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.

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.

Initial presentation in psychiatry emergency room led to diagnosis of many urinary bladder stones in a male patient.

The first case of man who presented to psychiatry emergency room for evaluation of abnormal behavior because of urinary stones was reported. Careful evaluation of patient led to a diagnosis of 37 urinary bladder stones in an Egyptian man with obstructive uropathy and metabolic defects in the form of hyperoxaluria and hypocitraturia. Knowledge of the differential diagnosis of metabolic defects can lead to successful outcome in preventing reformation of urinary tract stones after surgery. A 61-year-old Egyptian man presented to psychiatry emergency room because he was found lying on floor in bathroom to urinate by his wife who thought her husband needed psychiatric evaluation. Patient gave history of frequent urination and dysuria on and off for 3 years. In the last 3 months before his presentation to emergency room, he got into a habit of lying down on his left side when he went to bathroom to urinate because it was easier for him to pass urine. Renal consultation requested because of presence of red blood cells in urinalysis. Computed tomography of the abdomen and pelvis showed bilateral hydronephrosis and multiple bladder stones. Twenty-four-hour urine collection showed low urinary citrate and high oxalate. Patient underwent open vesicolithotomy and removal of 36 stones. Stone analysis showed 75% uric acid and 25% calcium oxalate. Patient did very well after surgery, and 1 month later, he underwent transuretheral resection of prostate without any complications. Now patient has no difficulty passing urine and he has no recent attack of urinary tract infection. Knowledge of the differential diagnosis of metabolic defects in men with urinary bladder stones would hopefully provide clinicians with the proper diagnostic tools to more specifically treat such patients with improved success in preventing reformation of urinary tract stones after surgery.

[Urinary trace elements excretion in patients with cystine calculosis]. / Wydalanie pierwiastków sladowych z moczem u chorych z kamica cystynowa.

UNLABELLED: Not all cystinuric patients develop urinary calculi. There is the need to identify modulating factors. In recent years the role of trace elements in the mechanism of stone formation has received a steadily increasing attention. The aim of our study was to determine urinary excretion of trace elements in patients with recurrent cystine calculosis. MATERIAL AND METHODS: 11 patients with recurrent cystine calculosis and 15 healthy volunteers participated in the study. Urinary zinc, copper, chromium, boron and arsenic concentrations were determined by use of inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: There was a statistically significant difference between the patients with cystinuria and controls in mean arsenic urinary excretion (0.625+/-0.6 microg/kg body weight/24 h vs. 1.264+/-0.635 microg/kg b.w./24 h, p=0.02). There was also a noticeable difference in urinary chromium, the mean concentration of which was higher in case of the patients with cystinuria (3.32+/-3.59 microg/kg/24 h) than in the controls (1.38+/-0.69 microg/kg/24 h). The mean urinary excretion of zinc, copper, and boron (in microg/kg b.w./24 h) were respectively 7.40+/-7.98; 1.33+/-0.48; 26.55+/-15.81 in the patients with cystinuria and 9.84+/-5.33; 1.16+/-0.84; 35.87+/-13.76 in the controls. CONCLUSION: It seems that a decreased excretion of arsenic may contribute to the development of cystine calculosis.

[Misdiagnosis of urinalysis due to in vivo formation of urinary stones]. / Fehlinterpretation von Urinanalysen durch in vivo wachsende Harnsteine.

The occurrence of biochemically unaltered urinalyses in patients with severe recurrent stone formation is not a rare observation in practice. The possible reasons for that phenomenon are manifold. We show that stone growth-related urinary depletion of lithogenic constituents caused by acute growth of urinary calculi in vivo can be an important reason for the observed phenomenon. The described process which can strongly influence the urinary composition occurs in any stone-bearing patient. Thus, it is strongly recommended that stone-related alterations be taken into account when interpreting the urinalyses of these patients. Based on simplified model assumptions, the extent of the expected chemical depletion effect can be calculated for any stone patient's urine sample. In two easy-to-use nomograms, we have combined the key parameters which govern the process, allowing the user a fast and easy estimation.

The effect of Phyllanthus niruri on urinary inhibitors of calcium oxalate crystallization and other factors associated with renal stone formation.

