Involvement of renal ACE activity in proteinuria-associated renal damage in untreated and treated adriamycin nephrotic rats.

Proteinuria is assumed to play a pathogenetic role in progressive renal damage. Angiotensin-converting enzyme (ACE) inhibition reduces proteinuria and provides renoprotection. This suggests that ACE activity might play a pathogenetic role in the development of proteinuria-induced renal structural damage. We investigated this hypothesis in untreated and treated established adriamycin nephrosis, a model of proteinuria-induced renal damage. In a time-course experiment, the development of renal structural damage in untreated adriamycin nephrotic rats was paralleled by a significant rise in renal ACE activity. Moreover, on cross-sectional analysis, a consistent positive correlation between renal, but not plasma, ACE activity and proteinuria, focal glomerulosclerosis and interstitial injury was present. Notably, these associations were present, not only in the untreated condition, but also during intervention with either ACE inhibition or AT(1)-receptor antagonism. Interestingly, we found that higher renal ACE activity is associated with more severe renal damage for a given amount of proteinuria, suggesting that renal ACE activity may be either a permissive or a promoting factor in the processes by which proteinuria eventually leads to renal structural damage. This relationship was abolished by renin-angiotensin system (RAS)-blockade, suggesting that RAS-mediated effects are involved in the relationship between renal ACE activity and proteinuria-induced renal damage. In conclusion, in untreated as well as treated adriamycin nephrotic rats, renal ACE activity is closely associated with renal outcome. This association appears to be independent of the specific mode of blockade of the RAS. Renal ACE activity is a consistent marker of individual differences in proteinuria-associated renal damage: further studies are needed to investigate a possible pathogenetic role in renal damage.

RT-PCR and immunohistochemical evaluation of sentinel lymph nodes after in vivo mapping with Patent Blue V in colon cancer patients.

OBJECTIVE: Lymph node status is the most important predictive factor in the treatment of colorectal cancer. As sentinel lymph node (SLN) biopsy might upstage stage II colon cancer, it could have therapeutic consequences in the future. We investigated the feasibility of in vivo SLN detection with Patent Blue V dye and evaluated nodal microstaging and ultrastaging using cytokeratin immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIAL AND METHODS: In 30 consecutive patients operated on for colon cancer, subserosal injection with Patent Blue dye was used for SLN detection in four different hospitals under the supervision of one regional coordinator. In searching for occult micrometastases, each SLN was examined at three levels. In tumor-negative SLNs at routine hematoxylin-eosin (H&E) examination (pN0) we performed CK8/CK18 immunohistochemistry (IHC) and RT-PCR for carcinoembryonic antigen (CEA). RESULTS: The procedure was successful in 29 out of 30 patients (97%). The SLN was negative in 18 patients detected by H&E and IHC. In 16 patients the non-SLN was also negative, leading to a negative predictive value of 89% and an accuracy of 93%. Upstaging occurred in 10 patients (33%) - 7 by IHC and 3 by RT-PCR. Aberrant lymphatic drainage was seen in 3 patients (10%). CONCLUSIONS: The SLN concept in colon carcinoma using Patent Blue V is feasible and accurate. It leads to upstaging of nodal status in 33% of patients when IHC and PCR techniques are combined. Therefore, the clinical value of SLN should be the subject of further studies.

Vena cava filter behavior and endovascular response: an experimental in vivo study.

