Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.

Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.

Peptidome profiles in melamine diet-induced bladder stones in C57BL/6 mice.

The aim of this research was to detect potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6 mice. Magnetic bead-based weak cationexchange chromatography (MB-WCX) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were employed to detect serum biomarkers in 10 mice fed a melamine diet and 10 control mice. Seventeen peaks (fold change>1.5) with a mass to charge (m/z) value of 1000-10,000 Da were detected in the two groups. Among the significant peaks, five were upregulated and the other 12 were downregulated in the model group. Among the upregulated peaks, 2954.49 and 1710.49 were found to correspond to the peptide regions of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8(Ndufα8) and basigin, respectively, by liquid chromatography with electrospray ionization and tandem triple quadrupole mass spectrometry(LC-ESI-MS/MS). Western blot analysis was used to detect the expression of Ndufα8 and basigin in another 10 model mice and 10 control mice. The western blot results confirmed the LC-ESI-MS/MS data. The expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence. In conclusion, Ndufα8 and basigin may be potential serum biomarkers for the detection of melamine diet-induced bladder stones in C57BL/6 mice.

Divertículo vesical adquirido. Caso clínico / Acquired bladder diverticulum. A clinical case

El divertículo vesical (DV) puede ser congénito o adquirido. Consiste en una herniación de la mucosa vesical. Los adquiridos se deben a un mecanismo de hiperpresión endovesical generalmente en pacientes con obstrucción infravesical secundaria a hiperplasia benigna de próstata (HBP), patología del cuello vesical o uretral. Cuando los DV son sintomáticos o producen complicaciones se debe plantear tratamiento quirúrgico. En los últimos años la diverticulectomía laparoscópica ha presentado muy buenos resultados aunque la cirugía abierta está indicada en algunos casos más complejos. En DV adquiridos de tamaño pequeño y mediano está indicado el tratamiento transuretral. Se presenta un caso clínico y una revisión de diferentes aspectos diagnósticos y quirúrgicos del DV (AU)

The bladder diverticulum (DV) can be congenital or acquired. It consists of a herniation of the bladder mucosa. The acquired is due to a mechanism of intravesical hyperpressure usually in patients with bladder outlet obstruction secondary to BPH, cervical or urethral pathology. When DV are symptomatic or produce complications surgical treatment should be considered. In recent years laparoscopic diverticulectomy has shown very good results although open surgery is indicated in some more complex cases. In small and medium acquired DV transurethral treatment is indicated. One clinical case and a review of various diagnostic and surgical aspects of DV are presented (AU)

Preventive and therapeutic effects of sodium bicarbonate on melamine-induced bladder stones in mice.

The actual preventive and therapeutic effects of alkalinizing urine on melamine-induced bladder stones (cystolith) are not completely known. Using an ideal model, two experiments were conducted in Balb/c mice. The mice were fed a normal diet in controls and a melamine diet in the other groups. The first day was set as experiment-day 1. In "Experiment 1", either low-/mid-/high-dose sodium bicarbonate (SB) or sterile water was administered by intragastric perfusion (once daily) to the mice for 14 days. Relative to the model group, the mean pH of the urine in the SB groups was significantly elevated at 3 h after SB administration, with a significant decrease in cystolith incidence on experiment-day 14. In "Experiment 2", on experiment-day 12, the melamine diet was replaced by a normal diet in 4 groups with melamine withdrawal (MW). Meanwhile, either mid-/high-dose SB or sterile water was administered by intragastric perfusion (once) to the mice in the corresponding groups. On experiment-day 12, after an additional 8 h, the cystolith incidence was significantly reduced in the high-SB, MW + mid-SB and MW + high-SB groups than in the model group. In conclusion, low urinary pH is one of the main determinants of the formation of melamine-associated stones, urinary alkalinization can be achieved by a proper dose of oral SB, and SB acts to prevent and treat melamine-induced cystoliths in mice.

Proteome of melamine urinary bladder stones and implication for stone formation.