OBJECTIVE: To evaluate the effect of an aqueous extract of Phyllanthus niruri (Pn), a plant used in folk medicine to treat lithiasis, on the urinary excretion of endogenous inhibitors of lithogenesis, citrate, magnesium and glycosaminoglycans (GAGs). MATERIALS AND METHODS: The effect of chronic (42 days) administration of Pn (1.25 mg/mL/day, orally) was evaluated in a rat model of urolithiasis induced by the introduction of a calcium oxalate (CaOx) seed into the bladder of adult male Wistar rats. The animals were divided into four groups: a sham control (16 rats); a control+Pn (six); CaOx+water instead of Pn (14); and CaOx+Pn (22). Plasma and urine were collected after 42 days of treatment for biochemical analysis and the determination of urinary excretion of citrate, magnesium and GAGs. The animals were then killed and the calculi analysed. RESULTS: The creatinine clearance or urinary and plasma concentrations of Na+, K+, Ca2+, oxalate, phosphate and uric acid were unaffected by Pn or the induction of lithiasis. Treatment with Pn strongly inhibited the growth of the matrix calculus and reduced the number of stone satellites compared with the group receiving water. The calculi were eliminated or dissolved in some treated animals (three of 22). The urinary excretion of citrate and magnesium was unaffected by Pn treatment. However, the mean (sd) urinary concentration of GAGs was significantly lower in rats treated with CaOx+Pn, at 5.64 (0.86) mg/g creatinine, than when treated with CaOx + water, at 11.78 (2.21) mg/g creatinine. In contrast, the content of GAGs in the calculi was higher in the CaOx + Pn rats, at 48.0 (10.4) g/g calculus, than in the CaOx + water group, at 16.6 (9.6) g/g calculus. CONCLUSION: These results show that Pn has an inhibitory effect on crystal growth, which is independent of changes in the urinary excretion of citrate and Mg, but might be related to the higher incorporation of GAGs into the calculi.

Excretion of tamm-horsfall protein in patients with uric acid stones.

The cause of reduced Tamm-Horsfall protein excretion in patients suffering from uric acid diathesis is still unknown. Our investigation was conducted based on the hypothesis that the solubility of uric acid is increased by Tamm-Horsfall protein and that an increased uric acid content in the urine might cause a decrease in Tamm-Horsfall protein. In 20 patients with uric calculi the excretion of Tamm-Horsfall protein, uric acid, calcium, and citrate was measured. 65% of the patients had pure uric acid stones (group I) and 35% showed mixed stones with at least 30% of uric acid (group II). Reduced Tamm-Horsfall protein excretion was found in 63% of the patients of group I and in 43% of the patients of group II. The excretion of Tamm-Horsfall protein was significantly reduced in pure uric acid stone formers compared to normal subjects (p < 0. 0001). The excretion of uric acid was elevated in 61% of the patients of group I and in 86% of the patients of group II. There was no significant correlation between Tamm-Horsfall protein excretion and uric acid excretion (r = 0.2139). Calcium excretion was elevated in 57% of the patients with mixed stones. The excretion of citrate was reduced in almost all of the patients of groups I and II. Our results do not support the hypothesis that an increased content of uric acid in the urine causes a decrease in Tamm-Horsfall protein. In our opinion the lower excretion of Tamm-Horsfall protein in some of the stone patients might be caused by damage in the distal tubular epithelium. Moreover, it has to be supposed that there are defects both in the distal and the proximal tubule in patients prone to develop uric acid calculi.

Study of calcium oxalate crystalluria on renal and vesical urines in stone formers and normal subjects.

OBJECTIVE: The aim of this study is to compare vesical and renal calcium oxalate crystalluria in an attempt to correlate crystal formation with chemical composition and calcium oxalate saturation of renal urine. MATERIAL AND METHODS: Urine specimens were directly collected from the bladder and the kidney, of 11 stone formers and 11 control subjects under general anesthesia. The type of crystals present in urine as well as their size, number by cubic millimeter and state of aggregation were determined. In addition, calcium, magnesium, sodium, chloride, phosphate, citrate, oxalate, pyrophosphate and uric acid were measured in order to evaluate the calcium saturation status (EQUIL V program). RESULTS: Calcium oxalate crystals were detected in 3 stone formers (27%) and 2 control subjects (18%) in vesical urine and in 4 stone formers (36%) and 3 control subjects (27%) in renal urine. Only 2 stone formers presented with simultaneous renal and vesical crystalluria. Subjects of the two groups with and without renal crystalluria were compared in terms of chemical composition and calcium oxalate saturation of renal urine. Crystalluric subjects (n = 7) had significantly higher uricosuria (p = 0.02), calciuria (p = 0.04), magnesiuria (p = 0.04) and calcium oxalate molar product (p = 0.05) than noncrystalluric (n = 15); calcium oxalate saturation was similar (p = 0.5). CONCLUSIONS: Beyond theorical considerations on lithogenesis, our observations and in particular the apparent discrepancy between renal and vesical crystalluria pose the problem of the clinical interest of the evaluation of calcium oxalate crystalluria based on freshly voided urine in the assessing the lithogenic risk or in the follow-up of patients who are particularly prone to stone recurrence.

Rodent bladder tumors do not always predict for humans.