PURPOSE: To evaluate the behavior and endovascular response of a new nitinol permanent vena cava filter, the TrapEase. METHODS: Percutaneous implantation of the filter was performed in six goats, with inferior vena cava (IVC) diameter close to that of man. Radiologic data concerning the IVC, filter diameter, patency and stability were collected. At 2, 4, 20 and 26 weeks post-implantation, histopathologic analysis of the IVC wall was performed at the site of filter distension, and distal and proximal to the filter. RESULTS: All filters remained patent. There was no migration and no signs of biological incompatibility. Signs of neointimalization were seen at 2 weeks, with well-developed neointima at 4 weeks. No acute vessel wall perforation was detected by cavography at implantation. During follow-up histologic analysis at 26 weeks, perforation of some of the small fixation barbs was seen, causing minimal damage to the vessel wall and adjacent organ tissue without impairing organ function. These events were well tolerated, probably due to the gradual nature of the penetration of fixation barbs allowing reactive fibrous tissue development. At 26 weeks the parallel filter struts were well covered with neointima and did not perforate the vessel wall. There were no complications associated with the filter implantation. CONCLUSIONS: The TrapEase vena cava filter was well tolerated and is suitable for incorporation into the IVC wall of healthy animals without any apparent deleterious reaction due to biological incompatibility.

Vessel wall reaction after vena cava filter placement.

PURPOSE: To evaluate the interaction between the Cordis Keeper vena caval filter and vessel wall in a porcine model. METHODS: Implantation of the filter was performed in five pigs. Radiologic data concerning inferior vena cava (IVC) diameter and filter patency, filter leg span, and stability were collected. At 2 or 6 months post-implantation, histopathologic analysis of the IVC wall was performed. RESULTS: All filters remained patent with no evidence of migration. However, at 6 months follow-up, two legs of one filter penetrated the vessel wall and were adherent to the liver. These preliminary results suggest that with the observed gradual increase in the filter span, the risk of caval wall penetration increases with time, especially in a relatively small IVC (average diameter 16 mm). CONCLUSION: The Cordis Keeper filter was well tolerated, but seems to be prone to caval wall penetration in the long term.

Clinical relevance of immunohistochemical staining for ecto-AMPase and ecto-ATPase in chronic allograft nephropathy (CAN).

INTRODUCTION: Chronic allograft nephropathy (CAN) is a major cause of deterioration of kidney function in transplanted patients. It is thought that glomerular ischaemia may contribute to glomerular dysfunction and proteinuria in these subjects. As reduced expression of glomerular ecto-ATPase concurrent with upregulation of glomerular ecto-AMPase activity is associated with local ischaemia, we compared the expression of these glomerular ecto-enzymes in kidney biopsies from subjects with CAN or with acute rejection vs normal human kidney tissue. METHODS: Kidney biopsies in comparison with normal kidney tissue samples (n = 10) were studied from subjects with CAN (n = 6), acute interstitial rejection (n = 13), acute vascular rejection (n = 3), or subjects whose biopsies were histologically difficult to classify (n = 8). Cryostat sections (4 micro m) were stained for ecto-ATPase using standard procedures. For the demonstration of ecto-AMPase activity, conventional enzyme histochemistry was used. Reaction product of individual sections was quantified using computerized image analysis. RESULTS: The results clearly show decreased expression of glomerular ecto-ATPase in combination with increased glomerular ecto-AMPase in all biopsies from subjects with CAN vs normal kidney tissue (P < 0.001). Although to a lesser extent, this staining pattern was also observed in patients with vascular rejection as well as in subjects whose biopsies were histologically difficult to classify (P < 0.01), while biopsies from subjects with interstitial rejection and normal control tissue stained negative for glomerular ecto-AMPase. CONCLUSION: In CAN diminished glomerular ecto-ATPase expression occurs in association with significantly enhanced activity of glomerular ecto-AMPase. This is a strong indication for ischaemic injury of the glomerular microvasculature. As positive staining for ecto-AMPase in acute rejection episodes may be an important sign for long-term prognosis, we feel that screening of biopsies from individual subjects for glomerular ecto-AMPase activity should be considered.

Expression of inducible and endothelial nitric oxide synthases, formation of peroxynitrite and reactive oxygen species in human chronic renal transplant failure.

