Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b^0,+AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S³⁵) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P = 0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b^0,+ amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches. Gunnar Wendt-Nordahl, Sreedhar Sagi contributed equally to this work.     

Transarterial nephrectomy: the current status of experimental and clinical studies

INTRODUCTION: Open nephrectomy is associated with significant morbidity. For several years, minimally invasive alternatives have been developed such as laparoscopic nephrectomy or transarterial renal ablation. This paper focuses on the different principles of vaso-occlusion and further improvements of the technique such as capillary chemoembolization in experimental as well as clinical studies. MATERIALS AND METHODS: Based on own in vitro studies, the principle of capillary embolization with occlusion of the entire arterial system up to the capillaries by a precipitating corn protein (Ethibloc) has been developed in animal studies (i.e., rat and canine kidney model). The precipitation speed of Ethibloc can be prolonged by 40% glucose per injection. The organ-ablative efficacy was evaluated in models of unilateral hypertension and chemically induced renal tumors (i.e., dimethyl-nitrosamine). Further studies using the model of unilateral transarterial implantation of Yoshida-sarcoma cells compared capillary chemoembolization using Ethibloc/mitomycin C (MMC) versus chemoperfusion and capillary embolization. Before starting clinical trials, the optimal mixture of Ethibloc and MMC was determined in vitro and in vivo. Prior to the vaso-occlusion, the volume of the arterial system of the kidney is determined by perfusion of the kidney with contrast-dye via a blocked balloon-catheter. Then 25% of the determined volume of 40% glucose is pre-injected followed by Ethibloc/MMC being injected with 1-cm(3) syringes. Once the capillary bed and tumor sinusoids are reached, the balloon catheter is emptied by postinjection of 40% glucose. RESULTS: Capillary embolization proved to be significantly superior to a central (i.e., ligation of renal artery) or peripheral type of occlusion resulting in complete coagulation necrosis of the normal rat and canine kidney with reduction of the elevated blood pressure, similar to nephrectomy in the model of renal hypertension. In the model of chemically induced renal tumors, complete necrosis of T2 stages could be achieved in 83% using Ethibloc compared to only 63% with Gelfoam particles, and 17% after ligation. In T3/T4 stages, the response rate was only 60% versus 0% after central and peripheral occlusion. In the highly aggressive Yoshida-sarcoma model, capillary chemoembolization yielded an 80% complete response rate compared to only 75% after capillary embolization and 70% after chemoperfusion. The optimal mixture of Ethibloc and MMC ranged between 1 and 2 mg of MMC to 1 cm(3) of Ethibloc, therefore for clinical trials 10 mg MMC was added to the 7.5 cm(3) syringe of Ethibloc. Clinical studies included 68 preoperatively as well as 62 palliatively embolized patients with renal cell carcinoma. The procedure was relatively well tolerated and usually associated with a mild postembolization syndrome. After an interval of up to 28 days, complete necrosis of the renal tumor could be achieved in tumors up to 9 cm in diameter. Hematuria ceased in all cases, and in selected cases long-lasting responses of very large tumors (i.e., vena cava involvement) could be achieved. DISCUSSION: Capillary chemoembolization represents an effective concept for ablation of malignant renal tumors. It offers control of tumor growth in case of temporary inoperability as well as cessation of hematuria in a palliative situation. Because of the local ablative efficiency, it may still represent a minimally invasive option in advanced stages of renal carcinoma (i.e., in combination with immunochemotherapy or targeted therapy).     

Absolute bioavailability of oral immediate and slow release fluphenazine in healthy volunteers

Objective: The present study was conducted with the aim of investigating the absolute bioavailability of fluphenazine in healthy volunteers after administration of immediate and slow release oral formulations. Methods: The oral dose was 12 mg fluphenazine hydrochloride. The intravenous bolus dose was 2.5 mg. Fourteen healthy volunteers of both sexes were enrolled in this randomised, crossover trial. Twelve volunteers completed the trial according to protocol. Results: The concentration maxima after administration of the slow release formulation were approximately half those measured after the immediate release formulation and were recorded later by a factor of 2 (immediate release: C_max = 2.3 ng⋅ml⁻¹, t_max = 2.8 h; slow release: C_max = 1.2 ng⋅ml⁻¹, t_max = 4.6 h). The concentrations measured 10 min after intravenous bolus administration of 2.5 mg fluphenazine hydrochloride were approximately 100 times higher (261 ng⋅ml⁻¹). The geometric means for the absolute bioavailability of fluphenazine were 2.7% for the immediate release formulation and 3.4% for the slow release formulation. The absolute bioavailability of fluphenazine is thus much lower than previously generally accepted. Received: 14 December 1995/Accepted in revised form: 26 March 1996     

Penile revascularization in nonresponders to intracavernous injections using a modified microsurgical technique.

