Basolateral LAT-2 has a major role in the transepithelial flux of L-cystine in the renal proximal tubule cell line OK

During renal reabsorption, the amino acid transporters b(o,+) and y(+)L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y(+)L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y(+)LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans-epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption.     

Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy

BACKGROUND: Certain cancers depend for growth on uptake of cystine/cysteine from their environment. Here we examined advanced human prostate cancer cell lines, DU-145 and PC-3, for dependence on extracellular cystine and sensitivity to sulfasalazine (SASP), a potent inhibitor of the x(c)(-) cystine transporter. METHODS: Cultures were evaluated for growth dependence on exogenous cystine, x(c)(-) transporter expression, response to SASP (growth and glutathione content). In vivo, effect of SASP was determined on subrenal capsule xenograft growth. RESULTS: Cystine omission from culture medium arrested DU-145 and PC-3 cell proliferation; both cell lines expressed the x(c)(-) transporter and were growth inhibited by SASP (IC(50)s: 0.20 and 0.28 mM, respectively). SASP-induced growth inhibition was associated with vast reductions in cellular glutathione content - both effects based on cystine starvation. SASP (i.p.) markedly inhibited growth of DU-145 and PC-3 xenografts without major toxicity to hosts. CONCLUSIONS: SASP-induced cystine/cysteine starvation leading to glutathione depletion may be useful for therapy of prostate cancers dependent on extracellular cystine.     

Investigations on the early diagnosis of cystinuria in children

The procedure described confirms that the Ni2+/S2O4(2-) tablet test is a suitable quick cystine screening test for large groups. In 16 (0.67%) of 2,395 one- to three-year-old crèche children significantly enhanced cystine levels of 0.60-0.96 mmol/l were found. The corresponding creatinine values (mmol/l) did not reveal any significant deviations from the normal values. In a preliminary group of 9 children, enhanced lysine levels support the suspicion of the existence of cystinuria without clinical symptoms. This also forms the basis for further necessary family histories as well as for prophylactic measures.     

Altered status of glutathione and its metabolites in cystinotic cells.

BACKGROUND: Cystinosis is an autosomal recessive disorder, caused by mutations of the lysosomal cystine carrier cystinosin, encoded by the CTNS gene (17p13). The concomitant intralysosomal cystine accumulation leads to multi-organ damage, with kidneys being the first affected. Altered mitochondrial oxidative phosphorylation has been demonstrated in animal proximal tubules loaded with cystine dimethyl ester, mimicking cystine accumulation in cystinosis, but has not been confirmed in cells of patients with cystinosis. Furthermore, the link between cystine accumulation and mitochondrial damage is also missing. We hypothesized that cytosolic cysteine deficiency resulting in intracellular glutathione (GSH) shortage might be involved in cellular dysfunction in cystinosis. METHODS: Components of the gamma-glutamyl cycle were measured in cultured skin fibroblasts (n = 9) and polymorphonuclear (PMN) leukocytes (n = 15) derived from patients with cystinosis and compared with the values in cultured fibroblasts (n = 9) and PMN cells (n = 18) of healthy controls. RESULTS: Cystine content in cystinotic fibroblasts and PMN cells was significantly elevated compared with the controls, consistent with the lysosomal cystine accumulation in these cells. Although no reduction of total intracellular GSH content was found in cystinotic cells, it inversely correlated with cystine levels. Furthermore, GSH disulfide (GSSG) was elevated in cystinotic cells, resulting in an increased GSSG/total GSH (%) ratio. No relationship between intracellular cystine and GSH was found in control fibroblasts and PMN cells. CONCLUSION: An elevated GSSG/total GSH (%) ratio might indicate increased oxidative stress present in cystinotic cells. Inverse correlation between cystine accumulation and intracellular GSH content indicates that under stress conditions such as intensive energy demand or increased oxidative insult, cystinotic cells may be more prone to GSH depletion.     

