Surgical Atlas. Percutaneous nephrolithotomy: the Mannheim technique.
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Anatomy, Artistic↗
Biomedical subjects
Thomas Knoll
Publications and source records attributed to Thomas Knoll.
Impact of hypoxia and hypercapnia on calcium oxalate toxicity in renal epithelial and interstitial cells.
Although there is an ongoing controversy about the primary site of calcium oxalate stone (CaOx) formation, there is some evidence for extratubular crystallization. However, the mechanisms leading to such interstitial calcifications are not clear. Anatomical studies have demonstrated a close association between the renal vasculature and renal tubules. It has been hypothesized that disorders of the vasculature may contribute to renal stone formation. The exceptional papillary environment with low oxygen and high carbon dioxide is of interest in this context and its impact on CaOx toxicity to renal cells has to be evaluated. LLC-PK1, Madin-Darby canine kidney (MDCK), human umbilical vein endothelial (HUVEC) and fibroblast cell lines were exposed to hypoxia (3% O2) alone, hypercapnia combined with hypoxia (3% O2, 18% CO2) or standard culture conditions (20% O2) for 72 h. Cell survival rates were determined microscopically after 4 h of incubation with CaOx at final concentrations of 1, 2 and 4 mM. DAPI staining and western blot were used to evaluate the induction of apoptosis. We confirmed that CaOx leads to concentration-dependent effects on the viability of the cell lines. HUVECs were most vulnerable to CaOx among the four cell lines. Incubation under hypoxia alone had no impact on CaOx toxicity to any of the cell lines in terms of survival. However, under combined hypoxic and hypercapnic conditions, all cell lines displayed a significant reduction of cell survival compared to room air incubation. Again, this effect was most pronounced for HUVECs. The induction of apoptosis could not be demonstrated in any experimental setting. Combined hypoxia and hypercapnia clearly aggravate CaOx toxicity to renal cell lines. As we could not demonstrate the induction of apoptosis, this effect may be a result of toxic necrosis. Especially the CaOx effect on interstitial cell lines might be of interest in the chronic ischemic papillary environment. An increased toxicity may lead to recurrent stone formation, and vice versa, diseases of the vasculature, like arteriosclerosis, may further promote stone formation by induction of local ischemia. This issue has to be clarified by further studies.
Animals↗
Bipolar and multipolar radio frequency ablation with resistance controlled power output: standardized ex vivo kidney tissue evaluation.
PURPOSE: We investigated a newly developed bipolar and multipolar RF ablation system with an internally cooled electrode and resistance controlled power output in a standardized model of perfused ex vivo kidney tissue. MATERIALS AND METHODS: RF energy was applied at different power levels (20, 30 and 60 W) for 1, 3, 5 and 9 minutes. Each treatment parameter was repeated 5 times. For the 20/30 W levels a bipolar electrode with an active conducting part of 20/30 mm was selected. At 60 W 2 bipolar electrodes with an active conducting part (30 mm each) were connected. Lesion volumes and shapes were calculated by measuring the maximum vertical, long axis and short axis diameters of the macroscopic lesion. RESULTS: Lesion volume increased significantly with the treatment time and generator power applied (p < 0.0001). Lesion size in multipolar ablated zones was larger than that in bipolar ablated zones. A reliable dose-effect relationship existed between the generator power/applied treatment time and ablated tissue lesion size. All lesions were elliptical. CONCLUSIONS: Bipolar and multipolar RF ablation with an internally cooled electrode and tissue resistance control represent an interesting advance in RF technology. The development of lesion size and volume is predictable, while a uniform lesion shape can be achieved in perfused ex vivo kidney tissue. Further in vivo trials are required to test whether complete and reliable tumor tissue ablation is possible with this system.
Animals↗
Urothelial carcinoma of the ureter, giant rectal stone and sigmoid carcinoma 55 years after ureterosigmoidostomy.
We present the case of a unique accumulation of complications 55 years after ureterosigmoidostomy for bladder exstrophy and discuss possible implications for follow-up strategies.
Adenocarcinoma↗
Low-power holmium:YAG laser urethrotomy for treatment of urethral strictures: functional outcome and quality of life.
