cortical lipid peroxidation (41%; P < 0.05) compared with vehicle-treated ADR rats. In contrast NAC had no effect on NF-kappaB activation, tubulointerstitial injury, or UpV in ADR rats., Conclusion: The activation of NF-kappaB may have an important role in mediating cortical interstitial monocyte infiltration and tubular injury in nonimmune proteinuric tubulointerstitial inflammation.

The transcription and translation of monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, are increased in proximal tubule epithelial cells (PTC) stimulated with pathophysiologically relevant concentrations of albumin. The purpose of this study was to investigate whether nuclear factor kappaB (NFkappaB)/Rel proteins play a role in albumin-induced MCP-1 transcription. Confluent monolayers of rat PTC in primary culture were stimulated with delipidated bovine serum albumin. NFkappaB, the NFkappaB inhibitory protein (IkappaB), and MCP-1 transcription were assessed using electrophoretic mobility shift assays, Western immunoblotting, semiquantitative reverse transcription-PCR, and ribonuclease protection assays. Activation of NFkappaB by delipidated bovine serum albumin (15 mg/ml) was detectable within 2 h, maximal after 8 h, and maintained for at least 16 h of continuous exposure. Supershift analysis showed that the activated proteins were composed of p50/p50, p50/p65, and p50/c-Rel dimers. dimers. Cytoplasmic IkappaBalpha levels were decreased 30 min after stimulation and returned to unstimulated levels by 4 to 8 h. IkappaBbeta levels were decreased at 2 h and there was no recovery until 8 h. Inhibition of NFkappaB with pharmacologic agents (N-tosyl-phenylalanine chloromethyl ketone and dexamethasone) and an antisense oligonucleotide to the rat p65 subunit of NFkappaB significantly reduced MCP-1 transcription. The 3.6-kb 5' flanking region of the rat MCP-1 gene was cloned and sequenced, and two putative kappaB binding sites were identified within the enhancer region. Therefore, albumin increased NFkappaB and reduced IkappaB levels in PTC, and MCP-1 expression was dependent on NFkappaB activation. It is concluded that the activation of NFkappaB/Rel proteins modulates chemokine production in PTC in response to albumin and is likely to have an important role in the mediation of tubulointerstitial injury in proteinuric renal disease.

In a comparison of different methods for estimating Verticillium dahliae in soil, 14 soil samples were analyzed in a blinded fashion by 13 research groups in seven countries, using their preferred methods. One group analyzed only four samples. Twelve soil samples were naturally infested, and two had known numbers of microsclerotia of V. dahliae added to them. In addition, a control was included to determine whether transport had an effect on the results. Results differed considerably among the research groups. There was a 118-fold difference between the groups with the lowest and highest mean estimates. Results of the other groups were evenly distributed between these extremes. In general, methods based on plating dry soil samples gave higher numbers of V. dahliae than did plating of an aqueous soil suspension. Recovery of V. dahliae from samples with added microsclerotia varied from 0 to 59%. Most of the variability within each analysis was at the petri dish level. The results indicate the necessity to check the performance of detection assays regularly by comparing recoveries with other laboratories, using a common set of soil samples. We conclude that wet plating assays are less accurate than dry plating assays.

The potential role of nitric oxide (NO) in iron-induced toxicity was studied in proximal tubule cells in primary culture. NO production (NO2-/NO3-) was significantly increased in iron-treated compared with control cells (3.43 +/- 0.08 vs. 1.56 +/- 0.08 nmol/dish, P < 0.01). NO synthase (NOS) activity was also induced by iron treatment (16.2 +/- 2.0 vs. 0.4 +/- 0.2 nmol of [3H]citrulline/mg protein, P < 0.01). L-Arginine, a substrate for NOS, augmented iron-induced NO production and cell damage [lactate dehydrogenase (LDH) leakage], whereas aminoguanidine, an inhibitor of NOS, reduced iron-induced NO production and LDH leakage. Sodium nitroprusside, an exogenous NO donor, induced LDH leakage in a dose-dependent manner, but no effect on lipid peroxidation (malo-ndialdehyde bis[dimethyl acetal] (MDA) production) was observed. Superoxide dismutase and catalase decreased iron-induced MDA production but did not affect LDH leakage or NO production. Dimethylpyrroline N-oxide and desferal prevented MDA production, LDH leakage, and NO production. We concluded that NO is one of the mediators of iron-induced toxicity in proximal tubule cells. NO-induced toxicity is not dependent on lipid peroxidation. This may explain the variable effect of different antioxidants on cell damage and lipid peroxidation in iron-induced cytotoxicity.

