Wayne C. Waltzer

Effect of freezing parameters (freeze cycle and thaw process) on tissue destruction following renal cryoablation.

BACKGROUND AND PURPOSE Renal cryoablation is a successful nephron-sparing treatment alternative for selected patients with small renal tumors. The purpose of this study was to compare the effects of the number of freeze cycles (one v two) and the thaw process (active v passive) on renal tissue following cryodestruction. MATERIALS AND METHODS Sixteen female mongrel dogs (19.9 +/- 2.1 kg) were randomly divided into four groups and underwent transabdominal laparoscopic access by standard techniques. Tissue freezing was performed using argon gas following interstitial cryoprobe (3 mm) placement into the upper and lower poles of the left kidney. Single active (SA), single passive (SP) double active (DA) or double passive (DP) 15-minute treatment cycle(s) were carried out via the CRYOcare Cryosurgical Unit (Endocare, Irving, CA) on eight kidneys each. An active thaw process with helium gas or a passive thaw process was initiated after each freeze period. The cryoprobe was removed when the temperature reached 0 degrees C. Four weeks following cryosurgery, animals were sacrificed, and the renal tissue was evaluated grossly and histologically. RESULTS Interstitial cryoprobe temperatures decreased from 31.3 degrees C +/- 1.4 degrees C to -142 degrees C +/- 1.0 degrees C following the 15-minute freeze cycle. The temperature reached 0 degrees C significantly faster following active thaw than with the passive process (2.13 +/- 0.24 min/freeze cycle and 15.18 +/- 2.97 min/freeze cycle, respectively; P < 0.0001). Grossly, each lesion consisted of a central area of necrosis surrounded by a rim of white tissue. On microscopic examination, each lesion consisted of a central area of liquefaction necrosis (LN) surrounded by various degrees of fibrosis and granulation tissue admixed with residual parenchyma. The size of the LN was significantly different in tissues subjected to double and single freeze cycles when compared across both thaw processes (active and passive). There was no significant difference in the overall lesion volume following DA, DP, SA, or SP. CONCLUSIONS Renal cryodestruction via laparoscopic access achieves complete tissue ablation without complications. The double freeze cycle produced significantly larger areas of LN than the single freeze regardless of the thaw process. The type of thaw process did not affect the amount of tissue damage. Utilizing a double 15-minute freeze cycle with the faster active thaw process will effectively cryoablate renal tissue as well as significantly reduce overall operative time.

In vivo bladder imaging with microelectromechanical-systems-based endoscopic spectral domain optical coherence tomography.

We report the recent technical improvements in our microelectromechanical systems (MEMS)-based spectral-domain endoscopic OCT (SDEOCT) and applications for in vivo bladder imaging diagnosis. With the technical advances in MEMS mirror fabrication and endoscopic light coupling methods, the new SDEOCT system is able to visualize morphological details of the urinary bladder with high image fidelity close to bench-top OCT (e.g., 10 mum12 mum axial/lateral resolutions, >108 dB dynamic range) at a fourfold to eightfold improved frame rate. An in vivo animal study based on a porcine acute inflammation model following protamine sulfate instillation is performed to further evaluate the utility of SDEOCT system to delineate bladder morphology and inflammatory lesions as well as to detect subsurface blood flow. In addition, a preliminary clinical study is performed to identify the morphological features pertinent to bladder cancer diagnosis, including loss of boundary or image contrast between urothelium and the underlying layers, heterogeneous patterns in the cancerous urothelium, and margin between normal and bladder cancers. The results of a human study (91% sensitivity, 80% specificity) suggest that SDEOCT enables a high-resolution cross-sectional image of human bladder structures to detect transitional cell carcinomas (TCC); however, due to reduced imaging depth of SDEOCT in cancerous lesions, staging of bladder cancers may be limited to T1 to T2a (prior to muscle invasion).

