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Biomedical subjects

P N Furness

Publications and source records attributed to P N Furness.

At least 19 recordsLinked to original sources
PubMed1999

Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy.

BACKGROUND: Proteinuria and tubular atrophy have both been closely linked with progressive renal failure. We hypothesized that apoptosis may be induced by tubular cell exposure to heavy proteinuria, potentially leading to tubular atrophy. Apoptosis was studied in a rat model of "pure" proteinuria, which does not induce renal impairment, namely protein-overload proteinuria. METHODS: Adult female Lewis rats underwent intraperitoneal injection of 2 g of bovine serum albumin (BSA, N = 16) or sham saline injections (controls, N = 8) daily for seven days. Apoptosis was assessed at day 7 in tissue sections using in situ end labeling (ISEL) and electron microscopy. ISEL-positive nuclei (apoptotic particles) were counted in blinded fashion using image analysis with NIH Image. Cell proliferation was assessed by detection of mRNA for histone by in situ hybridization, followed by counting of positive cells using NIH Image. RESULTS: Animals injected with saline showed very low levels of apoptosis on image analysis. BSA-injected rats had heavy proteinuria and showed both cortical and medullary apoptosis on ISEL. This was predominantly seen in the tubules and, to a lesser extent, in the interstitial compartment. Overall, the animals injected with BSA showed a significant 30-fold increase in the number of cortical apoptotic particles. Electron microscopy of tubular cells in a BSA-injected animal showed a progression of ultrastructural changes consistent with tubular cell apoptosis. The BSA-injected animals also displayed a significant increase in proximal tubular cell proliferation. This increased proliferation was less marked than the degree of apoptosis. CONCLUSION: Protein-overload proteinuria in rats induces tubular cell apoptosis. This effect is only partially balanced by proliferation and potentially provides a direct mechanism whereby heavy proteinuria can induce tubular atrophy and progressive renal failure.

Animals↗
PubMed1998

New model of renal warm ischaemia-reperfusion injury for comparative functional, morphological and pathophysiological studies.

BACKGROUND: Renal warm ischaemia-reperfusion injury is pertinent to vascular and transplant surgery. While established models provide functional and morphological data the authors wanted to be able to correlate this with the underlying pathophysiology at any chosen time point, thus allowing future interventional effects on reperfusion injury to be evaluated. METHODS: In a rodent model bilateral renal warm ischaemia (15-60 min) and then reperfusion (20 or 80 min) before nephrectomy allowed for analysis of early reperfusion pathophysiology. The remaining kidney provided functional data (glomerular filtration rate (GFR)) at days 2 and 7 before nephrectomy for late analysis and morphology using a new grading system. RESULTS: Acceptable survival rate (ten of 12 animals) was seen with up to 45 min of warm ischaemia. Renal function was impaired at day 2 following 30-60 min of warm ischaemia (P< 0.01) and day 7 in the 45- and 60-min groups (P < 0.05 and P < 0.01 respectively). Strong correlation existed between duration of ischaemia and GFR at day 2 (r2=0.88) and day 7 (r2=0.95). Histological damage in the cortical tubules was evident in the 45- and 60-min groups (P< 0.01). CONCLUSION: This new model allowed comparative functional, morphological and pathophysiological studies while minimizing the number of animals required. Overall 45 min of warm ischaemia gave significant, recoverable injury and is recommended for investigating renal reperfusion injury.

Animals↗
PubMed1998

Nitric oxide generation is increased in experimental renal warm ischaemia-reperfusion injury.

BACKGROUND: Nitric oxide has a clearly defined place in normal renal homoeostasis while there is a continuing debate as to its role under pathophysiological conditions. This study investigated the role of nitric oxide in a model of renal warm ischaemia-reperfusion injury. METHODS: Groups of rats underwent bilateral renal warm ischaemia (for 15-60 min) followed by reperfusion (20 or 80 min) before unilateral nephrectomy for measurement of renal nitric oxide (as nitroxides) and oxidative damage. Renal function was measured on days 2 and 7 before killing and nephrectomy. A further group received the nitric oxide synthase inhibitor N(G)-nitro L-arginine methyl ester (L-NAME; 50 mg per kg body-weight) before induction of warm ischaemia. RESULTS: In early reperfusion there was a correlation between the duration of warm ischaemia (15-45 min) and renal nitrate (r2=0.97) which increased from a mean(s.e.m.) baseline value of 95(5.9) to 208(17.3) nmol per mg protein following 45 min of warm ischaemia. Levels were further raised at 80 min and maintained through to day 7 (241(12.5) nmol per mg protein in 45-min group). This rise was attenuated by L-NAME (P< 0.01) as was the early rise in oxidative damage seen otherwise. By day 7, however, oxidative damage was increased (all P< or = 0.01). CONCLUSION: Renal nitric oxide increased early in recoverable warm ischaemia-reperfusion injury and remained raised to day 7. Nitric oxide synthase inhibition ameliorated early but exacerbated late damage suggesting that the early burst of nitric oxide is cytotoxic but that overall nitric oxide may exert a cytoprotective effect.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Diagnosis of early acute renal allograft rejection by evaluation of multiple histological features using a Bayesian belief network.