Dr. David Clayson, 20 years ago, suggested that chemicals which lead to the formation of calculi in rodents might pose an artifact with respect to extrapolation to potential carcinogenic risk to humans. We reviewed what has been learned about the role of calculi in urinary bladder carcinogenesis in the ensuing 20 years, along with several examples. Formation of microcrystalluria and amorphous precipitate also poses problems in interpretation and examples are described. The chemicals producing these solid urinary materials are non-genotoxic, with marked increase in cell proliferation being the mode of action by which they are able to produce cancer in long-term rodent bioassays.

Evaluation of Ammannia baccifera Linn. for antiurolithic activity in albino rats.

Ethanolic extract of A. baccifera was tested for its antiurolithic activity in male albino rats. Urinary stones were induced by implantation of zinc discs in the urinary bladder. The stones formed were mainly of magnesium ammonium phosphate with traces of calcium oxalate. Ethanolic extract of A. baccifera (2g/kg/day, po) was found to be effective in reducing the formation of stones as also in dissolving the pre-formed ones. There was a significant increase in the urinary excretion of calcium, magnesium and oxalate, four weeks after implantation of zinc discs. Treatment with A. baccifera has significantly reduced calcium and magnesium levels in the prophylactic group while it has reversed the levels of these ions to normal values in the curative group.

Ascorbic acid is an abettor in calcium urolithiasis: an experimental study.

Two sets of animal experiments using guinea pigs were planned to evaluate the effect of ascorbic acid supplementation on the lithogenic process. In the first set of experiments, 10, 40, and 60 mg doses of ascorbic acid/100g body weight/day were given for 105 days. Neither of the ascorbic acid doses given induced crystalluria, calcification or stone formation, thereby confirming our previous findings that ascorbic acid in the doses used by clinicians does not cause urolith formation. In the second set of experiments, ascorbic acid was supplemented in hypercalciuric (induced by calcium carbonate feeding) and hyperoxaluric (induced by sodium oxalate feeding) animals for 45 days. The results indicated that it exacerbated the calcification process in renal and bladder tissue.

Canine cystinuria: an extended study on the effects of 2-mercaptopropionylglycine on cystine urolithiasis and urinary cystine excretion.

A clinical study covering 1 to 6 years was undertaken during which 25 cystinuric dogs were orally treated with 2-mercaptopropionylglycine (2-MPG). The drug was effective at dissolving uroliths at a dose of approximately 40 mg kg-1 body weight. In 15 dogs with bladder uroliths, complete urolith dissolution was achieved on 9/17 occasions (53%). When 2-MPG was administered prophylactically at 30 mg/kg body weight, uroliths did not reform in 14 dogs (56%). In four dogs, uroliths re-formed during treatment, but dissolved when the dose of 2-MPG was raised to 40 mg kg-1 body weight. Six dogs were surgically treated, and in two of these animals the uroliths were found to consist of magnesium ammonium phosphate. Euthanasia was performed on six dogs during the study; three because of recurrent uroliths with urethral obstruction, and three because of aging. In one dog, uroliths were present in the bladder throughout the study. The purpose of the study was to propose a new strategy for individual treatment of cystinuric dogs. This was accomplished by measuring the urinary free cystine concentration and the mixed cysteine-2-MPG disulphide in a subgroup of 15 of the 25 dogs. To evaluate cystine excretion, morning samples of urine were used, and the cystine concentration was related to the creatinine concentration. For dose adjustment it was difficult to evaluate the effect of 2-MPG on urinary cystine excretion, especially when cystine uroliths were present. However, this variable was studied in order to identify dogs with a strong tendency for urolith formation during 2-MPG treatment. In some cases, urinary cystine excretion returned to normal with time, and in three dogs, 2-MPG treatment could be stopped after 1.5 to 3.5 years. In spite of no further treatment, urinary cystine was almost undetectable up to 2 years later, and the dogs did not develop any new uroliths. It was concluded that 2-MPG is a satisfactory alternative treatment for cystinuric dogs. It has a good prophylactic effect, shown as a change in the rate of urolith formation from on average 6 months before to 17 months during 2-MPT treatment. The drug was shown to have few side effects, and the dog owner drug compliance can be followed by measurement of the mixed 2-MPG-cysteine disulphide.

Possible role for chondroitin sulfate in urolithiasis: in vivo studies in an experimental model.

The effect of chondroitin sulfate upon the growth of calcium oxalate crystals was measured in vivo by using an experimental model in rats. Adult male Wistar rats were treated by chronic i.p. injections of chondroitin sulfate solutions (1, 5 or 10 mg in 0.3 ml of saline, every 2 days). This treatment led to a dose-dependent increase in the urinary chondroitin sulfate concentration. Urolithiasis was induced by the introduction of a calcium oxalate seed into the bladder of the animals. Urine samples were collected and the calculi formed were removed after 42 days. The chondroitin sulfate concentration have decreased in the lithiasic urines, as compared to controls and higher chondroitin sulfate doses correlated with larger calculi. The presence of chondroitin sulfate in the matrices of stones obtained from chondroitin sulfate-treated animals suggested that there was some adsorption of chondroitin sulfate on to the growth sites of the calcium oxalate crystals. In contrast to the chondroitin sulfate effect observed in vitro, which inhibits the growth of calcium oxalate crystals, our results suggest that in vivo chondroitin sulfate promotes the growth of stones in the urinary tract.

