keith Stewart

High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) i...

The presence of cytotoxic HLA antibodies (Ab1) against donor lymphocytes in pretransplant sera is almost always associated with rapid rejection of the renal transplant. We have investigated the possibility that antiidiotypic antibodies (Ab2) to cytotoxic HLA antibodies might modulate the immune response and favorably influence renal allograft outcome. The role of antibodies (Ab3) which potentiate the cytotoxic effect of Ab1 was also studied. Pretransplant sera from 63 patients were tested for inhibitory or potentiating activity in the short antiidiotypic assay. Inhibitory activity was detected in 30 patients and in 28 the transplant survived more than a year. Of patients without antibody activity 11 of 17 had grafts surviving more than one year, and of those showing potentiating activity 11 of 16 were functioning at a year. The difference in transplant survival between the first group and the other two groups was statistically significant (P less than 0.05). There was no significant difference in survival rates between the latter two groups. Potentiating activity is therefore not an independent predictor of transplant failure, whereas the presence of antiidiotypic antibody activity did correlate with improved allograft survival.

Culture of isolated kidney glomerular cells has been employed for almost four decades as a tool to dissect pathophysiological effects of individual cell types in renal disease. This chapter aims to highlight in detail the available techniques to isolate, culture, and characterize human glomerular epithelial and mesangial cells. To establish primary culture of these cells, glomeruli are isolated from the cortex of kidney by differential sieving and cellular outgrowths from cultured glomeruli further subcultured in appropriately coated tissue culture plates/flasks. Methods used for characterization of isolated glomerular mesangial and epithelial cells (podocytes) are described as are the phenotypic markers useful for identification. Other sources of isolated glomerular cells such as immortalized cell lines are briefly discussed.

We have previously shown that the presence in pretransplant recipient sera of Fc receptor blocking antibodies detected by the EA inhibition assay is correlated with improved allograft survival. Twenty-four such sera were assessed for the presence of autoantibodies by the EA inhibition and lymphocytotoxicity assays. No autolymphocytotoxic antibodies were found, and autologous EA inhibition was noted in only one case. EA-inhibiting alloantibodies did occur, and their presence was correlated with improved allograft survival. Sera from 37 dialysis patients were also studied, and neither autologous EA inhibiting nor autologous lymphocytotoxic antibodies were present. Thus Fc receptor blocking alloantibodies that were correlated with improved renal transplant survival were not autoantibodies.

Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients.

Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the mechanism of cisplatin nephrotoxicity in vivo and in an in vitro model system. Nephrotoxicity was induced in rats (6 mg/kg cisplatin i.p.) and mice (10 mg/kg cisplatin i.p.). Cisplatin administration significantly elevated blood urea nitrogen (BUN) and serum creatinine in male Sprague Dawley rats day 5 post-treatment (BUN Delta+28+/-5 micromol/ml; serum creatinine Delta+108+/-4 nmol/ml, P<0.05) and in male C57BL6 mice day 4 post-treatment (BUN Delta+21+/-4 micromol/ml; serum creatinine Delta+81+/-5 nmol/ml, P<0.05). Nephrotoxicity was confirmed by histological analysis that revealed significant damage to the proximal tubules of cisplatin- versus saline vehicle-treated animals. Inhibition of gamma glutamyltranspeptidase prevented cisplatin nephrotoxicity in Sprague Dawley rats (day 5 BUN Delta+1+/-2 micromol/ml; serum creatinine Delta+8+/-4 nmol/ml) and C57BL6 mice (day 4 BUN Delta+1+/-0.8 micromol/ml; serum creatinine Delta-1+/-2 nmol/ml), but not cellular toxicity in rat proximal tubular (RPT) or human proximal tubular (HPT) cultures. Inhibition of aminopeptidase N (AP-N) or renal dipeptidase (RDP) in male Sprague Dawley rats, or in RPT and HPT cell cultures, did not reduce cisplatin toxicity. In contrast to published findings inhibition of C-S lyase did not prevent the nephrotoxicity of cisplatin in vivo or cellular toxicity in vitro. These data demonstrate that the biotransformation enzymes AP-N, RDP and C-S lyase are not implicated in the metabolism of cisplatin to a nephrotoxic metabolite as has been previously hypothesised. Instead, our data demonstrate that gamma glutamyltranspeptidase is a key enzyme involved in mediating cisplatin nephrotoxicity, which potentially acts to cleave cisplatin-GSH conjugates to a toxic metabolite.

Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a number of strains and experimental models, but the inter-relationship between the two is not clear. The present study was undertaken to compare the pattern of glomerular injury in these two types of GS. One- and two-shot Thy1 glomerulonephritis (GN) was induced at 2 months of age and followed for 12 months. At 12 months histological injury in proteinuric rats was characterized by segmental hyaline lesions. Two-shot Thy1 GN resulted in accelerated, but morphologically identical injury at 8 months. Histological lesions predictive of subsequent accelerated GS were evaluated at 1, 2, 4 and 6 months. In this regard, glomerular hypercellularity, rather than hypertrophy or matrix increase, was the most consistent histological index of later accelerated disease. The profibrotic cytokines transforming growth factor (TGF)-beta(1) and -beta(3) were localized distinctly to segmental hyaline lesions, but not to areas of matrix increase within the glomerular tuft. This study reveals that GS after Thy1 GN represents acceleration of an age-related disease, presents evidence for use of prolonged glomerular hypercellularity as the best histological index of future disease progression, and correlates the key lesion of GS in these animals, the segmental hyaline lesion, with the presence of TGF-beta peptides.

Macrophages are intimately involved in the development of immune-mediated inflammation, including glomerulonephritis. We have transduced primary cultures of macrophages to express IL-10 and tested the ability of these cells to control rat nephrotoxic nephritis (NTN), a model of human glomerulonephritis. Ad-IL-10-transduced bone-marrow-derived macrophages (BMDM) produced large amounts of IL-10 in culture, and their TNF-alpha production was decreased in response to interferon-gamma and LPS. Transduced macrophages were injected into the renal artery of rats, 6 h after the induction of NTN, where they localized efficiently to inflamed rat glomeruli. Delivery of IL-10-expressing macrophages to nephritic rats produced a marked reduction in albuminuria compared with unmodified NTN or injection of Ad-null-transduced BMDM. IL-10 treatment decreased the number of glomerular ED1- and ED3-positive cells, MHC class II expression, and the number of fibrinoid lesions. Interestingly, anti-inflammatory changes in the Ad-IL-10-injected kidney were mirrored by changes in the contralateral kidney. These results highlight that Ad-IL-10-transduced macrophages infiltrate inflamed glomeruli and reduce the severity of glomerular inflammation, emphasizing the value of local delivery of genetically modified macrophages in the manipulation of inflammatory disease.

Diagnostic bronchial biopsy samples from lung cancer patients may be used for molecular biologic analyses to help select therapy and provide prognostic information. Some have suggested that direct molecular analysis of bronchial biopsy fragments may be feasible, bypassing histologic examination. We analyzed a series of 100 bronchial biopsy specimens in lung cancer patients to assess the frequency and quantity of tumor present in biopsy samples. The proportion of tumor in bronchial biopsy specimens was assessed by measuring the tumor area in histologic sections using computer-aided morphometry. In only 48% of cases did all the biopsy fragments contain some tumor. The median number of fragments obtained at bronchoscopy was 4; median number actually containing tumor was 3. The mean total surface area of tumor (as a percentage of the total sample area) in biopsy fragments was, for all cases, 33.4%; median area 28%. Biopsies with small cell carcinoma had more tumor (mean area 46.5%, median 49%; p = 0.0006) than all other non-small cell carcinoma cases. Malignant bronchial biopsy samples frequently contain limited amounts of primary carcinoma. Often, one or more of the biopsy fragments will not contain tumor. This has important implications for the storage and use of bronchial biopsy samples for genetic analysis.

Caspase activation has been implicated in the development of ischemia-reperfusion injury. Here, we investigate the effects of different caspase inhibitors on the renal dysfunction and injury caused by ischemia-reperfusion of the rat kidney. Bilateral clamping of renal pedicles (45 min) followed by reperfusion (6 h) caused significant renal dysfunction and marked renal injury. Caspase-1 inhibitor II (N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-1-(carboxymethyl)-3-chloro-2-oxo-propyl]-L-alaninamide, Ac-YVAD-CMK, 3 mg/kg, administered i.p.) significantly reduced biochemical and histological evidence of renal dysfunction and injury. However, although caspase-3 inhibitor I (N-acetyl-L-aspartyl-L-glutamyl-N-(2-carboxyl-1-formylethyl]-L-valinamide, Ac-DEVD-CHO, 3 mg/kg, administered i.p.) produced a significant improvement of renal (glomerular) dysfunction (reduction of serum creatinine levels), it was not able to reduce tubular dysfunction and injury. Furthermore, the pan-caspase inhibitor caspase inhibitor III (N-tert-butoxycarbonyl-aspartyl(OMe)-fluoromethylketone, Boc-D-FMK, 3 mg/kg, administered i.p.) did not reduce renal dysfunction and injury. Both caspase-1 and -3 inhibitors markedly reduced the evidence of oxidative and nitrosative stress in rat kidneys subjected to ischemia-reperfusion. Overall, these results demonstrate that inhibition of caspase-1 reduces renal ischemia-reperfusion injury to a greater extent than caspase-3 inhibition, supporting the notion that the mode of acute cell death in our model of renal ischemia-reperfusion is primarily via necrosis. Furthermore, our finding that a pan-caspase inhibitor did not reduce the renal dysfunction and injury suggests that activation of some caspases during ischemia-reperfusion could provide protection against acute ischemic renal injury. Overall, these results demonstrate that inhibition of caspase-1 activity reduces renal ischemia-reperfusion injury and that this therapeutic strategy may be of benefit against ischemic acute renal failure.