PURPOSE: To evaluate the efficacy of endourethrotomy with the holmium:YAG laser as a minimally invasive treatment for urethral stricture. PATIENTS AND METHODS: Between January 2002 and January 2004, 32 male patients with symptomatic urethral strictures (8 bulbar, 9 penile, 9 combined) were treated with Ho:YAG-laser urethrotomy in our department. The stricture was iatrogenic in 60% (N = 18), inflammatory in 16.6% (N = 5), traumatic in 13.3% (N = 4), and idiopathic in 7% (N = 3). The stricture was incised under vision at the 12 o'clock location or the site of maximum scar tissue or narrowing in asymmetric strictures. Laser energy was set on 1200 to 1400 mJ with a frequency of 10 to 13 Hz. Postoperatively, drainage of the bladder was performed for 4 days using a 18F silicone catheter. Triamcinolone was instilled intraurethrally after removal of the catheter in all patients. Patients were followed up by mailed questionnaire, including International Prostate Symptom Score and quality of life. RESULTS: Retrograde endoscopic Ho:YAG laser urethrotomy could be performed in all 32 patients. Most patients (22; 68.7%) did not need any reintervention. Ten patients developed recurrent strictures that were treated by another laser urethrotomy in 4 patients (12.5%), while 6 patients (18.7%) needed open urethroplasty with buccal mucosa. Including 2 patients treated with repeat laser urethrotomy, 24 patients (75%) were considered successful after a mean follow-up of 27 months (range 13-38 months). No intraoperative complications were encountered, although in 5% of patients, a urinary-tract infection was diagnosed postoperatively. No gross hematuria occurred. CONCLUSIONS: The Ho:YAG laser urethrotomy is a safe and effective minimally invasive therapeutic modality for urethral stricture with results comparable to those of conventional urethrotomy. Further data from long-time follow-up are necessary to compare the success rate with that of conventional urethrotomy and urethroplasty. Nevertheless, the Ho:YAG laser urethrotomy might at least be an alternative to urethroplasty in patients with high comorbidity who are not suitable for open reconstruction.
Adult↗
Risk of collateral damage to endourologic tools by holmium:YAG laser energy.
BACKGROUND AND PURPOSE: Today, the holmium:YAG laser is the gold standard in endourologic stone treatment because of its high efficacy. However, guidewires and stone-extraction tools often are close to the fiber and may be damaged accidentally by the laser. The aim of our study was to evaluate the duration of laser application required to disrupt wires at different energy settings. MATERIALS AND METHODS: Two standard wires (0.035-inch guidewire and 0.025-inch hydrophilic Terumo wire) and two baskets (1.9F Nitinol tipless and 3F stone extraction) were investigated. We used a holmium:YAG laser (Auriga; Wavelight Laser Technologie AG, Erlangen, Germany) and two fibers (230 and 365 microm) at 800 and 2000 mJ and a 5-Hz pulse rate. The laser was brought into direct contact with the wires. RESULTS: The average time required for transection was 55 to 103 seconds for a safety wire. The Terumo wire broke after 20 to 40 seconds of direct laser application; 15 to 34 seconds was required for a Dormia basket. Thin Nitinol basket wires were disrupted after only 1 to 4 seconds. One- and two-factorial variance analysis showed a high degree of significance (P < 0.0001) of the energy level and type of wire. CONCLUSION: The disruption of stone-extraction tools occurs in a remarkably short time with laser exposure, especially in case of Nitinol baskets, whereas guidewires are more resistant. Attention should be paid to avoiding contact of laser energy with wires and baskets during stone fragmentation.
Aluminum↗
Ablation of clinically relevant kidney tissue volumes by high-intensity focused ultrasound: Preliminary results of standardized ex-vivo investigations.
PURPOSE: To investigate strategies to achieve confluent kidney-tissue ablation by high-intensity focused ultrasound (HIFU). MATERIALS AND METHODS: Our model of the perfused ex-vivo porcine kidney was used. Tissue ablation was performed with an experimental HIFU device (Storz Medical, Kreuzlingen, Switzerland). Lesion-to-lesion interaction was investigated by varying the lesion distance (5 to 2.5 mm), generator power (300, 280, and 260 W), cooling time (10, 20, and 30 seconds), and exposure time (4, 3, and 2 seconds). The lesion rows were analyzed grossly and by histologic examination (hematoxylin-eosin and nicotinamide adenine dinucleotide staining). RESULTS: It was possible to achieve complete homogeneous ablation of a clinically relevant tissue volume but only by meticulous adjustment of the exposure parameters. Minimal changes in these parameters caused changes in lesion formation with holes within the lesions and lesion-to-lesion interaction. CONCLUSIONS: Our preliminary results show that when using this new device, HIFU can ablate a large tissue volume homogeneously in perfused ex-vivo porcine tissue under standardized conditions with meticulous adjustment of exposure parameters. Further investigations in vivo are necessary to test whether large tissue volumes can be ablated completely and reliably despite the influence of physiologic tissue and organ movement.