Periurethral Mass Formations Following Bulking Agent Injection for the Treatment of Urinary Incontinence

PURPOSE Durasphere is gaining popularity as a bulking agent for treating women with stress urinary incontinence. We present a series of patients with periurethral mass formation following Durasphere injection. MATERIALS AND METHODS The charts of 135 women with a mean age of 69.4 years (range 46 to 83) who underwent Durasphere periurethral injections were retrospectively reviewed. Patients who had a periurethral mass were identified and their clinical data were collected and analyzed. RESULTS Four patients (2.9%) were diagnosed with periurethral mass formation 12 to 18 months (average 14.7) following a Durasphere injection. Clinical presentation varied, including irritative voiding symptoms, pelvic pain and urinary incontinence. All patients were found to have a tender and tense periurethral mass. A radiopaque mass was revealed during videourodynamic study in 1 patient. Incision, and transvaginal and endoscopic drainage or transvaginal excision were used to treat these masses. Intraoperative and pathological findings as well as operative outcomes are presented. CONCLUSIONS Irritative or obstructing voiding symptoms, pelvic pain or a periurethral mass in patients with a history of Durasphere or other periurethral bulking agent injection should alert the physician to the possibility of periurethral mass formation. The true incidence of this late complication remains to be determined.

Etiology and Pathogenesis of Hypertension following Renal Transplantation

Hypertension is a common feature of end-stage renal disease (ESRD) and constitutes a major risk factor for accelerated cardiovascular disease and premature death. While a reduction in the incidence and severity of hypertension is one potential benefit of kidney transplantation, hypertension often remains as a problem after transplantation, even in recipients of wellfunctioning allografts. Despite many studies of the underlying etiologic factors, the mechanisms implicated in post-transplant hypertension are still incompletely understood. A number of variables, including status of the diseased native kidneys, steriod therapy, rejection, recurrence of original disease, activation of the renin-angiotensin system, sodium and calcium metabolism, and transplant renal artery stenosis may play a role in the etiology of hypertension after transplantation. Patients with persistent high blood pressure for more than 3 months after transplantation should be thoroughly investigated, with controlled sodium intake so that proper medical or surgical therapy can be implemented. Since sodium restriction, radionuclide renal scanning, renin levels, or responses to saralasin or captopril may not provide a clear index of various possibilities, an accurate differential diagnosis my also require invasive procedures such as arteriography and/or renal biopsy.

Prostate Cancer Cell Adhesion to Bone Marrow Endothelium The Role of Prostate-Specific Antigen

Bone metastasis is the most frequent complication of prostate cancer (PC). Elucidation of the biological basis of this specificity is required for the development of approaches for metastatic inhibition. We investigated the possibility that the preferential attachment of PC cells to bone marrow endothelium (as opposed to endothelium from other organs) affects this specificity. We selected, from peptide phage-displayed libraries, peptide ligands to surfaces of PC cells (C4-2B) attenuated (30–40%) binding of C4-2B cells to bone marrow endothelial cells (BMECs). We then determined the molecules on the surface of C4-2B cells interacted with the selected peptides using column affinity chromatography and a cDNA expression phage-displayed library generated from C4-2B cells in T7 phage. We identified a phage from the cDNA library that specifically bound to one of the selected peptides-L11. This phage displayed the amino acid sequence homologous for the COOH-terminal portion of prostate-specific antigen (PSA). To examine the possible direct involvement of PSA in the interactions between PC and BMECs, we performed a cell–cell adhesion assay. Antibodies to PSA attenuated PC cells adhesion to BMECs. In addition, exogenous proteolytically active PSA modulated this adhesion. Finally, inactivation of mRNA coding PSA by a small interfering RNA (siRNA) diminished C4-2B cell adhesion to BMECs. These results indicate that PSA expressed as secreted and surface-associated molecules in C4-2B cells is involved in cell–cell interactions and/or digests components of bone marrow endothelium for preferential adhesion and penetration of PC cells. The suggested experimental approach is a promising strategy for identification of cell surface molecules involved in intercellular interactions.