At certain centers, microsurgical penile revascularization, using different surgical techniques, has gained importance throughout the past years. In general, only patients classified as intracavernous injection nonresponders are subjected to this kind of surgery. Since 1988, revascularization surgery has been performed at our clinic on 19 intracavernous injection nonresponders. The Hauri technique was carried out on the first 6 patients. The last 10 patients underwent modified anastomosis surgery. The inferior epigastric artery and the dorsal penile artery are anastomosed, one behind the other, end-to-side, to the dorsal penile vein. This results in a more simple procedure with assurance of flow. The Virag technique was performed on 3 patients. 18 patients achieved erections with or without the aid of intracavernous injections (at a mean follow-up of 13.4 months). 11 patients were capable of spontaneous erections, whereby it was particularly noted that 8 of the 10 patients undergoing the modified technique achieved spontaneous erections. The results demonstrate that intracavernous injection nonresponders benefit from revascularization surgery.     

Progress in Management of Ureteric Stones

Most symptomatic urinary stones are found within the ureter. Depending on stone localisation and size, a substantial portion is able to pass the upper urinary tract spontaneously. This process may take days to weeks and the patient has to receive sufficient analgetic and spasmolytic medication. There is some evidence, that alpha-adrenoreceptor blockade supports expulsion of praevesical stones.Standard and least invasive treatment for ureteric stones is extracorporeal shockwave lithotripsy (SWL). However, stone free rate after SWL of ureteric stones after single SWL is lower than for kidney stones and many patients require more than one shockwave application. Recent developments in ureteroscopy (URS) like small-calibre scopes and Ho:YAG laser lithotripsy have changed treatment algorithms as URS is highly efficient, has lower costs than SWL and is still minimally invasive. Especially for larger stones (>10 mm), URS seems to have advantages compared to SWL. Today, open or laparoscopic ureterolithotomy are reserved for special indications and are not used routinely.In summary, SWL offers minimal-invasiveness but a higher risk of treatment failure compared to URS which reaches immediate high stone free rates. Treatment decisions have to be drawn individually taking into account patients preference, personal experience and local equipment.     

The mainz-pouch (mixed augmentation ileum 'n zecum) for bladder augmentation and continent diversion

Summary The ideal urinary reservoir constructed from bowel material should be a low-pressure system with a high capacity, capable of preventing upper tract deterioration resulting from ureteral obstruction or reflux. It should achieve reliable control of continence and assure easy emptying of the reservoir. In the Mainz-pouch, the combination of cecum and ileum, the latter of which is able to absorb pressure waves created by the cecum, produces a low-pressure system with a high capacity immediately postoperatively. By incorporating large bowel in our pouch, ureteral implantation can be done using a simple and reliable standard antireflux technique with a submucosal tunnel. The Mainz-pouch has been done since 1983 in 26 patients. Of these 11 were for bladder augmentation after subtotal cystectomy and 15 for continent urinary diversion. All of the patients with bladder augmentation are completely dry day and night; 2 patients with myelomeningocele are on intermittent catheterization for bladder evacuation. The remainder void spontaneously without significant residual urine. Of 15 patients with Mainz-pouch urinary diversion, 4 had an alloplastic stomal prosthesis implanted for control of continence and 11 have isoperistaltic ileo-ileal invagination, where by the invagination valve can easily be fixed to the intussuscepting ileum by sutures or staples. Of the 4 alloplastic stomal prostheses, 2 have been removed because of infection. In 1 of these patients, an ileo-ileal invagination was performed in the same operation to achieve continent closure. All patients with the invagination valve, as well as the 2 patients with an alloplastic stomal prosthesis, are completely continent, but in 3 cases, revision of the ileo-ileal invagination became necessary due to prolapse of the valve.     

Karyotype analysis and marker chromosomes of renal cell carcinoma

Karyotyping was performed in 46 human renal cell carcinomas of various histological grades. Controls included chromosome analysis of normal renal parenchymal cells from the same patients. Various numerical chromosome aberrations were found as well as marker chromosomes. They are specific of the individual tumor but no single marker was identified occurring in all tumors tested. Only trisomy 3 was found in different tumors (31%), suggesting a more general character of this aberration. Histological dedifferentiation and large tumor size correlate with focusing of the karyotype towards distinct chromosomal modes indicative of distinct tumor cell subclones within these tumors. Their rapid growth, reflecting biological aggressiveness, is most probably responsible for the patients' poor prognosis.     

Extracorporeal shock wave lithotripsy (ESWL): alternatives and adjuvant procedures

Summary Within a short time, extensive statistics on ESWL have documented its efficiency in the treatment of most renal and ureteral stones. Approximately 20% of all stone patients, however, require additional or other forms of therapy, such as URS, PNL, or surgery. Up to now, the differential indications for these procedures have not been completely established. A crucial factor for successful application of ESWL is stone volume and localization. Large stones with a central stone mass may be successfully treated by combining ESWL and PNL, while surgery is still preferred in those with a peripheral stone mass.