Animals↗
Technical characterization of a new bipolar and multipolar radiofrequency device for minimally invasive treatment of renal tumours.
OBJECTIVE: To investigate the technical characteristics of a newly developed device for bipolar and multipolar radiofrequency ablation (RFA) of kidney tissue with a resistance-controlled power output. MATERIALS AND METHODS: The standardized model of the isolated perfused ex vivo porcine kidney was used. Two different applicators (20 and 30 mm active length) were selected for bipolar RFA, and one pair of applicators (2 x 30 mm active length) for multipolar RFA. RF energy was applied at different power levels (20, 30, 60 W) depending on the total active length of the electrodes. Treatment times were 1, 3, 5 and 9 min. The ablation cycles were recorded in continuous digital real-time and displayed on a monitor showing pre-set power, actual applied power, applied energy, tissue resistance, and impedance. Lesion sizes were measured macroscopically. A coagulation coefficient (coagulated tissue volume per applied energy unit) was calculated. RESULTS: There was a dosage-effect relationship between the generator power/treatment time and the sizes of the lesions. With increasing treatment time, less tissue volume was coagulated per unit of applied energy. The actual applied energy was lower than that calculated theoretically. The resistance and impedance values for the 30-W applicator were lower than those of the 20-W applicator. CONCLUSIONS: The technical features of this RFA device, with internally cooled bipolar and multipolar applicators and a resistance-controlled power output, represents an innovative improvement in RF technology. In vivo studies are needed to confirm the expected advantages and the suitability of this device for complete and reliable ablation of renal tumours.
Animals↗
Intravenous contrast media in uroradiology: evaluation of safety and tolerability in almost 50,000 patients.
OBJECTIVE: To evaluate the frequency of adverse events and possible risk factors after the administration of an intravenous contrast medium (CM), iobitridol, in a large multicentre postmarketing surveillance study. SUBJECTS AND METHODS: A total of 49,975 patients undergoing intravenous urography were included in this study. A water-soluble, non-ionic CM (iobitridol, 1 ml/kg body weight) was administered. Age, sex, indication for the actual examination and adverse events were documented. Also, high-risk patients were identified. RESULTS: Of the 49,975 patients, 28,336 (56.7%) were males and 21,639 (43.3%) females. Indications for urography were mainly urolithiasis (39.1%), inflammatory diseases (25.6%) and tumours (13.9%). Additionally, 7.4% were risk patients: 1.9% with creatinine >1.5 mg/dl (0.1% were on haemodialysis); 0.8% had a history of previous allergic reactions to CM, 3.7% pre-existing asthma or allergies and 2% other risk factors against CM examination. Only 0.9% of the patients experienced acute adverse events that were non-serious and transient. Less than 0.1% of the patients experienced vomiting, dizziness or cardiovascular problems. Only 1 patient developed an anaphylactic shock but recovered fully under treatment. A chi(2) analysis revealed that adverse reactions occurred in patients with pre-existing renal insufficiency or allergies. Significantly more females had contrast-agent-related symptoms compared to men. Patients undergoing urography for urolithiasis had significantly fewer symptoms compared to patients with other indications. CONCLUSION: Iobitridol is clinically safe and well tolerated in urography as demonstrated in this study of a large patient population, producing mainly minor symptoms as adverse events. However, caution is advised when administering iobitridol to high-risk patients.
Acute Disease↗
5-year-follow-up of patients with clinically insignificant residual fragments after extracorporeal shockwave lithotripsy.
