Urethral proteomic analysis in ovariectomized mice administered 17ß-oestradiol replacement therapy.

The molecular mechanism by which 17ß-oestradiol (E2) increases urethral tone is unclear. As human tissue is limited in availability, we explored changes in the urethras of ovariectomized (OVX) mice. Twenty-four virgin female mice were randomised into three groups: mice with a sham operation only (control), OVX mice without E2 replacement (OVX), and OVX mice with E2 replacement (OVX + E2). Two weeks after the ovariectomy, mice received either E2 or placebo for 4 weeks. Leak point pressure (LPP) and maximum urethral closure pressure (MUCP) were assessed in these mice at 6 weeks after OVX, under anaesthesia. After measurements were recorded, the animals were sacrificed and the urethras were removed for proteomic and further analyses. LPP and MUCP values were significantly higher in OVX + E2 group than in OVX group. Fourteen differentially expressed proteins within the urethras of mice from OVX and OVX + E2 groups were identified; six proteins were upregulated and eight proteins were down-regulated. Most E2-induced proteins are involved in proteolysis, development, neurophysiological processes, transcription, and the cell cycle. Expression of survival motor neuron (SMN) protein in the urethra was significantly increased in OVX + E2 group compared to OVX group. OVX can impair urethral tone in female mice. E2 supplementation in OVX mice rescued urethral tone. E2-mediated increase in urethral tone in OVX mice involves overexpression of SMN, decreased proteolysis and promotion of development, neurophysiological processes, and transcription in the urethra. The urethra actively undergoes multiple biological processes in response to OVX and OVX with E2 stimuli. Impact statement Estrogens are known to modulate lower urinary tract trophicity. Although treatments with 17ß-oestradiol (E2) result in an increase in urethral tone, the mechanism by which E2 increases urethral tone is still not completely understood. Ovariectomy can impair urethral tone in female mice. E2 supplementation in ovariectomized mice rescued urethral tone. E2-mediated increase in urethral tone in ovariectomized mice involves overexpression of survival motor neuron, decreased proteolysis and promotion of development, neurophysiological processes, and transcription in the urethra. This information will offer clues about pathogenesis of stress urinary incontinence after menopause and will open additional avenues for novel research and potential therapies.

Emodin, a Natural Anthraquinone, Increases Uric Acid Excretion in Rats with Potassium Oxonate-Induced Hyperuricemia.

The treatment of hyperuricemia and gout is mostly based on lowering serum uric acid levels using drugs, such as allopurinol, or increasing urinary excretion of uric acid. However, some patients still experience adverse reactions to allopurinol and turn to Chinese medicine as an alternative. Therefore, it is crucial to design a preclinical study to obtain more convincing data on the treatment of hyperuricemia and gout with Chinese medicine. This study aimed to explore the therapeutic effect of emodin, a Chinese herbal extract, in a rat model of hyperuricemia and gout. In this study, we used 36 Sprague-Dawley rats, which were randomly divided into six groups for experimentation. Hyperuricemia was induced in rats by intraperitoneal injections of potassium oxonate. The efficacy of emodin in reducing serum uric acid levels was demonstrated by comparing the positive control group with groups treated with three different concentrations of emodin. The inflammatory profiles, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α levels, were unaffected by emodin treatment. In the experimental results, it was observed that the serum uric acid concentration in the vehicle control group was 1.80 ± 1.14, while the concentrations in the moderate and high concentration emodin groups were 1.18 ± 0.23 and 1.12 ± 0.57, resulting in no significant difference in uric acid concentration between these treatment groups and the control group, indicating that emodin has a therapeutic effect on hyperuricemia. The increase in the fractional excretion of uric acid (FEUA) demonstrated that emodin promoted urinary uric acid excretion without significantly affecting the inflammatory profile. Thus, emodin reduced the serum uric acid concentration to achieve effective treatment of hyperuricemia and gout by increasing urinary excretion. These results were supported by the measured serum uric acid and FEUA levels. Our data have potential implications for the treatment of gout and other types of hyperuricemia in clinical practice.

Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model.

Approximately 1 in 20 people develops kidney stones at some point in their life. Although the surgical removal of stones is common, the recurrence rate remains high and it is therefore important to prevent the occurrence of kidney stones. We chose Astragalus membranaceus (AM), which is a traditional Chinese medicine, to study the prevention of urolithiasis using a Drosophila model based on our previous screening of traditional Chinese herbs. Wild-type Drosophila melanogaster Canton-S adult fruit flies were used in this study. Ethylene glycol (EG, 0.5%) was added to food as a lithogenic agent. The positive control agent (2% potassium citrate (K-citrate)) was then compared with AM (2, 8, and 16 mg/mL). After 21 days, the fruit flies were sacrificed under carbon dioxide narcotization, and the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination to observe calcium oxalate (CaOx) crystallization. Then, the ex vivo dissolution of crystals in the Malpighian tubules was compared between K-citrate and AM. Survival analysis of the EG, K-citrate, and AM groups was also performed. Both 2% K-citrate and AM (16 mg/mL) significantly inhibited EG-induced CaOx crystal formation. Mean lifespan was significantly reduced by the administration of EG, and the results were significantly reversed in the AM (8 and 16 mg/mL) groups. However, AM extract did not directly dissolve CaOx crystals in Drosophila Malpighian tubules ex vivo. In conclusion, AM extract decreased the ratio of CaOx crystallization in the Malpighian tubules and significantly ameliorated EG-induced reduction of lifespan. AM prevented CaOx crystal formation in the Drosophila model.