BACKGROUND AND AIMS: The development of the Banff classification of renal transplant pathology has allowed the standardisation of approaches to transplant biopsy histology and reduced interobserver and interdepartmental variation. The usefulness of the Banff classification in the diagnosis of acute rejection has previously been tested by sending sections from 21 "difficult" biopsies to almost all of the renal transplant pathologists in the UK. Although the Banff classification improved reproducibility, the accuracy of diagnosis of early acute rejection was unchanged from the "conventional" approach. Perhaps this is because in making a diagnosis of acute rejection, the Banff classification uses only two features: tubulitis and intimal arteritis. To include more features on a systematic basis would be laborious for a human observer. Therefore, a Bayesian belief network was developed for this task. METHODS: The network was initialised with observations from 110 transplant biopsies. Its performance was then tested on 21 biopsies that had been seen by 37 different renal transplant pathologists in an earlier study. These biopsies had been selected to represent histologically difficult problems but, in retrospect, they all had clear diagnoses of rejection or non-rejection on clinical grounds. RESULTS: Using the Bayesian belief network, a relatively inexperienced pathologist made 19 of 21 correct diagnoses, better than had been achieved by any of the pathologists who had seen the same sections previously (17 of 21), and considerably better than the average proportion of correct diagnoses provided by all 37 renal transplant pathologists (65%). Application of the system by a second pathologist produced a tendency to overdiagnosis of acute rejection, illustrating the consequences of interobserver variation. CONCLUSIONS: In the diagnosis of acute rejection, further useful information can be extracted from features that are currently not considered in the Banff classification. Integration of data by a computer can give a more reliable diagnosis of early acute rejection, but routine application will require the development of a more sophisticated system that can also accommodate clinical data, perhaps one that can continue to "learn" as more data are entered.

Acute Disease↗
PubMed1998

Reproducibility in the quantification of mRNA levels by RT-PCR-ELISA and RT competitive-PCR-ELISA.

The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.

DNA Primers↗
PubMed1997

Basement membrane synthesis and degradation.

The biological importance of complex interactions between cells and extracellular matrix has become widely recognized. For normal epithelial cells, contact with the matrix is limited to the basement membrane. Our understanding of the composition and assembly of basement membranes is increasing, as is our understanding of the mechanisms by which synthesis and degradation of basement membranes are controlled. Basement membrane abnormalities may result from disease and may cause disease. Papers in this edition of the Journal of Pathology discuss changes in basement membrane composition in disease, and add yet another link to the many connections between basement membranes, fibrosis and the control of cell proliferation.

Basement Membrane↗
PubMed1997

The use of digital images in pathology.

Digital images are routinely used by the publishing industry, but most diagnostic pathologists are unfamiliar with the technology and its possibilities. This review aims to explain the basic principles of digital image acquisition, storage, manipulation and use, and the possibilities provided not only in research, but also in teaching and in routine diagnostic pathology. Images of natural objects are usually expressed digitally as 'bitmaps'--rectilinear arrays of small dots. The size of each dot can vary, but so can its information content in terms, for example, of colour, greyscale or opacity. Various file formats and compression algorithms are available. Video cameras connected to microscopes are familiar to most pathologists; video images can be converted directly to a digital form by a suitably equipped computer. Digital cameras and scanners are alternative acquisition tools of relevance to pathologists. Once acquired, a digital image can easily be subjected to the digital equivalent of any conventional darkroom manipulation and modern software allows much more flexibility, to such an extent that a new tool for scientific fraud has been created. For research, image enhancement and analysis is an increasingly powerful and affordable tool. Morphometric measurements are, after many predictions, at last beginning to be part of the toolkit of the diagnostic pathologist. In teaching, the potential to create dramatic yet informative presentations is demonstrated daily by the publishing industry; such methods are readily applicable to the classroom. The combination of digital images and the Internet raises many possibilities; for example, instead of seeking one expert diagnostic opinion, one could simultaneously seek the opinion of many, all around the globe. It is inevitable that in the coming years the use of digital images will spread from the laboratory to the medical curriculum and to the whole of diagnostic pathology.

Humans↗
PubMed1997

A quantitative immunofluorescence study of glomerular cell adhesion proteins in proteinuric states.