OBJECTIVES: After SWL treatment, many patients have residual fragments in the kidney or ureter. Fragments </=4mm have high probability to pass spontaneously and are normally not further treated. The term clinically insignificant residual fragment (CIRF) has been established for these stones. However, this term is still controversial, as persisting fragments might be important risk factors for stone growth and recurrence. Aim of this study was to evaluate the impact of CIRF on stone recurrence. METHODS: We evaluated 173 patients (age 3-89 years) who have been treated by SWL and released with CIRF. Mean follow up was 4.9 years (4.5-5.3). 78% of the patients were recurrent stone formers with >2 stones episodes. These patients and their referring urologist received follow-up questionnaires which contained questions about stone clearance, late complications, auxillary measures and dietary or drug metaphylaxis. RESULTS: Most residual stone fragments were located within the lower calyx (17%) and the renal pyelon (14%). Stone analysis was available in 142 patients with CIRF and revealed calciumoxalate calculi in 93.6% of the cases. In 78.6%, CIRF cleared spontaneously within few weeks and did not recur within 5 years. However, residual stones led to stone recurrence and need of re-treatment in 21.4%. Renal pyelon (23%) and calices showed comparable growth of former CIRF (lower calices 26.5%, middle calices 27%, upper calices 26%). Only 48% of the patients with recurrent stone formation followed dietary metaphylaxis. However, a significant correlation between a general or specific metaphylaxis and stone growth of CIRF could not be demonstrated. CONCLUSIONS: Most of the CIRF after SWL pass spontaneously without any complications. But considering that one fifth of the patients developed new stones at the side of residual fragments, it is obvious that close follow-up is required. Although we could not demonstrate a relation between metaphylaxis and stone re-growth, it is conceivable that adequate metaphylaxis can reduce stone recurrences.
Adolescent↗
Minimally invasive treatment of renal cell carcinoma: comparison of 4 different monopolar radiofrequency devices.
OBJECTIVES: Radiofrequency Ablation is an investigational treatment option for RCC. The aim of our study was to test the ablation algorithms of four different RF systems in a standardized ex vivo perfused porcine kidney model. MATERIALS AND METHODS: A multiline monopolar dry electrode (impedance-based system), a multiline monopolar dry electrode (temperature-based system), a single monopolar wet electrode (impedance-based system) and a single monopolar dry, internally-cooled electrode (impedance-based system) were selected. RF energy was applied at different treatment parameters (power with and without control, tissue temperature, saline enhancement) for 1, 3, 5 and 9 minutes in healthy perfused ex vivo porcine tissue. Each treatment parameter was repeated 5 times. Maximum vertical, long-axis and short-axis diameters of the macroscopic lesion were measured and lesion volumes/ shapes were calculated. RESULTS: Lesion volumes increased significantly with the pre-selected tissue temperature and saline enhancement. Saline enhancement created larger, but irregular shaped lesions. The impedance-based system created lesion volumes that were predictable by treatment time and generator power. Lesions were unpredictable when uncontrolled generator power was applied. The created lesion shape was dependent on the selected electrode configuration. CONCLUSIONS: The currently available monopolar RFA systems offer different specific technical features to control tissue ablation. Detailed knowledge of the specific characteristics of each RF system is necessary to provide a higher chance of successful clinical outcome by complete and reliable ablation.
Animals↗
New bipolar resection device for transurethral resection of the prostate: first ex-vivo and in-vivo evaluation.
BACKGROUND AND PURPOSE: Bipolar resection systems for transurethral resection of the prostate (TURP) offer some advantages over conventional monopolar devices. As both the active and the return electrode are placed on the tip of the resectoscope, current flow is locally limited, and negative effects caused by the current flow through the patient's body in the monopolar array can theoretically be reduced. Furthermore, bipolar resectoscopes use 0.9% sodium chloride solution as irrigation fluid, and therefore, the risk of TUR syndrome is theoretically eliminated. In this trial, we evaluated a new bipolar resectoscope in an ex-vivo model and in vivo and compared it with a standard monopolar resection device. MATERIALS AND METHODS: The modified model of the isolated blood perfused porcine kidney was used to determine cutting qualities, ablation rate, blood loss, and coagulation depth of the bipolar resection device compared with a monopolar resectoscope. A measurement system placed in the line between the generator and the patient was used to determine electric parameters when both resectoscopes were used in vivo. RESULTS: The ex-vivo evaluation showed worse cutting ability of the bipolar resection device compared with the monopolar resectoscope, with essential difficulties in starting a cut. The ablation rate was equal, as both resectoscopes had the same loop diameter. The bleeding rate was lower in bipolar (15.16+/-3.31 g/min) than monopolar (20.78+/-1.52 g/min) resection, whereas bipolar cutting resulted in marginally deeper coagulation zones (236.25+/-36.69 microm 216.0+/-42.25 microm). The in-vivo measurements made it clear that more power and more current is delivered to the patient during bipolar resection. The reason lies in a period at the beginning of each cut, when the patient's impedance is low and current flow high. CONCLUSION: Our results suggest that bipolar resection offers an alternative to conventional monopolar TURP. The advantage of a more localized energy field is at least in part compensated for by the higher generator output power during the procedure. Our ex-vivo results indicate a reduced bleeding rate compared with monopolar resection. Clinical studies have to be performed to prove the significance of our findings for patient treatment.