Mercuric conjugates of cysteine are transported by the amino acid transporter system b(0,+): implications of molecular mimicry

Humans and other mammals continue to be exposed to various forms of mercury in the environment. The kidneys, specifically the epithelial cells lining the proximal tubules, are the primary targets where mercuric ions accumulate and exert their toxic effects. Although the actual mechanisms involved in the transport of mercuric ions along the proximal tubule have not been defined, current evidence implicates mercuric conjugates of cysteine, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys), as the most likely transportable species of inorganic mercury (Hg(2+)). Because Cys-S-Hg-S-Cys and the amino acid cystine (Cys-S-S-Cys) are structurally similar, it was hypothesized that Cys-S-Hg-S-Cys might act as a molecular mimic of cystine at one or more of the amino acid transporters involved in the luminal absorption of this amino acid. One such candidate is the Na(+)-independent heterodimeric transporter system b(0,+). Therefore, the transport of Cys-S-Hg-S-Cys and cystine was studied in MDCK II cells that were or were not stably transfected with b(0,+)AT-rBAT. Transport of Cys-S-Hg-S-Cys and cystine across the luminal plasma membrane was similar in the transfected cells, indicating that Cys-S-Hg-S-Cys can behave as a molecular mimic of cystine at the site of system b(0,+). Moreover, only the b(0,+)AT-rBAT transfectants became selectively intoxicated during exposure to Cys-S-Hg-S-Cys. These findings indicate that system b(0,+) likely contributes to the nephropathy induced by Hg(2+) in vivo. These data represent the first direct molecular evidence for the participation of a specific transporter in the luminal uptake of a large divalent metal cation in proximal tubular cells.     

Evaluation of Synchronous Twin Pulse Technique for Shock Wave Lithotripsy: Determination of Optimal Parameters for In Vitro Stone Fragmentation

PurposeThe Twinheads extracorporeal shock wave lithotriptor (THSWL) is composed of 2 identical shock wave generators and reflectors. One reflector is under the table and the other is over the table with a variable angle between the axes of the 2 reflectors. The 2 reflectors share a common second focal point, making it possible to deliver an almost synchronous twin pulse to the targeted stone. We studied the optimal parameters for in vitro stone fragmentation.Materials and MethodsTwo types of 1 cm artificial stones were used, namely Bon(n)-stones of 3 compositions (75% calcium oxalate monohydrate [COM] plus 25% uric acid, struvite and cystine) and plaster of Paris. The parameters tested were shock wave number (100, 500 and 1,000), shock wave power (8, 11 and 14 kV) and angle between the reflector axes (67, 90 and 105 degrees). After the optimal parameters were determined we studied the disintegrative efficacy of THSWL for 3 types of human urinary calculi, including COM, calcium hydrogen phosphate (brushite) and cystine. Each stone received 1,000 twin shock waves at 14 kV with an angle of 90 degrees between the reflectors. All experiments were done using a rate of 60 twin shock waves per minute. Following lithotripsy stone fragments were processed and sized. The ratio of the weight of fragments greater than 2 mm-to-total weight of all fragments was calculated.ResultsOptimal stone fragmentation results for THSWL were obtained with the maximum number of shock waves (1,000) and full power (14 kV). There was no significant statistical difference in fragment size or the ratio of fragments greater than 2 mm with the use of different angles except for cystine and plaster of Paris calculi, for which the right angle was most effective. At application of the optimal parameters to human stones THSWL produced small fragment size for COM and cystine stones, while brushite stones were not fragmented to the same extent.ConclusionsThe efficacy of synchronous twin pulse technology improves as the number of shock waves and power increase. A 90-degree angle between the shock wave reflectors is advantageous for certain stones (that is cystine and plaster of Paris) but it is not a factor for other stone compositions. THSWL has satisfactory disintegrative efficacy for human stones, especially COM and cysteine calculi.     