Nitric oxide (NO.) is produced by NO synthases (NOS) and can interact with reactive oxygen species (ROS) to form peroxynitrite, which induces protein damage by formation of nitrotyrosine. NO. has a promotional effect on acute rejection. To investigate the role of NO. during chronic renal transplant failure (CRTF), we studied the expression of eNOS and iNOS in conjunction with H2O2 production and the formation of nitrotyrosines. Nephrectomy material from 10 patients and 10 control kidneys was used in this study. Expression of iNOS, eNOS, nitrotyrosine and the presence of ROS-producing cells and macrophages were determined using immunohistochemistry. INOS expression in nonsclerosed glomeruli and interstitium was significantly increased in patients with CRTF (p < 0.05). Glomerular eNOS expression was decreased in patients with CRTF compared with glomeruli of control kidneys (p < 0.01). Nitrotyrosine and ROS positive cells were significantly increased in CRTF in the interstitium (p < 0.05), but not in glomeruli. In summary, we found a marked interstitial increase in iNOS protein expression together with a decrease in glomerular eNOS expression in CRTF patients, associated with a significant increment in ROS and nitrotyrosine-positive cells in the interstitium. Our results suggest that loss of NO. production by glomerular eNOS in conjunction with an increased NO. production by interstitial iNOS, together with the formation of ROS and nitrotyrosine, is involved in the pathogenesis of CRTF.

Addition of AT1 blocker fails to overcome resistance to ACE inhibition in adriamycin nephrosis.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors provide renoprotection, but there is considerable interindividual variability in therapeutic efficacy, with residual proteinuria and progressive renal function loss in many individuals. This requires additional strategies to optimize therapy response, particularly for individuals with a poor response to ACE inhibition. We studied whether co-treatment with an angiotensin II subtype 1 (AT1) receptor antagonist (AII-A) improves the individual antiproteinuric response of maximal ACE inhibition in established adriamycin nephrosis. METHODS: Rats were instituted on lisinopril (75 mg/L) six weeks after disease induction. After two weeks rats were re-stratified for residual proteinuria to continue this regimen, to a higher dose of lisinopril (150 mg/L) or to co-treatment with the AII-A L 158,809 for another four weeks. Groups on monotherapy AII-A and vehicle served as controls (all groups N=15). RESULTS: Lisinopril lowered proteinuria by 63% from 741 to 246 g/day (range of percentage change -90 to +2%). Neither increasing the dose of the ACE inhibitor nor addition of AII-A to ACE inhibition improved the antiproteinuric efficacy on a group or individual level: non-responders remained non-responders. All drug categories reduced hard end-points of focal glomerulosclerosis to a similar degree. CONCLUSIONS: ACE inhibition has variable renal protective efficacy in the adriamycin model. Neither increasing the dose of the ACE inhibitor beyond the optimal level nor co-treatment with AII-A overcome the individual therapy resistance. Thus, in established adriamycin nephrosis, blockade of the renin-angiotensin system at two different levels offers no additional benefit over ACE inhibition alone, either on the group or individual level.

Therapy evaluation of laryngeal carcinomas by tyrosine-pet.

INTRODUCTION: One of the major problems in head and neck oncology is determination of tumor status after radiotherapy. Physical examination and conventional imaging by CT and MRI do not always accurately differentiate between residual or recurrent tumor and posttreatment inflammation, fibrosis, edema, or scarring. The feasibility of positron emission tomography (PET) with L-[1-(11)C]-tyrosine (TYR) for therapy evaluation of laryngeal squamous cell carcinomas by identification of residual or recurrent disease after radiotherapy was investigated. PATIENTS AND METHODS: Nineteen patients with laryngeal carcinomas had standard workups with endoscopy and conventional imaging. All subjects underwent a TYR PET scan (PET1) before definitive treatment. For determination of tumor status, a second TYR PET scan (PET2) was performed 3 months after radiotherapy. At the time of scanning, seven patients were clinically suspected of having residual disease, and in these cases, additional CT imaging and biopsies during endoscopy were performed. During the minimal follow-up period of 29 months, six patients had clinical suspicion of recurrent disease. In these six cases, a third TYR PET (PET3), CT imaging, and biopsy were performed. RESULTS: All pretreatment tumors were depicted by TYR PET (PET1). Three months after radiotherapy, sensitivity and specificity of TYR PET (PET2) for discrimination between residual tumor and benign posttreatment tissue changes were both 100%, and for CT, 50% and 67%, respectively. For detection of recurrent tumor during follow-up, sensitivity and specificity of TYR PET (PET3) were also 100%, and CT, 75% and 50%, respectively. CONCLUSIONS: Dynamic TYR PET is an accurate imaging modality for therapy evaluation in detection of residual and recurrent disease with higher sensitivity (100%) and specificity (100%) for discrimination of tumor status by TYR PET compared with conventional imaging.