Whenever there is heavy proteinuria, the glomerular epithelial cells, the podocytes, show dramatic morphological changes which clearly demonstrate changes in cell adhesion. However, there is little information on the types of cell adhesion molecules expressed in the normal human glomerulus. Assessments of changes in cell adhesion molecules in human proteinuria have been confined to semi-quantitative immunostaining for integrins, and the results have not been entirely consistent. This study sought first to define which cell adhesion molecules are present in the normal glomerulus, using indirect immunofluorescence and a panel of antibodies directed against transmembrane adhesion proteins and against several cytoplasmic proteins which are known to be involved in adhesion. A wide variety of integrins were detected, the dominant form being alpha 3 beta 1. The cytoplasmic focal adhesion proteins vinculin, talin, paxillin, p130CAS, and pp125FAK were detected, although vinculin appeared to be confined mainly to the mesangium. The only intercellular adhesion molecule detected in the vicinity of the slit diaphragm was ZO-1; the results imply that the slit diaphragm does not bear a close relationship to any other form of intercellular junction. Changes in these adhesion components were also studied in proteinuria, using 18 cases each of minimal change nephropathy, 'early' membranous nephropathy, and normal controls. Fluorescence intensity was measured by image capture using a low light video camera and subsequent digital image analysis, an approach which demonstrated acceptable reproducibility. The most striking changes were an increase in phosphotyrosine and p130CAS in the nephrotic patients. Contrary to previous reports, little change was found in the expression of the most abundant integrins, nor did overall glomerular staining for ZO-1 alter. These results imply a controlled alteration in glomerular cell adhesion in nephrotic states in man, probable representing increased turnover of cell adhesion structures rather than the decrease which has been reported in short-term animal models. This is the first report of increased glomerular phosphotyrosine in man, which is associated with less stable adhesions and may be related to the loss of foot processes. Using human biopsy material, it was not possible to determine which proteins were phosphorylated, but the probable relationships to changes in cytoskeletal structure and slit diaphragm permeability justify further study.

Cell Adhesion↗
PubMed1997

A UK-wide trial of the Banff classification of renal transplant pathology in routine diagnostic practice.

BACKGROUND: The Banff classification of renal transplant pathology has gained wide support since its introduction in 1993. There have been several studies which have tested its usefulness in the context of research-oriented centres. We sought to evaluate its use in a wider context. METHODS: We recruited pathologists from all but one of the renal transplant centres in the UK. Sections were circulated from 21 selected, 'difficult' cases, in all of which the clinical question was confirmation or exclusion of acute rejection, and in all of which a definite diagnosis had been obvious from the subsequent clinical course. Participants were asked first to diagnose or exclude acute rejection by their usual approach, then to apply the Banff classification. No clinical information was given beyond the time since engraftment, in order to confine the evaluation to the morphological features present in the sections. At the end of the study the subjective impressions of the participants were sought using a structured questionnaire. RESULTS: Using the Banff classification produced no detectable difference in the number of 'correct' diagnoses when compared with a conventional approach, irrespective of whether the 'correct' diagnosis is based on retrospective clinical information or on the consensus opinion of the pathologists involved, and irrespective of where in the Banff schema one applies a 'cut-off' for the diagnosis of acute rejection. However, the reproducibility of the diagnoses was improved. The results suggest that in the Banff classification the best 'cut-off' for the diagnosis of acute rejection is between Banff category 3 and category 4, although in this difficult area we found a large improvement in diagnostic accuracy if input of clinical information occurs. CONCLUSIONS: The improved reproducibility justifies the use of the Banff classification to harmonise approaches between centres, especially in research projects. While there are good reasons also to adopt it in routine diagnostic practice, further refinement is necessary before an improvement in the accuracy of diagnosis can be demonstrated.

Diagnostic Errors↗
PubMed1997

D-penicillamine reduces renal injury in the remnant model of chronic renal failure in the rat.

BACKGROUND: Glomerulosclerosis and interstitial fibrosis, which are cardinal features of the end-stage kidney, result from accumulation of extracellular matrix proteins, particularly collagen, in the glomerular mesangium and renal interstitium. This study examined the effect of D-penicillamine (DPC), which inhibits collagen deposition, on disease progression in the remnant kidney. METHODS: Two groups of 10 rats underwent two-thirds nephrectomy and were pair-fed 20% casein paste (Gp 1) or the same paste supplemented with 90 mg/kg body wt per day of DPC (Gp 2). Two further groups of five non-nephrectomized animals also received 20% casein paste either alone (Gp 3) or supplemented with DPC (Gp 4). In a further experiment, systolic blood pressure was compared at 1 and 4 weeks after nephrectomy in eight DPC-treated remnants and eight untreated controls. RESULTS: Gp 2 developed significantly less proteinuria than Gp 1 (41 +/- 9 vs 142 +/- 33 mg/24 h at 6 weeks, P < 0.005; 136 +/- 36 vs 282 +/- 59 mg/24 h at 12 weeks, P < 0.05). At sacrifice after 12 weeks, glomerular filtration rates were higher (1.34 +/- 0.08 vs 1.07 +/- 0.1 ml/min, P < 0.05), kidney total collagen content was lower (14.9 +/- 1.5 vs 26.9 +/- 5.4 mg/kidney, P < 0.05) and glomerular abnormalities, interstitial fibrosis and lymphocytic infiltration were less marked in Gp 2 compared with Gp 1. DPC had no effect on protein excretion, total kidney collagen or GFR in non-nephrectomized rats, and did not influence the early rise in blood pressure seen after two-thirds nephrectomy. CONCLUSIONS: These findings demonstrate that DPC reduces renal injury in the remnant kidney, and raise the possibility of a therapeutic role for DPC in the treatment of patients with chronic renal failure.