Downregulation of tight junction protein zonula occludens-2 and urothelium damage in a cyclophosphamide-induced mouse model of cystitis.

OBJECTIVES: The cyclophosphamide (CYP)-induced model of cystitis in mice closely fits the symptoms of chronic bladder inflammation. Cystitis was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in protein expression and composition in the bladder of CYP-treated mice. MATERIALS AND METHODS: Administration of CYP induced cystitis-related symptoms in mice. Cystometry was assessed and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured. Voiding interval values (time between voids) were assessed in mice under anesthesia. The bladders were removed for proteomic analysis using label-free quantitative proteomics and liquid chromatography-mass spectrometry. Additionally, immunochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. RESULTS: Compared to the control group, the voiding interval values and urothelial thickness in the bladder in the CYP-treated group were significantly decreased. Additionally, we identified 105 differentially expressed proteins in the bladder of CYP-treated mice with proteomic analysis. These proteins were involved in cell-cell tight junctions, exocytosis, muscle development, contraction, and regulation, immune responses, proteolysis, and cell adhesion. IHC and Western blot confirmed the downregulation of the tight junction protein ZO-2 in the urothelium of bladder. CONCLUSIONS: Our results suggest that downregulation in tight junction protein ZO-2 and urothelium damage may have a role in cystitis-related OAB. These changes could be related to the molecular mechanism of cystitis-related OAB.

Toward a new insight of calcium oxalate stones in Drosophila by micro-computerized tomography.

We previously developed an animal model of calcium oxalate (CaOx) deposition on the Malphigian tubules of Drosophila melanogaster as a model of urolithiasis. Here, we introduce a new tool for the study of anatomical structure for Drosophila. As a consequence of technical development, the invention of micro-computerized tomography (CT) has been introduced to the small animal, such as rat and mice. We used Drosophila as a model organism and fed the flies 0.5% lithogenic agent ethylene glycol for 3 weeks. Samples were simply prepared for further scanned by micro-CT to scan samples at 800 nm resolution. CT scanning was performed at 40 kVp of voltage, 250 µA of current, and 1750 ms of exposure time and without filter. Reconstruction of sections was carried out with the GPU-based scanner software. Specific region of interests was further analyzed by DataViewer software. Area with high radiologic density level was defined as CaOx deposition for further 3D analysis. Image of whole lithogenic Drosophila was compared with control. High radiologic density level was detected in the region of Malphigian tubules which can be identified as CaOx stones. There was no stone image in the control group. The image was the same as human non-contrast CT for the diagnosis of stone disease. Micro-CT clearly demonstrated the calcium oxalate calcifications in the Malphigian tubules of fruit fly. The image system provides that a new vision on study animal will facilitate further study of stone disease. With the development of new technology on micro-CT, more delicate and advanced image will be presented in the future.

A promising protein responsible for overactive bladder in ovariectomized mice.

OBJECTIVE: Ovariectomy (OVX) in mice is a model mimicking a neuro-electronic proof of an overactive bladder in postmenopausal women. Overactive bladder (OAB) was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in cell junction protein expression and composition in the bladder of OVX mice. MATERIALS AND METHODS: Thirty-six virgin female mice were randomized into three groups: mice with a sham operation only (control), OVX mice without estradiol (E2) replacement, and OVX mice with E2 replacement (OVX + E2). Cystometry assessment was conducted and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured after 8 weeks. Voiding interval values (time between voids) were assessed in mice under anesthesia. After measurements, the bladders were removed for proteomic analysis using the label-free quantitative proteomics and liquid chromatography-mass spectrometry technology. Lastly, immunohistochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. RESULTS: We identified 73 differentially expressed proteins in the bladder of OVX mice. The OVX mice showed significantly lower voiding interval values. Voiding interval values were significantly higher in the OVX + E2 group than in the OVX group. Urothelial thicknesses in the bladder were also significantly lower in the OVX group than in the control group. E2 replacement reversed the urothelium layers. Additionally, the expression of ZO-2, a tight junction protein, was the most affected by OVX treatment. IHC and Western blot confirmed the downregulation of ZO-2 in the bladder of OVX mice. Expression of ZO-2 protein was significantly increased in OVX + E2 group compared with OVX group. CONCLUSION: This exploratory study estimated changes in protein expression and composition in the bladder of OVX mice. These changes may be associated with the molecular mechanisms of OAB.