Diet-Induced Metabolic Syndrome Altered Bladder Urothelium in Adult Female Rats †

: In recent years, the prevalence of chronic non-communicable diseases has increased. In females, there is a close relationship in the development of these diseases after menopause to estrogenic signaling occurring in various tissues; such is the case for the bladder, compromising its physiology in females. We sought to analyze the effect of diet-induced metabolic syndrome on the bladder epithelium. Eighteen 12-week-old Wistar rats were divided into an intact control group (C, n = 6), a cafeteria diet SMet group (CAF, n = 6), and a high-fat/high-sugar diet SMet group (HF/HS, n = 6). Atrophy and hyperplasia in the bladder epithelium were observed in the case of the CAF diet, while only inﬂammation was observed in the other schemes.


Introduction
In recent years, there has been a constant growth in the prevalence of chronic noncommunicable diseases, which even appear increasingly at an earlier age [1].However, there is a close relationship in the development of these diseases in females, and the role of estrogen fluctuation in reproductive stages such as gestation and menopause has been widely discussed [2,3], compromising its physiology in females.Urinary incontinence (UI) is a common experience throughout a woman's life and has a significant impact on well-being and quality of life [4].The prevalence of urinary incontinence worldwide is reported to be 8.5%; it is the most common urinary tract disease affecting adult women.The main risk factors associated with stress UI include age, pregnancy and parity, a history of hysterectomy, obesity, and pelvic radiation [5].Most related studies focus on analyzing the morphophysiology of the pelvic floor and bladder musculature, neglecting effects on the urothelium [6,7].Metabolic syndrome (MetS) is a set of disorders characterized by low-grade inflammation that alters various systems and is associated with type 2 diabetes mellitus and cardiovascular disease [8].Diet plays a crucial role in the development of MetS, with the combination of high caloric intake, poor nutrient quality, and unhealthy food choices contributing to its negative impact on overall health [9].Considering that women are more affected by bladder diseases such as UI, it is of relevance to analyze the effect of metabolic syndrome (MS) models of diet, using a cafeteria diet (CAF) or high-fat/high-sugar diet (HF/HS), on the bladder urothelia of female rats.

Materials and Methods
Eighteen 12-week-old Wistar rats were divided into an intact control group (C, n = 6), a cafeteria diet SMet group (CAF, n = 6), and a high-fat/high-sugar diet SMet group (HF/HS, n = 6).The rats were housed with controlled temperature and artificial illumination (20 ± 2 C; light/dark from 7 AM to 9 PM).This light condition was used.All animal procedures followed the Guidelines of Mexican Law of Production, Care, and Use of Laboratory Animals (NOM-062-ZOO-1999) and were approved by the research committee of the division de Ciencias de la Salud from Universidad Cristóbal Colón (registration code: COVID-100).
The control group had access to water and feed (23% protein, 50% carbohydrates, and 27% lipids) ad libitum; the cafeteria group had a designed diet (approximately 11% protein, 60% carbohydrates, and 29% lipids) in which ultra-processed feeds were used; the high-fat/high-sugar diet (HF/HS) was designed, and pellets were prepared with a composition of 18% protein, 55% carbohydrates, and 27% lipids.In the case of the CAF group, the animals had access to chow and water along with the diet, which consisted of bread rolls, French fries, soft drinks, and sausages, among others.The duration of the treatment was 10 weeks.All rats used in the experimental procedures were euthanized with an overdose of sodium pentobarbital (60 mg/kg).Blood was obtained via cardiac puncture for biochemical measures.
The bladders were fixed in formaline, histologically processed, and then embedded in Paraplast X-tra (Sigma-Aldrich).Each bladder was transversally cut at a thickness of 5 µ using a microtome (Thermo Scientific, Model 325).Tissue sections were mounted on gelatin-coated slides.Each tissue section was stained with Masson's trichrome and PAS, and photographs were taken at 10×, 40×, and 100×.Data were analyzed statistically, and differences were considered when p < 0.05, using graph Pad v.6 statistical packages.

Results
The cafeteria diet was effective at generating metabolic syndrome, with the presence of hyperglycemia, elevated cholesterol and triglycerides, as well as higher body weight gain (Table 1), while the HF/HS diet generated increased body weight and hypercholesterolemia.With respect to the bladder epithelium in the case of the HF/HS diet, it generated a detachment of umbrella cells and areas of desquamation, and it can be observed that there are foci of inflammation.In the case of the CAF diet, it generated an important disarrangement of the epithelium, with many pyknotic nuclei and changes in basal cells, as well as blank spaces that do not appear in the other groups (Figure 1).

Discussion
Several studies have shown that different diet patterns can emulate the clinical manifestations of MetS.In the case of high-fat/high-sugar (HF/HS) diets, we find similarities to what was shown previously in which the diet appears to be less effective in females [10].In the case of the cafeteria diet, it was shown to be an excellent model with the classical manifestations of MetS: hyperglycemia, dyslipidemia, and higher adiposity.The treatment time was adjusted to 10 weeks, derived from the fact that as indicated above, pathogenesis in females is slower due to the protective effect of estrogens [8].The manifestations observed in the bladder coincide with problems such as cystitis [9], in which there is desquamation and a loss of umbrella cells.In the case of the CAF diet, in addition, disarray and many pyknotic nuclei were found, suggesting an increase in cell death.While histology alone does not indicate urinary incontinence, other studies show that the cafeteria diet leads to changes in the electrophysiological profile [11,12].The results show that the cafeteria diet is a model that could be more useful for analyzing metabolic syndrome in females than other diet-generated models.Further studies are required to analyze the relationship of bladder alterations in females.

Discussion
Several studies have shown that different diet patterns can emulate the clinical manifestations of MetS.In the case of high-fat/high-sugar (HF/HS) diets, we find similarities to what was shown previously in which the diet appears to be less effective in females [10].In the case of the cafeteria diet, it was shown to be an excellent model with the classical manifestations of MetS: hyperglycemia, dyslipidemia, and higher adiposity.The treatment time was adjusted to 10 weeks, derived from the fact that as indicated above, pathogenesis in females is slower due to the protective effect of estrogens [8].The manifestations observed in the bladder coincide with problems such as cystitis [9], in which there is desquamation and a loss of umbrella cells.In the case of the CAF diet, in addition, disarray and many pyknotic nuclei were found, suggesting an increase in cell death.While histology alone does not indicate urinary incontinence, other studies show that the cafeteria diet leads to changes in the electrophysiological profile [11,12].The results show that the cafeteria diet is a model that could be more useful for analyzing metabolic syndrome in females than other diet-generated models.Further studies are required to analyze the relationship of bladder alterations in females.

Table 1 .
Parameters measured to identify metabolic syndrome.

HF/HS n = 6 CAF n = 6
bData are means ± SEMs.Different letters indicate significant differences between groups.