Animals↗
PubMed1997

Semiautomatic quantitation of macrophages in human renal biopsy specimens in proteinuric states.

AIMS: To develop and validate a rapid and economical semiautomated approach to the measurement of immunostainable tissue components which is applicable to routine diagnostic practice. To apply this approach to the measurement of macrophages in renal biopsy specimens in nephrotic states, as protein in the renal tubules may induce macrophage infiltration, and the morphology of macrophages in tissue sections does not lend itself to cell counting. METHODS: Macrophages were identified by immunostaining with a pan-macrophage marker, followed by digital image capture and analysis using a macro procedure written for the freeware image analysis program NIH-Image. RESULTS: The method was rapid, robust and accurate to within the limits imposed by sampling error inherent in the use of small needle biopsy specimens. Very few macrophages are found in normal kidney (mean volume fraction (+/- 95% confidence limits) 0.04% (0.02%)) but infiltration of macrophages was detected in minimal change nephropathy (0.29% (0.12%)) and in membranous glomerulonephritis (0.42% (0.11%)). A statistically significant correlation was found between macrophage volume fraction and weight of proteinuria in minimal change nephropathy but not in membranous glomerulonephritis. Correlations were found in both diseases between macrophage volume fraction and serum creatinine at time of biopsy. CONCLUSIONS: The equipment is inexpensive and measurement takes less than one minute per biopsy specimen. The results indicate that macrophage infiltration is part of the pathological process in minimal change nephropathy and membranous glomerulonephritis. The correlation with creatinine at time of biopsy suggests that renal impairment in minimal change nephropathy may result from infiltration by immunologically active cells and not merely from haemodynamic changes in nephrons. However, the correlation is not close, indicating that the relation between macrophage infiltration and disease severity is not a simple one.

Biopsy↗
PubMed1997

A questionnaire-based survey of errors in diagnostic histopathology throughout the United Kingdom.

AIMS: To obtain a crude estimate of the rate at which consultant histopathologists become aware of errors in their work, and to gather information about the circumstances under which errors are made. METHODS: A postal questionnaire was sent to all consultant histopathologists (1021) in the United Kingdom. RESULTS: There was a 19.8% response to the questionnaire (202 returned). 119 pathologists reported 244 errors, 82 from within the preceding year. 42% of these errors had affected patient management. Pathologists usually blamed human error or excessive workload; however, data analysis did not reveal an excess of responses from laboratories with heavy workloads. A disproportionately large proportion of errors appear to be made in samples from the lymphoreticular system and by locum consultant pathologists. CONCLUSIONS: This study undoubtedly suffered from reporting bias, but the figures suggest that a typical pathologist probably becomes aware of having made a serious diagnostic error approximately once per year. A larger, more systematic study of the circumstances under which errors are likely to occur appears to be justified but would require significant resources because of the large sample size required.

Consultants↗
PubMed1996

The effect of various forms of heparin on the release of immune complexes from the surface of cultured mesangial cells.

Heparin is capable of enhancing the rate of release of antigen from nephritic rat kidneys. It also interferes with the binding of immune complexes by cultured glomerular mesangial cells. Postulating that these two effects might be related, we sought to determine what basic aspects of the molecular structure of heparin are responsible for the interference with binding in vitro. After cultured mesangial cells had bound radiolabelled synthetic immune complexes, heparin or a variety of structurally related molecules were added to the supernatant. De-N-sulphated heparin, heparan sulphate, low molecular weight heparin, and low molecular weight dextran sulphate had no effect on immune complex binding. High molecular weight dextran sulphate was able, like heparin, to dislodge immune complexes from mesangial cells, suggesting that high molecular weight and high sulphation are required. These results differ from previous findings in vivo, suggesting that the effect of heparin in vivo is not due to interaction at the mesangial cell surface. Alternative explanations for the effect of heparin in the intact animal include destabilization of the immune complex structure or, more probably, an effect at the boundary between the immune complex deposit and the basement membrane.

Animals↗