Carbon-11 tyrosine PET for visualization and protein synthesis rate assessment of laryngeal and hypopharyngeal carcinomas.

Accurate assessment of tumour extent and lymph node involvement in squamous cell carcinomas of the head and neck region is essential for therapy planning. Unfortunately, conventional diagnostic examination and imaging techniques, which monitor tumours on the basis of anatomical parameters, have drawbacks in clinical practice. The aim of this study was to investigate the feasibility of L-[1-(11)C]-tyrosine (TYR) positron emission tomography (PET) for visualisation of squamous cell carcinoma of the larynx and hypopharynx and quantification of tumour activity by assessment of protein synthesis rate (PSR). Dynamic TYR PET was performed on 31 patients with T1-T4 laryngeal or hypopharyngeal carcinoma before therapy. Plasma activity of TYR, (11)CO(2) and (11)C-protein levels were measured, and PSRs were calculated for primary malignancies. All 31 laryngeal and hypopharyngeal tumours were visualised as a hotspot (sensitivity 100%). The median PSR of the tumours (2.06 nmol ml(-1) min(-1); range 0.72-6.96) was significantly higher ( P<0.001) than that of non-tumour (background) tissue (0.51 nmol ml(-1) min(-1); range 0.22-0.89). L-[1-(11)C]-Tyrosine PET appears to be a potential method for visualisation of primary laryngeal and hypopharyngeal tumours. In vivo quantification of tumour activity by assessment of PSR is possible and may have a future role in the therapy planning and therapy evaluation of laryngeal and hypopharyngeal tumours.

Detection of micrometastatic breast cancer by means of real time quantitative RT-PCR and immunostaining in perioperative blood samples and sentinel nodes.

The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein-2 (EGP-2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Fifty-eight breast cancer patients, 52 primary tumors, 75 sentinel nodes (SN) and 149 peripheral blood (PB) samples (from before, during and 4 days after operation) were examined. Immunostaining was performed with antibodies directed against EGP-2 and CK19. Detection limits were one Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line cell/2.10(6) leukocytes (immunostaining) and one MCF-7 cell/10(6) leukocytes qRT-PCR. Control noncancer lymph nodes (n = 10) showed nonspecific CK19 staining, but were qRT-PCR negative; control healthy volunteer PB (n = 11) was always negative. Primary tumor samples, all positive with immunostaining, showed a wide variation of EGP-2 (>10(4) fold) and CK19 mRNA expression (>10(3) fold). SN (n = 19) from 16 patients were tumor-positive with routine haematoxylin-eosin (H&E) and/or immunostaining. SN tumor presence was positively correlated to qRT-PCR expression, but 3 tumor-positive SN were false negative with qRT-PCR. Three SN were qRT-PCR positive, while tumor negative with H&E and/or immunostaining. No immunostaining positive PB was observed, but 19 patients (33%) had one or more qRT-PCR positive PB samples. We concluded that primary tumors have varying expressions of EGP-2 and CK19 mRNA. Both markers can be used in qRT-PCR to obtain adequate sensitivity for single tumor cell detection. In SN, immunostaining appears more sensitive/specific than H&E or qRT-PCR for tumor detection. No immunostaining positivity was found in PB, while 33% of patients had qRT-PCR positive PB. The clinical value of these findings will have to be clarified.