Investigations of the efficacy of ascorbic acid therapy in cystinuria

Summary We investigated ascorbic acid therapy for cystinuria in a study of seven healthy control persons and seven cystinuric patients. The study lasted 9 days. During the first period, we collected 24-h urine specimens from all subjects on 3 consecutive days. Starting on day 4, all were given 5 g ascorbic acid/day for a period of 6 days. On the last 3 days, 24-h urine specimens were again collected. Quantitative amino acid determination was performed using an HPLC method described elsewhere. During ingestion of ascorbic acid, the mean excretion of cysteine by the control group increased from 134.1 to 159 mol/ day, whereas the excretion of cystine decreased from 107.1 to 82 mol/day. The corresponding values for the cystinuric patients increased from 352.4 to 452.1 mol/ day for cysteine and decreased from 4,131.6 to 3,663.2 mol/day for cystine. Thus, ascorbic acid seems to have only mild reducing properties in respect to cystine.     

Nutrition and somatomedin. XXIII. Molecular regulation of IGF-I by amino acid availability in cultured hepatocytes

The poor growth associated with protein-calorie malnutrition occurs despite circulating growth hormone levels that are normal or elevated and is thought to be mediated partly by blunted generation of insulinlike growth factor I (IGF-I) in the liver. To explore underlying mechanisms, we asked whether altered availability of amino acids could regulate hepatic IGF-I release independent of the contributions of regulatory hormones. Normal rat hepatocytes were isolated by collagenase digestion and maintained in serum-free medium with fixed concentrations of insulin and dexamethasone. Levels of immunoassayable albumin and IGF-I accumulation in daily changes of medium were sustained for 3-5 days, and all studies were performed within this period. Cellular viability and content of DNA were unaffected by deprivation of the essential amino acids lysine or tryptophan and the nonessential amino acids cysteine and/or cystine. However, deletion of tryptophan or lysine from the culture medium led to 63 and 76% declines in IGF-I release, respectively (both P less than 0.001 vs. complete medium), although omission of cysteine or cysteine plus cystine produced no significant change. Over 5 days of culture, release of albumin was maintained in complete medium, but omission of tryptophan depressed albumin release over days 2-5 (P less than 0.001). In complete medium, IGF-I release rose for 3 days and then declined. In tryptophan-deficient medium, IGF-I levels were comparable to control values after 24 h but did not rise at 48 h and then fell rapidly after 72 h in culture, with values significantly below levels in complete medium (all P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Experiences with acetylcysteine in cystinuric patients.

Acetylcysteine seems to be beneficial in treating cystinuric stone-forming patients. It probably is effective in reducing cysteine to cystine disulfide, which is more soluble. Six brief case reports demonstrate its use and its lack of toxicity. The results merit a larger clinical trial.     

Cysteine analogues potentiate glucose-induced insulin release in vitro

In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhance insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.     

A quantitative assay for telomerase enzyme activity predicts severity of disease in neuroblastoma

PurposeTo determine whether telomerase enzyme activity correlates with tumor stage, grade, and prognosis using a quantitative telomerase assay.MethodA new semiquantitative assay for telomerase enzyme activity (Telomerase Repeat Amplification Protocol–enzyme-linked immunosorbent assay) was employed on 21 neuroblastoma specimens. Enzyme-linked immunosorbent assay results were compared to tumor stage, patient age, and overall tumor aggressiveness. The principle investigator was blinded to all clinical data. A Student’s t test was used to determine significance (p < 0.05).ResultsTelomerase activity correlated with stage of tumor (p < 0.007). Age of patient did not correlate with telomerase activity independently.ConclusionsTelomerase enzyme activity was predictive for neuroblastoma stage. This implies that increased telomerase activity is involved in tumor dedifferentiation. The semiquantitative telomerase assay may be used as a rapid prognostic indicator and serve as a guide to initiating treatment in childhood neuroblastoma.     