An Approach To Organ Salvage From Non-heartbeating Cadaver Donors Under Existing Legal And Ethical Requirements For Transplantation

Effective utilization of nonheartbeating cadaver donor organs is limited by the time required to obtain the necessary family consent prior to organ retrieval (a delay of at least 4-6 hr); this exceeds by far the maximum tolerance of kidneys to warm ischemia. Measures that could theoretically permit use of such organs include: (1) rapid in situ flush cooling; (2) continued in situ kidney cooling until permission for donation is secured; and (3) cell-membrane stabilization of vital organs, with only minimal invasion of the donor body. These measures were tested experimentally in dogs. Hemorrhagic shock was produced in mongrel dogs. One hour after cessation of heartbeat, a rapid perfusion tube was placed into the femoral artery; it was advanced, and its balloon was inflated in the aorta above the renal vessels. The kidneys were then flushed in situ with 1000 cc of cold preservation solution containing a calmodulin inhibitor, trifluoperazine. Two other catheters were inserted percutaneously into the peritoneal cavity for continuous intraperitoneal cold perfusion. Core temperatures of 4 degrees C were maintained in situ in the kidneys for 5 hr. Six hours after cardiac arrest, the kidneys were removed and preserved ex vivo at 4 degrees C for 24 hr, and were then transplanted into their respective hosts (n = 11), where they sustained life uneventfully. This method requires a 2-inch incision in the groin of the prospective donor, and two small stab wounds of the abdomen; i.e., semi-invasive procedures which are commonly performed in emergency rooms. The perfused body could then be released to the family if donation is denied. The recently documented increased willingness of the public to donate organs when the termination of life support is not an issue, and court decisions that have authorized the performance of nondeforming diagnostic procedures in cadavers without consent, suggest that the salvage of transplantable semi-invasive procedures described in this study may be useful in helping to alleviate the current shortage of transplantable organs. This technique can provide the time needed for families to consider the option of organ donation from nonheartbeating cadaver donors in an unhurried and unpressured manner, while preserving the viability of vital organs during the decision-making process.

Immunohistologic analysis of human renal allograft dysfunction.

The value of percutaneous core needle biopsy in the immunohistological evaluation of renal allograft dysfunction was studied in 72 consecutive biopsies performed in 42 patients. The phenotypes of infiltrating cells mediating graft destruction were identified with monoclonal antibodies and immunoperoxidase staining techniques. Light microscopy, electron microscopy, and immunofluorescence staining were performed in all biopsies. Biopsies were divided into groups depending on their classification on the basis of standard histologic criteria, i.e., acute tubular necrosis (ATN), acute interstitial rejection, acute vascular rejection, chronic rejection and renal disease in native kidneys (RDNK) of nontransplant patients. Immunohistologic analysis of graft biopsies showed a significant increase in Leu 1 (pan-T cells), (P<0.001), Leu 2 (cytotoxic/suppressor cells) (P<0.001), and Leu 3 cells (P<.05) in acute interstitial rejection. The expression of BR antigen was significantly increased in both acute (P<.0.25) and chronic (P<.05) rejection, when compared with the findings in ATN biopsies. Leu M1 (monocytes/activated T cells) and Leu 10 (B cells/macrophages) were significantly increased (P<0.05 and P<.005, respectively) in acute interstitial rejection only. The helper/suppressor ratio of infiltrating cells showed no significant change in any clinopathologic category. There was no correlation between the cell populations infiltrating the graft and those monitored in the peripheral blood. Allograft mononuclear ceil infiltrates in cyclosporine (CsA) vs. azathioprine-treated patients revealed significantly fewer Leu 2 (P<.05) and Leu M1 (P<.05) cell populations in CsA patients during acute rejection. In 32 of these 72 biopsies (44.4%), the biopsy results provided a direct contraindication to the use of steroids, by allowing differentiation between allograft rejection and other causes of graft dysfunction. A total of 38% of the biopsies yielded a histological diagnosis that contradicted the clinical pre-biopsy diagnosis. All allografts showing evidence of severe small vessel disease and/or antibody-mediated rejection eventually were lost. These data highlight the usefulness of needle biopsy material as a guide to the study of intragraft immune events and to clinical management of recipients.