Melamine can cause urinary stones related to nephropathy of the kidney and hyperplasia or carcinoma of the bladder, but the mechanism of stone formation is not well understood. In this study, male rats were administered melamine for thirteen weeks to establish melamine bladder stone models and the stones were analysed by Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and western blot, respectively, for the composition and proteome, and to explore the implication of proteins for stone formation. The results showed bladder stones were composed of predominant melamine and a few amount of proteins. The proteins had a wide range of molecular weights and 1051 proteins were identified. Gene Ontology (GO) classification of the identified proteins showed most proteins were from injured cells, involved in various metabolic processes and had binding functions. Of the identified proteins, there were a few inflammatory proteins and urinary proteins. Physicochemical characteristics of the identified proteins showed that 67.1% proteins' isoelectric points (pI) value was below 7.0, 91.1% proteins' grand average of hydropathicity (GRAVY) scores were below 0 and nearly half of the proteins were stable. Our data indicated proteins might play an important role in melamine bladder stone formation.

The natural outcome of melamine-induced bladder stones with bladder epithelial hyperplasia after the withdrawal of melamine in mice.

The natural outcome of melamine-induced bladder stones (cystoliths) with bladder epithelial hyperplasia (BEH) after melamine withdrawn is unclear. Using an ideal dual-model system, three experiments were conducted in BALB/c mice. Each experiment included a control, model 1 and model 2 groups. The mice were fed a regular diet in controls or a 9373 ppm melamine diet in models, and the first day was designated as dosing day 1. The melamine diet was then replaced by the regular diet in the model 2 groups, and the first day was designated as post-dosing day 1. On dosing days 12, 35 and 49, the incidence of cystoliths and diffusely active BEH was 8/8 in the mice of three model 1 groups. On post-dosing days 1, 4 and 8, in the mice of three model 2 groups, the incidence of cystoliths was 2/8, 0/8 and 1/8, respectively, and the progressive regression of BEH was observed. In conclusion, both the stones and BEH have the natural property of rapid development and rapid regression, and melamine withdrawn plays a key role in the stone dissolution-discharge necessary for BEH regression. BEH may be reversible after the discharge of the stones. The conventionally conservative therapy is thus reasonable.

The rapid establishment and implications of a melamine-induced standardized bladder stone model in mice.

The key to establishing a standardized melamine-induced animal bladder stone (cystolith) model is to determine the most appropriate daily dose of dietary melamine, which is unknown. Based on golden section theory that is a well-known preferred proportion (0.618), and the 50% lethal dose (LD50) of mouse oral melamine [4550 mg/kg body weight (bw)], we proposed that the daily dose may be close to the LD50's golden section (i.e., 0.618 × 4550 mg melamine/kg bw). The latter as an average daily dose corresponds to 9373 ppm melamine diet in mice. In repeated experiments, a 100% incidence of cystoliths was observed on modeling day 14 in Balb/c and C57BL/6 mice fed the diet but not in mice fed similar diets containing 9842 (i.e., 9373 × 105%) or 8904 (i.e., 9373 × 95%) ppm melamine; the stones were relatively uniform and the difference in stone incidences between sexes or ages was not found in each 9373 ppm melamine group. In conclusion, 9373 ppm melamine diet is at least near the optimal dose diet or ideal for the rapid and stable establishment of a standardized cystolith model in the mice, and dietary melamine dose neither sex nor age is critical for stone formation.

The melamine excretion effect of the electrolyzed reduced water in melamine-fed mice.