Cystine analyses of separate day and night urine as a basis for the management of patients with homozygous cystinuria

Based on previous observations of the diurnal variation of urinary cystine excretion, the use of separate day and night urine collections was proposed. To improve the medical treatment of patients with cystinuria, this strategy was performed to guide the fluid intake and the administration of SH compounds (tiopronin, D-penicillamine, and MESNA).Twenty-six patients (19 treated with SH compounds and seven with alkalinization and hydration only) were followed during two 3.5-year periods. During Period 1, 24-h urine was collected and during Period 2, separate day and night urine was collected.There were 56 episodes of high urinary cystine supersaturation (> 1,200 micromol/l) during Period 2, 47% of which would have evaded detection with 24-h urine analysis. In comparison with Period 1, the urinary cystine concentration was lower (P < 0.05), and the urinary volume was higher (P < 0.05) during Period 2. Patients treated with tiopronin had reduced cystine excretion (P < 0.05) and at the end of Period 2, an increased dose of tiopronin, reflecting a more aggressive treatment. Furthermore, a reduced number of stone episodes and need of active stone removal (P < 0.05) was noted in the whole group of patients. Analyses of separate day and night urine samples can be used advantageously to reveal episodes of high supersaturation with cystine not detected in 24-h urine samples. Such a procedure might be useful for optimizing the treatment of patients with cystinuria.     

Percutaneous chemolitholysis of cystine stone

Although cystine stone accounts for only 1-3% of renal calculi, many of the pure cystine stones usually can not be fragmented by ESWL and the residuals after PCNL is quite common. Percutaneous dissolution alone or combined with ESWL and PCNL could successfully dissolve these stones including all the residuals. This paper reported seven patients (8 pieces of renal and 2 pieces of ureteral stones) with cystine calculi in whom percutaneous renal irrigation or per-ureteral catheter irrigation with tromethamine-E (THAM-E) were performed for a relatively short period of time. All the stones disappeared completely or near completely. The authors recommend that percutaneous irrigation alone (including irrigation with ureteral catheter) or combined with PCNL and ESWL are the method of choice in the treatment of pure cystine stone of the kidney.     

Chemolysis of artificial cystine stones (BON(N)-STONES) in vitro using a new dissolution device: first results

BACKGROUND AND PURPOSE: The treatment of cystine stones is a clinical problem. This in vitro study was performed to establish an experimental system that enables standardized and reproducible investigations on chemolysis of cystine stones to look for an improvement of dissolution strategies. MATERIALS AND METHODS: Artificial spherical stones made of cystine (BON(N)-STONES) with a diameter of 0.9 cm were used. A new dissolution device was developed simulating the physiological conditions in the upper urinary tract with computer-assisted online measurement of data. For chemolysis of artificial cystine stones, different solvents (artificial urine, physiologic sodium chloride solution, 2 % acetylcysteine, 8.4 % sodium bicarbonate solution, THAM, and combinations) were used. RESULTS: Chemolysis is an effective tool in the management of cystine stone disease. Statistical analysis showed significant differences (p < or = 0.05) for all solutions compared with artificial urine alone. A combination of THAM at pH 10 with acetylcysteine (2%) showed a 48-fold stronger ability to dissolve cystine calculi than did artificial urine. CONCLUSION: By performing standardized in vitro investigations, new basics to improve the dissolution of cystine stones have been developed. It is recommended to use artificial stones made of cystine and a dissolution device simulating physiological conditions for investigations on chemolysis in the future.     

Holmium: YAG lithotripsy: photothermal mechanism.