Aristolochic acid‑induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation

Abstract Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM–Chk2–p53–p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-l-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

Value of percutaneous core needle biopsy in the differential diagnosis of renal transplant dysfunction.

The value of percutaneous core needle biopsy in the differentiation of rejection from other causes of renal allograft dysfunction, and its subsequent effect on patient management were assessed in 64 consecutive biopsies performed on 34 patients in whom the clinical diagnosis was was uncertain. A complete clinical, biochemical and radiographic assessment was made in each patient before biopsy. Only 1 biopsy (1.6 per cent) yielded tissue inadequate for evaluation, while another biopsy caused a renal artery pseudoaneurysm that ruptured and resulted in graft loss. In 27 of these 64 biopsies (42 per cent) the results differed from the pre-biopsy diagnosis and directly affected patient management, particularly the use of steroids. The remaining biopsy specimens were helpful to confirm uncertain clinical impressions, and allowed accurate counseling for patients and family. Biopsies were of special usefulness in separating acute rejection from complications, such as acute tubular necrosis, cytomegalovirus infections, recurrence of original disease, cyclosporin toxicity and acute superimposed-upon chronic rejection. Of 64 biopsies 22 (34.3 per cent) demonstrated the absence of rejection and 8 demonstrated chronic rejection (12.5 per cent), thereby averting the use of steroids in 46.8 per cent of the patients. All patients with evidence of severe small vessel disease and/or antibody-mediated rejection eventually lost the grafts, including 2 with cytomegalovirus glomerulopathy who also suffered such vascular changes. These data highlight the extreme usefulness of needle biopsy in the evaluation and management of renal allograft dysfunction.

Early Detection of Carcinoma In Situ of the Bladder: A Comparative Study of White Light Cystoscopy, Narrow Band Imaging, 5-ALA Fluorescence Cystoscopy and 3-Dimensional Optical Coherence Tomography

PURPOSE We compared the efficacy and potential limitations of white light cystoscopy, narrow band imaging, 5-ALA fluorescence cystoscopy and 3-dimensional optical coherence tomography for early diagnosis of bladder carcinoma in situ. MATERIALS AND METHODS By expressing simian virus 40T antigen in the urothelium carcinoma in situ typically develops in SV40T transgenic mice in about 8 to 20 weeks and then frank high grade papillary urothelial carcinoma starts to emerge. A total of 18 control and 29 SV40T mice were examined during weeks 8 to 22 by white light cystoscopy, fluorescence cystoscopy, narrow band imaging and 3-dimensional optical coherence tomography. Results were validated by histology. Newly improved algorithms for computer aided detection were applied to acquired 3-dimensional optical coherence tomography images to enhance the quantitative diagnosis of carcinoma in situ in near real time. RESULTS Of 29 carcinoma in situ samples 27 were detected by 3-dimensional optical coherence tomography, 1 by white light cystoscopy, 26 by narrow band imaging and 13 by fluorescence cystoscopy. Of the 18 histologically confirmed benign cases 17 were detected by 3-dimensional optical coherence tomography, 14 by white light cystoscopy, 5 by narrow band imaging and 18 by fluorescence cystoscopy. The diagnostic sensitivity of white light cystoscopy (3.4%) and fluorescence cystoscopy (44.8%), and the specificity of narrow band imaging (27.8%) were significantly enhanced by 3-dimensional optical coherence tomography to 93.1% and 94.4%, respectively (p <0.01). CONCLUSIONS Three-dimensional optical coherence tomography with quantitative computer aided detection can significantly enhance the sensitivity of white light cystoscopy and fluorescence cystoscopy, and the specificity of narrow band imaging for early diagnosis of carcinoma in situ. This suggests the potential of narrow band imaging guided 3-dimensional optical coherence tomography for future clinical detection of carcinoma in situ when effective image guidance is desirable.