Our hypothesis is that the intake of functional water, electrolyzed reduced water (ERW) can excrete melamine in body was evoked by melamine-tainted feed (MTF). To address this issue, we investigated the effect of ERW in MTF-mice model by way of body weight gain, incidence of urinary crystals and bladder stone, biochemical and haematological examination, histopathologic finding of kidney and urinary bladder, and the evaluation of bladder stone. We found that the rate of body weight gain was significantly more increased in MTF+ERW group than MTF+PW group. Accordingly, the number of immunocytes such as leukocyte, neutrophil and monocyte as well as the mean weight of spleen was significantly increased in MTF+ERW group. The incidence of urinary crystals was significantly higher in MTF+ERW group, whereas the incidence of urinary bladder stones was lower in MTF+ERW group (52.4%) than in MTF+PW group (38.1%). Also, urinary crystals were more precipitated in MTF+ERW group than MTF+PW group, and urinary bladder stone consists of 100% melamine. Collectively, our data clearly show that ERW intake is helpful to excrete of melamine in MTF mice model and this is the first report on the melamine excretion and clinically implying the safer fluid remedy for melamine-intoxicated hosts.

Low dose estrogen supplementation reduces mortality of mice in estrogen-dependent human tumor xenograft model.

Breast cancer is one of the most frequent female cancers in the Western world. Perturbation of estrogen levels by hormone replacement therapy or pregnancy is associated with a variety of diseases, including breast cancer. Estrogen supplementation is required to establish appropriate animal models for estrogen-related diseases. In this report, we demonstrated that supplementation with high doses of 17beta-estradiol results in deaths in estrogen-dependent MCF-7 tumor xenograft model. Renal damage and bladder stone formation was implicated as a major cause of death. The mortality rate was significantly reduced when mice received a low dose of 17beta-estradiol. We also confirmed that low dose of 17beta-estradiol supplementation can support the growth of tumors in MCF-7 tumor xenograft model. These results suggest that low dose estrogen supplementation may be more appropriate in estrogen-dependent tumor xenograft models.

Are new-generation bisphosphonates effective for the inhibition of calcium oxalate stone formation in a rat model?

BACKGROUND: The effects of bisphosphonates on prophylaxis of stone formation are unclear. We evaluated the outcome of two new-generation bisphosphonates in a lithogenic rat model. METHODS: 36 male rats were divided into three groups of 12 animals each. Both calcium and creatinine levels of plasma and urine were measured. Zinc discs of about 40 mg each were surgically placed into the bladder. The first group received no treatment and the second and third groups were treated with an intraperitoneal injection of weekly clodronate (20 mg/kg) and zoledronic acid (7.5 microg/kg), respectively. At the end of the 8th week, the weight increase in discs and biochemical changes were analyzed comparatively. RESULTS: The mean weight of discs in the control, clodronate and zoledronic acid groups was 109.65 +/- 80.97, 79.82 +/- 17.99 and 72.91 +/- 19.29 mg, respectively (p > 0.05). The percentage increase in weight of discs was 164% for control, 90% for clodronate and 71% for the zoledronic acid group. The increase of urinary calcium level in the zoledronic acid group was lower than the others (p < 0.05). CONCLUSIONS: When considering the percentage increase in weight of discs, the difference between control and bisphosphonate groups support the idea that these drugs may have a preventive role in stone formation.

Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer.

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.

Effects of low-molecular-weight polyguluronate sulfate on experimental urolithiasis in rats.

Urinary macromolecules, especially glycosaminoglycans (GAGs), have attracted great interest as promising inhibitors of urinary stone formation. As an analogue of GAGs, low-molecular-weight polyguluronate sulfate (LPGS) with strong polyanionic nature was prepared by chemical modification of brown algae extract. The effects of LPGS both on ethylene glycol-induced nephrolithiasis and Zinc disc implant-induced urinary bladder stone formation in Wistar rats were evaluated, and its acute toxicity in Kunming mice and Wistar rats were also investigated. The contents of renal oxalate and calcium in ethylene glycol-induced nephrolithiasic rats were decreased significantly from 5.01 +/- 0.96 to 3.26 +/- 1.31 mumol/g kidney (P < 0.01) and 20.11 +/- 4.60 to 11.83 +/- 3.54 mumol/g kidney (P < 0.01), respectively, after oral administration of LPGS at dose-level of 100 mg/kg. The renal crystal depositions and histopathological changes were reduced also. The formation of zinc disc implant-induced urinary bladder stones in rats was inhibited considerably after oral administration of LPGS at dose-levels of 50 mg/kg (P < 0.05) and 100 mg/kg (P < 0.01). The intravenous LD(50) and the oral maximum tolerance value of LPGS in mice are 6.29 and 25 g/kg, and in rats are 2.25 and 10 g/kg, respectively. These data show that LPGS has significant prevention effects both on nephrolithiasis and urinary bladder stone formation in rats, and negligible oral toxicity both in mice and rats. LPGS is a safe and promising drug candidate for the prevention of urolithiasis.