It is unclear which patients with breast cancer benefit from anthracycline-based neoadjuvant chemotherapy and whether taxanes increase survival. Hsp70 and serpinB3 inhibit a lysosomal cell death pathway induced in anthracycline and taxane treated cells, which may be critical for breast cancer cell survival. Thus we evaluated serpinB3 and Hsp70 as putative prognostic biomarkers in breast cancer patients treated with neoadjuvant chemotherapy. SerpinB3 and Hsp70 were measured by immunohistochemistry in residual breast tumours of patients without a complete pathological response [pCR] (n = 250), from a retrospective cohort of 296 patients treated with anthracycline-based chemotherapy with or without sequential docetaxel prior to surgical resection. SerpinB3 (P = 0.02) and Hsp70 (P = 0.008) positivity in residual tumour were associated with a poor pathological response and serpinB3 was an independent prognostic biomarker (HR 2.1 (95% CI 1.2-3.8), P = 0.02). Docetaxel significantly improved overall survival of breast cancer patients treated with neoadjuvant chemotherapy. Furthermore, serpinB3 positivity predicted poor survival in patients treated with anthracycline-based chemotherapy alone (P = 0.02), but those with serpinB3 negative tumours had as equally good survival as those also treated with docetaxel (P = 0.7). Survival was independent of serpinB3 expression in patients who received sequential docetaxel. The Nottingham prognostic index (NPI), calculated at surgical resection, predicted overall survival in these neoadjuvantly treated patients (P < 0.001) and serpinB3 status segregated patients with a moderate NPI into distinct prognostic subgroups. The use of clinical (NPI) and molecular (serpinB3) biomarkers measured at surgical resection to provide accurate prognostication in patients who do not achieve a pCR following neoadjuvant chemotherapy could facilitate optimal post-operative clinical management of these patients and is of significant clinical value. Furthermore, serpinB3 status in residual tumour is a biomarker of neoadjuvant docetaxel benefit in patients not achieving a pCR and use of serpinB3 molecular subtyping for adjuvant docetaxel treatment planning warrants further investigation.

Recent evidence indicates that peroxisome-proliferator activated receptor (PPAR) agonists protect against ischemia/reperfusion (I/R) injury. Here we investigate the effects of the PPAR-gamma agonists, rosiglitazone and ciglitazone, on the renal dysfunction and injury caused by I/R of the rat kidney in vivo. Rosiglitazone or ciglitazone were administered to male Wistar rats prior to and during reperfusion. Biochemical indicators of renal dysfunction and injury were measured and histological scoring of kidney sections was used to assess renal injury. Expression of PPAR isoforms and intercellular adhesion molecule-1 during renal I/R were assessed using RT-PCR and Northern blot, respectively. Myeloperoxidase activity and activation of poly(ADP-ribose) polymerase (PARP) were used as indicators of polymorphonuclear (PMN) cell infiltration and oxidative stress, respectively. Expression of PPAR-alpha, PPAR-beta and PPAR-gamma 1 (but not PPAR-gamma 2) was observed in kidneys with down-regulation of PPAR-alpha expression during renal I/R. Rosiglitazone and ciglitazone significantly reduced biochemical and histological signs of renal dysfunction and injury. Renal expression of ICAM-1 caused by I/R was reduced by rosiglitazone and ciglitazone which was reflected by decreased PMN infiltration into reperfused renal tissues. Both rosiglitazone and ciglitazone reduced PARP activation indicating a reduction of oxidative stress. These results suggest that the PPAR-gamma agonists rosiglitazone and ciglitazone reduce the renal dysfunction and injury associated with I/R of the kidney. We propose that one mechanism underlying the protective effects involves inhibition of the expression of ICAM-1, a reduction of PMN infiltration into renal tissues and subsequent reduction of oxidative stress.

Wendita calysina is a Paraguayan herbaceous plant commonly known as burrito. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside. From W. calysina leaves, a standardized, water-soluble extract (WSE) rich in phenylpropanoid glycosides has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. In this study, we investigated the effect of the W. calysina WSE both in vitro in murine macrophage cell line J774.A1 stimulated with lipopolysaccharide (LPS) and, in vivo in an animal model of acute inflammation, carrageenan-induced pleurisy. Here we report that W. calysina WSE (0.05, 0.1, and 0.5 mg/ml) inhibited inducible nitric oxide synthase (iNOS) expression and activity in LPS-stimulated J774.A1. In vivo experiments showed that injection of carrageenan (2%) into the pleural cavity of rats elicited an acute inflammatory response characterized by iNOS expression, intercellular adhesion molecule-1 (ICAM-1) up-regulation, nitrotyrosine and poly (ADP-ribose) synthase (PARS) formation, and lung tissue damage-all parameters significantly reduced by W. calysina WSE (500 mg/kg per os). These results report, for the first time, that a treatment with W. calysina WSE exerts anti-inflammatory effects both in vitro and in vivo.