Animals↗
Validation of computer-based training in ureterorenoscopy.
OBJECTIVES: To evaluate the outcome of training both urological novices and experts, using the recently developed UroMentor (Simbionix Ltd, Israel) trainer, that provides a realistic simulation of rigid and flexible ureterorenoscopy (URS). SUBJECTS AND METHODS: Twenty experienced urologists (total number of previous flexible URSs 21-153) were monitored during simulated flexible URS for treating a lower calyceal stone, and the outcome was correlated with individual experience. A score was compiled based on the variables recorded, including total operation time, stone contact time, complications such as bleeding or perforation, and treatment success. A further five urological residents with no endourological experience were trained on the UroMentor in rigid URS for ureteric stone treatment. Their acquired clinical skills were subsequently compared to those of five urological residents who received no simulator training. RESULTS: All 20 experienced urologists disintegrated the stone on the simulator, and the score achieved was related to their personal experience; there was a significant difference in performance in those with < 40 and > 80 previous flexible URSs. For the five urological residents with no endourological experience, simulator training improved their skills, and comparison with urological residents who had received no simulator training showed advantages for the trained residents. After being trained on the simulator, the group performed better in the first four URSs on patients. CONCLUSIONS: Individual experience correlates with individual performance on the simulator. Simulator training was helpful in improving clinical skills. Although the distribution of computer-based simulators is limited by high prices, virtual reality-based training has the potential to become an important tool for clinical education.
Clinical Competence↗
Donor-site morbidity in buccal mucosa urethroplasty: lower lip or inner cheek?
OBJECTIVE: To evaluate donor-site complications of buccal mucosa urethroplasty and whether there is a difference in morbidity between harvesting the mucosa graft from the inner cheek or the lower lip. PATIENTS AND METHODS: Twenty-four consecutive patients with recurrent urethral strictures were treated with buccal mucosa urethroplasty in our department between September 2002 and April 2004. In 12 patients the graft was harvested from the lower lip or cheek and lower lip (group 1), and in 12 patients from the cheek (group 2). The mean (range) age of patients was 51 (26-66) years in group 1 and 53 (32-75) years in group 2. The mean (range) graft length was 6.2 (2-16) cm in group 1 and 5.7 (2-13) cm in group 2. All patients were followed up using a mailed questionnaire that asked about pain, numbness, difficulties in mouth opening or ingestion, and satisfaction, monthly for the first 3 months and then every 6 months. The mean (range) follow-up was 12.5 (6-23) months. RESULTS: There were no bleeding complications or disturbances in wound healing. All of the patients reported numbness in the area of the mental and buccal nerves, and graft-site tenderness after surgery. In group 1, the pain lasted for a mean (range) of 5.9 (0.5-22) months, compared to 1 (0.1-7) months in group 2 (P = 0.022). Perioral numbness lasted for a mean (range) of 10.3 (0.5-23) months in group 1 and 0.85 (0.1-3) months (P = 0.0027) in group 2. There were no statistically significant differences in problems with mouth opening or food intake between the two groups, but the patients in group 1 seemed to be less satisfied (6/12 patients satisfied) than those in group 2 (11/12 patients satisfied). CONCLUSIONS: Buccal mucosa graft harvesting from the lower lip and the inner cheek are both feasible, but harvesting from the lower lip resulted in a significantly greater long-term morbidity, which resulted in a lower proportion of satisfied patients. This seems to be due to a long-lasting neuropathy of the mental nerve. We therefore have changed our technique entirely from lower lip to inner cheek graft harvesting, whenever possible.