Possible contribution of indomethacin to the carcinogenicity of nongenotoxic bladder carcinogens that cause bladder calculi.

Nongenotoxic bladder carcinogens that form bladder calculi have been concluded to be of low carcinogenic risk to humans because bladder stones would be expelled or surgically removed before they had a chance to exert their carcinogenic effect. It is the aim of this report to examine the possible contribution of indomethacin to the carcinogenic risk posed by nongenotoxic bladder carcinogens that cause bladder stones. Indomethacin may act as a tumor promoter in the bladder by interfering with the synthesis of prostaglandins. Prostaglandins have a cytoprotective function in the gastric mucosa and possibly also in the urinary bladder. Diminished cytoprotection may be implicated in bladder carcinogenesis as beta-naphthylamine, a human bladder carcinogen, also inhibits prostaglandin synthesis in vitro. The presence of other tumor promoters in the bladder may further ensure that tumors would be formed even if bladder stones were expelled. People who are exposed to nongenotoxic bladder carcinogens that are present in the environment and that form bladder stones, therefore, may be at an increased risk for developing bladder cancer if they are also exposed to tumor promoters, such as indomethacin.

Parenteral ambroxol treatment causes xanthine and calcium oxalate stones in rats.

INTRODUCTION: Ambroxol (ABX) is known to promote bronchial secretion and is used as an expectorant. This study was undertaken to document the connection between ambroxol parenteral treatment and bladder stones in rats. MATERIAL AND METHODS: Forty-five wild rats (Rattus sp.) were divided into three equal groups. Rats from the first and second groups received ABX s.c. during 2 weeks in total doses of 30 mg/kg per 24 h and 60 mg/kg per 24 h, respectively. Rats from the control group received 1 mL of injection solution s.c. One month after the treatment termination, animals were sacrificed and urinary tracts without urethra were dissected. Stones found in the bladders were measured, weighed and chemically analysed. Voiding cystography was performed to exclude pathology of the lower urinary tract. Photo documentation was produced. RESULTS: From the first and second groups, 33% and 47% of rats, respectively, had solitary stones in the bladder. In one case from the second group, there was a huge stone in the bladder and urethra. There were no stones in rats from the control group. The mean length of stones was 1.38 +/- 0.23 mm and 1.41 +/- 0.60 mm in the first and second groups, respectively. Mean stone weight was 1.2 +/- 0.2 x 10(-3) g and 1.44 +/- 0.54 x 10(-3) g. Stones were composed of 67% of xanthine and 33% of calcium oxalate. CONCLUSIONS: Ambroxol parenteral treatment caused xanthine and oxalate stone formation. Attention should be paid to the possibility of urinary stone formation after long-term ABX treatment.

Elevated prostaglandin E2 level via cPLA2--COX-2--mPGES-1 pathway involved in bladder carcinogenesis induced by terephthalic acid-calculi in Wistar rats.