OBJECTIVE: A series of experiments were conducted to test the hypothesis that the mechanism of holmium:YAG lithotripsy is photothermal. METHODS AND RESULTS: To show that holmium:YAG lithotripsy requires direct absorption of optical energy, stone loss was compared for 150 J Ho:YAG lithotripsy of calcium oxalate monohydrate (COM) stones for hydrated stones irradiated in water (17+/-3 mg) and hydrated stones irradiated in air (25+/-9 mg) v dehydrated stones irradiated in air (40+/-12 mg) (P < 0.001). To show that Ho:YAG lithotripsy occurs prior to vapor bubble collapse, the dynamics of lithotripsy in water and vapor bubble formation were documented with video flash photography. Holmium:YAG lithotripsy began at 60 microsec, prior to vapor bubble collapse. To show that Ho:YAG lithotripsy is fundamentally related to stone temperature, cystine, and COM mass loss was compared for stones initially at room temperature (approximately 23 degrees C) v frozen stones ablated within 2 minutes after removal from the freezer. Cystine and COM mass losses were greater for stones starting at room temperature than cold (P < or = 0.05). To show that Ho:YAG lithotripsy involves a thermochemical reaction, composition analysis was done before and after lithotripsy. Postlithotripsy, COM yielded calcium carbonate; cystine yielded cysteine and free sulfur; calcium hydrogen phosphate dihydrate yielded calcium pyrophosphate; magnesium ammonium phosphate yielded ammonium carbonate and magnesium carbonate; and uric acid yielded cyanide. To show that Ho:YAG lithotripsy does not create significant shockwaves, pressure transients were measured during lithotripsy using needle hydrophones. Peak pressures were <2 bars. CONCLUSION: The primary mechanism of Ho:YAG lithotripsy is photothermal. There are no significant photoacoustic effects.     

In vitro dissolution of cystine urinary calculi

Dissolution of cystine urinary calculi was studied in vitro. Sodium hydroxide, acetylcysteine and tris(hydroxymethyl)aminomethane (tromethamine) were tested for effectiveness in dissolving cystine calculi. Calculi were mounted in a dissolution apparatus. Dissolution rates were calculated from the amount of cystine released into solution per unit time and were compared with linear regression techniques. Calculated dissolution rates of all the irrigating agents tested were similar at a pH of 7.5 and were significantly different at pH 10.0. The most effective agent in the promotion of cystine dissolution was 2 per cent acetylcysteine. Sprague-Dawley rats were used to test the effect of 2 per cent acetylcysteine, 0.3 molar tromethamine and 2 per cent acetylcysteine mixed with 0.3 molar tromethamine on the urothelium. A nephrostomy tube was placed in the left kidney of each rat. The kidneys were irrigated for 3 days with either 2 per cent acetylcysteine, 0.3 molar tromethamine or 2 per cent acetylcysteine mixed with 0.3 molar tromethamine. The rats then were sacrificed at various intervals after infusion. Irrigation with 2 per cent acetylcysteine at a pH of 10 caused an acute inflammatory response at 3 days that was healed almost completely in 4 weeks. The addition of tromethamine to the acetylcysteine did not prevent the inflammatory response.     

A comparison of the effects of potassium citrate and sodium bicarbonate in the alkalinization of urine in homozygous cystinuria

For many years, urine alkalinization has been one of the cornerstones in the treatment of homozygous cystinuria. Because of the relationship found between the excretion of urinary sodium and cystine, potassium citrate has emerged as the preferred sodium-free alkalizing agent. To evaluate the usefulness of potassium citrate for urine alkalization in cystinuric patients, sodium bicarbonate and potassium citrate were compared in 14 patients (10 on tiopronin treatment and four without treatment with sulfhydryl compounds). The study started with 1 week without the use of any alkalizing agents (Period 0) followed by 2 weeks with sodium bicarbonate (Period 1) and 2 weeks with potassium citrate (Period 2). Urinary pH, volume, excretion of sodium, potassium, citrate and free cystine, as well as the plasma potassium concentration, were recorded. Potassium citrate was shown to be effective as an alkalizing agent and, in this respect, not significantly different from sodium bicarbonate. Even though a normal diet was used, a significant increase in urinary sodium excretion was observed with sodium bicarbonate (Period 1). Urinary potassium and citrate excretion increased with potassium citrate (Period 2). A significant correlation was found between urinary sodium and cystine in the tio-pronin-treated patients. No significant differences in cystine excretion were recorded in Periods 0, 1 and 2. Plasma potassium was significantly higher during Period 2, but only one patient developed a mild hyperkalemia (5.0 mmol/l). The use of potassium citrate for urine alkalization in homozygous cystinuria is effective and can be recommended in the absence of severe renal impairment.     

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.