Adult↗
Percutaneous nephrolithotomy with ultrasonography-guided renal access: experience from over 300 cases.
OBJECTIVE: To report our experience with over 300 patients treated with percutaneous nephrolithotomy (PNL), for although PNL was established as a treatment in the 1970s, its use diminished with the introduction of extracorporeal shockwave lithotripsy (ESWL); clinical experience with ESWL showed its limitations, and the role of PNL for treating urolithiasis was redefined, which with improvements in instruments and lithotripsy technology has expanded the capability of percutaneous stone disintegration. PATIENTS AND METHODS: The study included 315 patients (156 males, 159 females, aged 13-85 years) treated with PNL in our department between 1987 and 2002. The mean (range) stone diameter was 27 (7-52) mm. The kidney was punctured under ultrasonography guidance via a lower-pole calyx whenever possible. The working channel was dilated using an Alken dilator under X-ray control. If necessary, a flexible renoscope was used. Ultrasonic, pneumatic and laser probes were used for lithotripsy. RESULTS: Four weeks after treatment the total stone-free rate was 96.5%; 45.7% of all patients were primarily stone-free, 21.3% had clinically insignificant residual stones that passed spontaneously within 4 weeks after PNL, and 33% of the patients needed auxiliary measures (a second PNL, ESWL, ureterorenoscopy). Overall, the early complication rate was 50.8%, the most common complications being transient fever (27.6%), clinically insignificant bleeding (7.6%) or both (3.2%); 3.5% of the patients developed urinary tract infections (with no signs of urosepsis), 3.2% had renal colic and 2.9% upper urinary tract obstruction. One patient (0.3%) developed acute pancreatitis after PNL; one died from urosepsis and one needed selective angiographic embolization of the punctured kidney due to bleeding. No patient required transfusions and there were no injuries to neighbouring organs. CONCLUSIONS: These results show that PNL causes no significant blood loss or major complications in almost all patients. Two aspects may especially reduce the potential complications: ultrasonography-guided renal puncture and using PNL in an experienced centre. PNL is a highly efficient procedure that provides fast and safe stone removal.
Adolescent↗
Cystinuria in childhood and adolescence: recommendations for diagnosis, treatment, and follow-up.
Cystinuria, an autosomal-recessive disorder of a renal tubular amino acid transporter, is the cause of about 10% of all kidney stones observed in children. Different genetic characteristics are not represented by different phenotypes. The stones are formed of cystine, which is relatively insoluble at the physiological pH of urine. Without any preventive measures, the patients will suffer from recurrent stone formation throughout their life. Even with medical management, long-term outcome is poor due to insufficient efficacy and low patient compliance. Many patients suffer from renal insufficiency as a result of recurrent stone formation and repeated interventions. However, regular follow-up and optimal pharmacotherapy significantly increase stone-free intervals. Medical management is mainly based on hyperhydration and urine alkalinization. Sulfhydryl agents such as tiopronin can be added. Recurrent stone formation necessitates repeated urological interventions. These mostly minimally invasive procedures carry the risk of impairment of renal function. In adults, extracorporeal shockwave lithotripsy (SWL) as well as intracorporeal lithotripsy is often unsuccessful. However, in children SWL shows excellent results for cystine stones. In cases with large stone burden, percutaneous nephrolithotripsy (PNL) or even open surgical nephrolithotomy are preferred. This review discusses the underlying pathogenetic mechanisms and provides guidance for the diagnosis, therapy, and management of cystinuria following the recommendations of the International Cystinuria Consortium and the European Association of Urology.
Adolescent↗
Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma.