Proteinuria has been invoked as a cause of tubulointerstitial injury in chronic renal disease, and in vivo studies have suggested indirectly the particular nephrotoxicity of one urinary protein holotransferrin (Tf-Fe). However, to date there has been no direct evidence for the nephrotoxicity of Tf-Fe. To examine the potential cytotoxicity of Tf-Fe and the mechanism involved, and to compare this to another urinary protein albumin, rat proximal tubule cells were studied in primary culture. Tf-Fe at pH 6.0 caused functional and ultrastructural injury, but no cytotoxicity was seen with cells exposed to albumin, apotransferrin (transferrin), or Tf-Fe at pH 7.4. The influence of pH on Tf-Fe-induced cytotoxicity was not due to pH per se, but could be explained by an effect on Tf-Fe uptake. At pH 6.0, uptake of 125I-Tf-Fe (3.55 +/- 0.05 versus 1.25 +/- 0.10 fmol/dish, P < 0.01) and intracellular iron concentration (1.14 +/- 0.25 versus 0.46 +/- 0.23 nmol/dish, P < 0.01) were increased compared with values at pH 7.4. In contrast, pH 6.0 did not increase iron uptake from FeCl3. Lysine (100 mM) inhibited Tf-Fe uptake, decreased intracellular iron concentration, and attenuated Tf-Fe-induced cytotoxicity. The iron chelator des-ferrioxamine (200 microM) and hydroxyl radical scavenger dimethylpyrroline N-oxide (32 mM) abolished lactate dehydrogenase leakage induced by Tf-Fe at pH 6.0. Lipid peroxidation, as assessed by production of malondialdehyde, preceded lactate dehydrogenase leakage. In summary, holotransferrin, but not albumin, is toxic to rat proximal tubule cells, a pH-dependent effect involving its uptake into tubule cells, its iron moiety, and its lipid peroxidation.

1. Proximal tubular cell dysfunction in chronic glomerular disease (CGD) has been ascribed, in part, to reabsorption of transferrin-iron from tubular fluid and subsequent cytosolic peroxidative injury. To investigate a possible role for altered mitochondrial function in tubular cell injury in CGD, renal cortical mitochondrial respiratory function was examined in rats with adriamycin nephrosis. 2. State 4 (resting) respiration was increased in adriamycin nephrosis in comparison with control (51 +/- 2 vs 43 +/- 2 ng atoms oxygen (O)/min per mg protein, respectively; P < 0.02). 3. Mitochondrial iron concentration was increased in nephrotic rats compared with control (9.52 +/- 0.70 vs 5.97 +/- 0.26 nmol Fe/mg protein, respectively; P < 0.001) and rates of state 3, state 4 and uncoupled respiration and the severity of proteinuria correlated with mitochondrial iron concentration. 4. To further define the relationship between mitochondrial iron accumulation and altered respiratory function, rats were loaded with iron. 5. In comparison with control, acute iron loading of normal rats impaired creatinine clearance (1.48 +/- 0.02 vs 0.40 +/- 0.29 mL/min), increased kidney weight (1.33 +/- 0.07 vs 1.74 +/- 0.14 g) and impaired mitochondrial enzyme activity (e.g. cytochrome oxidase 185.0 +/- 46.6 vs 362.0 +/- 32.8 delta log [cytochrome C]/min per mg protein; P < 0.05), but had no significant effect on rates of mitochondrial respiration or on mitochondrial fragility. 6. Mitochondrial iron concentration was not increased by iron loading, despite a similar increment in cytoplasmic iron to that seen in rats with adriamycin nephrosis. 7. In summary, resting mitochondrial respiration is increased in nephrotic rats in proportion to mitochondrial iron accumulation. Changes in mitochondrial oxygen consumption do not appear to be a primary event in the tubular cell injury of iron loading.