Xanthine urolithiasis: ultrastructure analysis of renal and bladder calculi.

The surface structure of a xanthine stone from a patient with hereditary xanthinuria was examined using scanning electron microscopy. This revealed that the xanthine stone was composed of at least two very different structures. One was a spherical protrusion with craters and a fine granular surface and the other consisted of a granular growth of plate-like crystals.

[An aged case of hereditary xanthinuria with xanthine urinary calculi].

A 74-year-old female with hereditary xanthinuria and xanthine stones is reported. She has a family history of consanguineous parents and a past history of right side nephrectomy due to a xanthine renal stone and vesicolithotomy of 3 bladder stones approximately 5 X 4 X 4 cm in size at the age of 58 and 71, respectively. Her young brother exhibited a slightly elevated urinary excretion of oxypurines. Laboratory examination showed a low serum level (0.3 mg/dl) and urinary excretion (1.56 mg/day) of uric acid, and high plasma and urine levels of oxypurines. No xanthine oxidase activity was detectably in duodenal mucosa by biopsy specimen obtained by duodenofiberscopy. Now she has another stone approximately 5 X 4 X 4 cm in her bladder. There have been are few elderly cases of hereditary xanthinuria with recurrent giant urolithiasis.

Relationships between some lithogenetic factors and vitamin B6-status in idiopathic calcium lithiasis.

Several researchers have shown that a reduced intake of vitamin B6 can induce increased oxalate urinary excretion leading to a higher incidence of calcium oxalate stones. Furthermore, the treatment with pyridoxine in patients with urinary stones and high oxalate excretion has led to contradictory results as the excretion of oxalate was either decreased, unchanged or increased. To verify if these divergent results were linked to a different B6 status of the patients undergoing the treatment, we studied the vitamin B6 and the main lithogenetic factor levels in patients with idiopathic calcium lithiasis as compared to normal subjects. The results showed that a high oxalate excretion is not necessarily coupled with a low vitamin B6 status and viceversa. However, some stone formers present a non homogeneous vitamin pattern that could be the consequence of an abnormal vitamin B6 metabolism.

Urinary constituents in an endemic area of stones and renal tubular acidosis in northeastern Thailand.

Nephrolithiasis and endemic renal distal tubular acidosis are common in northeastern Thailand. The etiology is still unknown. It is generally accepted that urine electrolytes influence the capacity of urine to inhibit or promote renal and also bladder stones. The purpose of this study was to analyse the composition of the urine in the indigenous population in the northeast area and compare their values with data obtained from a group of age matched adults, living in Bangkok. Twenty-four hour urine samples from 23 normal adult villagers from six villages within the province of Khon Kaen and 34 normal adults living in Bangkok were collected, and the daily excretion of creatinine, uric acid, calcium and inorganic phosphate, sodium, potassium, chloride, magnesium and oxalate were assayed. Daily urinary sodium, potassium, chloride and phosphate of the villagers were significantly lower than those of Bangkokians. No difference in the urinary excretion of calcium, magnesium, uric acid, oxalate and creatinine was found. The Na/Ca, and Ca/PO4 ratios of villagers were significantly lower than those of the Bangkok subjects. The villagers excreted significantly lower amounts of Na in the face of relatively higher urinary Ca. The above data, combined with our previous study showing the low values of urinary citrate in the villagers in the same areas, strongly indicate that the indigeneous population is at high risk in developing urolithiasis. The causes for these electrolyte abnormalities are still unknown. Low contents of the major electrolytes in their diets might play an important role. Low phosphate output indicates low protein diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Interdependence of urinary factors in calcareous bladder stone patients.

Twenty-four-hour urinary excretion of calcium, oxalic acid, inorganic phosphorus, magnesium and citric acid was examined in fifty-nine stone formers with bladder stones. Hypercalciuria and hyperoxaluria were present in 18.6% and 44.1%, respectively, while 11.9% of patients had both abnormalities. Hypomagnesuria and hypocitraturia were present in 67.8% and 69.5%, respectively, while 45.7% had both of these abnormalities. Normal urine chemistry in respect of parameters studied was observed only in 1.7% of cases. In 15.2% one risk factor was present, while 83.1% had two or more risk factors. "Path" analysis of the urinary parameters directly related to calcium lithiasis showed that magnesium and oxalic acid have substantial influence on calcium excretion, whereas citric acid had none. The influence of phosphorus did not provide any consistent trend.