New approaches in the treatment of transitional cell carcinoma (TCC) are using gene therapy to influence the disease at the genetic level. Technical advances in genomics, the availability of tissue-specific gene promoters and other developments have made this approach more realistic. Transporting the gene into the target cell is still the major problem. Several transfection techniques have been introduced. Transfection of naked DNA is one of the simplest to perform but transfection rates have been very poor. We investigated the influence of laser energy on transfection efficacy in urothelial cancer cells in vitro with two types of medical lasers. A suspension of human transitional cancer cells (UM-UC3; 3.5 million cells/ml) was mixed with 200 microg of plasmid DNA (pEGFP-N1). Two types of laser energy, neodymium:YAG (Nd:YAG) and holmium:YAG (Ho:YAG), were applied to the cell suspension in different energy settings. Twenty four hours after treatment, transfection rates were measured with FACS analysis. Energy setting parameters that determine the efficacy of laser were investigated. The significance of different transfection rates was estimated with the student's t-test. We demonstrated that the Nd:YAG laser was not suitable for achieving significant transfection of the reporter gene to the cells. In contrast, the Ho:YAG laser produced satisfactory transfection rates. There was an increase in transfection with increasing frequency of laser pulses, from 16% with 2 Hz up to 40% with 10 Hz (p < 0.0005). Pulse frequency was therefore stabilised at 10 Hz. Pulse energy (mJ) showed the same dependency: a transfection rate of 18.3% was achieved with 1,000 mJ and 53.8% with 2,000 mJ (p > 0.0005). Additionally, we investigated the impact of total pulse number (imp) with different pulse energies. At 1,000 mJ, a transfection rate of 18.3% was estimated with 200 imp and 48.56% with 750 imp, (p < 0.0005). At 2,000 mJ, a transfection rate of 53.8% was achieved with 200 imp and 58.26% with 500 imp. The optimal laser setting observed in this experiment was 10 Hz, 2,000 mJ and 500 imp. This study indicates that the efficacy of naked DNA delivery into TCC in vitro is improvable by application of Ho:YAG laser energy. The Nd:YAG laser did not increase transfection rates in our model. Our results with the Ho:YAG laser are encouraging for further studies to optimise DNA delivery. As TCC tissue is relatively easy to access, this method could become an effective and minimally invasive procedure in urothelial cancer treatment.
Carcinoma, Transitional Cell↗
The influence of oxalate on renal epithelial and interstitial cells.
Most renal stones in humans are composed of calcium oxalate. An increase in urinary oxalate levels has been shown to result in renal epithelial cell injury and crystal retention. However, the underlying mechanisms are unclear. Although the localization of primary stone formation and the associated cells playing the pivotal role in stone formation are still unknown, renal epithelial cells and interstitial cells seem to be involved in this process. The aim of this study was to evaluate the effects of oxalate on distinct renal epithelial and endothelial cells as well as fibroblasts. The first part focused on the toxicity of oxalate on the cells and a potential time- and dose-dependency. In the second part, renal epithelial cells were cultured in a two-compartment model to examine the vulnerability of the tubular or basolateral side to oxalate. LLCPK1, MDCK, renal fibroblast and endothelial cell lines were cultured under standard conditions. In part 1, cells were grown in standard culture flasks until confluent layers were achieved. Sodium oxalate was delivered at final concentrations of 1, 2 and 4 mM to either the apical or basolateral side (plain medium was delivered to the contralateral side). Cell survival was assessed microscopically by trypan blue staining after 1, 2 and 4 h. The influence of oxalate on proliferation and apoptosis induction was also investigated. In the second part, MDCK and LLCPK1 cells were grown in 6-well plates until confluent layers were achieved. Sodium oxalate at the above concentrations was applied, to either the apical or basolateral side and plain medium was delivered to the opposite side. The same protocol was then followed as in part 1. Part 1: sodium oxalate led to a time- and concentration-dependent decline in cell survival that was comparable in LLCPK1 and MDCK. Non-tubular cell lines like fibroblasts and endothelial cells were significantly more vulnerable to oxalate. These observations were reflected by significant impairment to cell proliferation. We could not demonstrate an induction of apoptosis in any cell line. Part 2: both cell lines were more vulnerable to oxalate on the basolateral side. This effect was more pronounced in MDCK cells at high oxalate concentrations (4 mM). Cells are apparently more resistant on the apical (tubular) side. Our results show that sodium oxalate has a negative effect on the growth and survival of renal epithelial cells and, to a greater extent, also fibroblasts and endothelial cells. We could not demonstrate any induction of apoptotic processes which implies a direct induction of cell necrosis. The finding of interstitial calcification and the proximity of tubules, vessels and interstitial cells make involvement of non-tubular renal cells in tissue calcification processes possible. Renal epithelial cells are apparently more vulnerable to oxalate on their basolateral side. Therefore, calcification processes within the interstitium may exert pronounced toxic effects to these cells, leading to inflammation and necrosis. These observations further support the idea of the interstitium as a site of primary stone formation.
Animals↗