To investigate the prostaglandin E2 (PGE2) biosynthetic mechanism in bladder carcinogenesis, we established Wistar rat model of bladder papilloma and transitional cell carcinoma (TCC) induced by 5% terephthalic acid (TPA) treatment. Then, the mRNA level of cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COX)-1 and -2, membrane-bound PGE2 synthases (mPGES)-1 and -2 was detected using reverse transcription polymerase chain reaction (RT-PCR). Immunoblotting was applied to detect the expression of COX-2 protein. Proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry. In addition, the level of PGE2 was measured by radioimmunoassay (RIA). Bladder papilloma (100%, 8/8) was examined in rats after 24-week treatment, and bladder TCC (80%, 16/20) was found after 48-week treatment. Histopathological changes were not found in control group rats. The incidence of bladder papilloma and TCC in test group was significantly higher than that in control group (P<0.01). The mRNA levels of cPLA2, COX-2 and mPGES-1 in the bladder papilloma and TCC were significantly higher than those in normal bladder (P<0.01), while the mRNA levels of COX-1 and mPGES-2 in TCC were unchanged compared with normal bladder. Bladder TCC exhibited a substantial expression of COX-2 protein. On the contrary, normal bladder tissue barely expresses COX-2 protein. PCNA labeling index (LI) and the level of PGE2 in bladder papilloma are much higher than those in normal bladder (P<0.01), but lower than those in bladder TCC (P<0.05). In conclusion, increasing PGE2 level via cPLA2--COX-2--mPGES-1 pathway may play an important role in rat bladder carcinogenesis. PGE2 may be a biomarker for the development of bladder TCC. cPLA2 and mPGES-1 may be targets for development of novel chemoprevention strategies for bladder TCC.

Modification of N-Methyl-N-Nitrosourea initiated bladder carcinogenesis in Wistar rats by terephthalic acid.

The effect of terephthalic acid (TPA) on urinary bladder carcinogenesis was examined. Male Wistar rats were initiated by injection of N-Methyl-N-Nitrosourea (MNU) (20 mg/kg b.w. ip) twice a week for 4 weeks, then given basal diet containing 5% TPA, 5% TPA plus 4% Sodium bicarbonate (NaHCO3) or 1% TPA for the next 22 weeks, and then euthanized. 5% TPA treatment induced a high incidence of urinary bladder calculi and a large amount of precipitate. Though 5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment did not induce urinary bladder calculi formation, they resulted in a moderate increase in urinary precipitate. Histological examination of urinary bladder revealed that MNU-5% TPA treatment resulted in a higher incidence of simple hyperplasia, papillary or nodular hyperplasia (PN hyperplasia), papilloma and cancer than MNU control. MNU-5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment increased slightly the incidence of simple hyperplasia and PN hyperplasia (not statistically significant). The major elements of the precipitate are phosphorus, potassium, sulfur, chloride, calcium and TPA. The present study indicated that the calculi induced by TPA had a strong promoting activity on urinary bladder carcinogenesis and the precipitate containing calcium terephthalate (CaTPA) may also have weak promoting activity on urinary bladder carcinogenesis.

Induction of bladder lesion by terephthalic acid and its mechanism.

OBJECTIVE: To provide more information for rational evaluation of potential risks of terephthalic acid (TPA), we studied the effects of TPA on rats' bladders in 90 days after TPA exposure. METHODS: Sprague Dawley rats were subdivided into five groups, ingesting 0%, 0.04%, 0.2%, 1%, and 5% TPA respectively for a sub-chronic feeding study lasting for 90 days. Urine, serum and samples of brain, liver, lung, kidney, bladder, etc. were collected and analyzed. RESULTS: TPA ingesting decreased the value of urinary pH, and increased the contents of Ca2+, Zn2+, Mg2+, Na+, K+ in urine. The volume of 24 h urine was significantly increased in male rats in the 1% and 5% TPA groups. Urinary white sediment was found in both sexes, and its formation in male rats seemed more susceptible than that in female rats. Alpha 2u-globulin (AUG) in serum and urine of male rats was markedly increased in a dose-dependent manner. Fifteen cases of hyperplasia (simple or atypical) were determined in the 5% TPA ingesting group, 14/52 in male rats and 1/23 in female rats. Among them 3 male rats had no stone or calculus. Those with either bladder stones or hyperplasia were accompanied with urinary white sediments. CONCLUSION: White sediment accompanied with elevated urine AUG is the basis of TPA induced urolith formation, and is also associated with TPA induced bladder epithelial cell proliferation. It can act as an early biomarker for the potential toxic effect of TPA.

Sulfadiazine-related obstructive urinary tract lithiasis: an unusual cause of acute renal failure after kidney transplantation.

We report on the first case of acute renal failure related to obstructive urinary tract lithiasis involving sulfadiazine crystals in a kidney transplant recipient. This patient had disseminated toxoplasmosis which was treated by sulfadiazine (4 g/day) and pyrimethamine (50 mg/day). In the fourth week of anti-toxoplasmosis therapy, he presented with obstructive acute renal failure: the plasma creatinine level increased from 220 micromol/l to 547 micromol/l. Apercutaneous pyelography was conducted showing the presence of a lithiasis located at the junction between the graft ureter and the bladder. Six days later, he underwent surgery to retrieve an orange-colored, friable stone. Its spectrophotometric analysis confirmed that the stone consisted of N-acetyl sulfadiazine crystals.

Bladder epithelial cell proliferation of rats induced by terephthalic acid-calculi.

OBJECTIVE: Urinary bladder hyperplasia associated with terephthalic acid (TPA) treatment was examined with concomitant use of sodium bicarbonate (NaHCO3) or hydrochlorothiazide to allow assessment of the relationship among bladder stones, epithelial hyperplasia, and corresponding cell cycle checkpoint gene expression in Sprague-Dawley (SD) rat. METHODS: A total of 112 weanling male SD rats that divided between six groups were given basal diet (control), diets containing 5% TPA or in combination with either 4% sodium NaHCO3 or 0.02% hydrochlorothiazide. After 90-day feeding, bladder samples were collected for histopathological diagnoses, and immunohistochemical method was used to characterize the expression of p16Ink4a cyclin D1, CDK4, EGFr and cyclin E in relation to that of proliferating cell nuclear antigen (PCNA). RESULTS: In TPA treatment groups, bladder stone incidence was 40% (21/52) with 14 cases of proliferative bladder. In control and other groups, neither stone nor epithelial cell proliferation was diagnosed. PCNA-positive focal hyperplasic lesions involved all epithelial layers. Overexpressions of cyclin D1, CDK4, EGFr are found in the corresponding lesion. p16Ink4a nuclear staining reduced in proliferative bladders especially with a great quantity of stone. In addition, no positive expression was detected on cyclin E. CONCLUSION: The present study provides a strong evidence of a link between induction of bladder hyperplasia, deregulation of the p16Ink4a-cyclin D1/CDK4 pathway, and abnormal EGFr mediated signal transduction pathway.

Urolithiasis associated with indinavir in a patient with spinal cord injury.

OBJECTIVE: To report a case of indinavir-induced urolithiasis, and the greater risk of this occurrence in individuals with spinal cord injury (SCI) who require fluid restriction for an intermittent catheterization program (ICP). METHODS: Case report. RESULTS: A 38-year-old man with a T4 ASIA A SCI (according to the American Spinal Injury Association classification scale) and human immunodeficiency virus (HIV) infection was using an ICP and taking indinavir (a protease inhibitor) as part of his antiviral regimen. Cystoscopy was performed to rule out recurrent urethral condylomata. He was found to have a bladder stone measuring 0.5 cm x 0.5 cm x 0.3 cm, which, on analysis, was composed of indinavir (100% exterior, 90% interior). The bladder stone was removed under direct visualization. The plain abdominal radiograph did not reveal any stones. CONCLUSION: Indinavir is a frequently used drug for the treatment of HIV that has the potential to induce urinary lithiasis. This is particularly problematic for individuals with SCI who are on fluid restriction and an ICP. Therefore, cystoscopy and monitoring for indinavir-induced urolithiasis should be undertaken in individuals with SCI who are taking indinavir. Considerations include switching to a different protease inhibitor or choosing an entirely new HIV drug cocktail with less potential for urolithiasis.