Vitamin A‐Enriched Diet Increases Urothelial Cell Proliferation by Upregulating Itga3 and Areg After Cyclophosphamide‐Induced Injury in Mice

ABSTRACT

Vitamin A (VitA) is an essential nutrient, affecting many cell functions, such as proliferation, apoptosis, and differentiation, all of which are important for the regeneration of various tissues. In this study, we investigated the effects of a VitA‐enriched diet on the regeneration of the urothelium of the urinary bladder in mice after cyclophosphamide (CP)‐induced injury. Female mice were fed VitA‐enriched and normal diet for 1 week before receiving an intraperitoneal injection of CP (150 mg/kg). Urinary bladders were removed 1 and 3 days after CP. On Day 1, RNA sequencing showed that VitA upregulated two Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways: the cell cycle and the PI3K‐Akt pathway. This was confirmed by qPCR, which showed significantly increased expression of the Itga3 and Areg genes. In addition, the effect of VitA on the proliferation of urothelial cells was analyzed by immunohistochemistry of Ki‐67, which confirmed an increased proliferation rate. No significant effects of the VitA‐enriched diet were observed on the expression of apoptosis‐related genes and on differentiation‐related markers of superficial urothelial cells. Our results suggest that a VitA‐enriched diet improves early urothelial regeneration after CP‐induced injury by promoting cell proliferation.

We investigated the effects of vitamin A (VitA)‐enriched diet on regeneration of urothelium after intraperitoneal injection (i.p.) of cyclophosphamide (CP). Control mice received i.p. of saline (S). Our results showed that VitA‐enriched diet on the first day after CP‐induced injury significantly increased the proliferation of urothelial cells, leading to improved regeneration. Proliferation was enhanced by upregulation of genes Itga3 and Areg.

1. Introduction

Vitamin A (VitA), synonymous with all‐trans retinol, comprises a group of derivatives known as retinoids—collectively classified as isoprenoids, that have similar properties to all‐trans retinol [1, 2]. These retinoids are essential nutrients that humans must obtain from the diet, as endogenous synthesis is not possible [2, 3, 4]. Although retinol itself is not biologically active, its metabolite, retinoic acid, is the form that can modulate the activity of transcription factors [5, 6]. VitA plays a crucial role in embryonic development, vision, immunity, reproduction, cell differentiation and proliferation, apoptosis, and the integrity of epithelial and immune cells [1, 2, 7, 8, 9, 10]. It also has antiinflammatory properties [11, 12, 13] and may have antioxidant properties [14, 15].

Retinoids are widely used in clinical practice, particularly in oncology (e.g., for the treatment of acute promyelocytic leukemia in adults and neuroblastoma in children) and in dermatology (for the treatment of psoriasis) [2, 7]. In psoriasis, VitA is an important part of the treatment as it can influence the regeneration process by reducing the proliferation rate of keratinocytes and promoting their differentiation [16, 17]. Similar effects of VitA have also been observed in the urothelium, the epithelium that covers the urinary bladder and maintains the blood–urine permeability barrier. Previous studies have established a link between the mechanisms of urothelial regeneration and retinoic acid signaling [18, 19, 20]. In particular, retinoic acid can influence the Bmp, Shh, and Wnt signaling pathways, which are involved in urothelial regeneration [18].

Furthermore, VitA has been shown to modulate the expression of uroplakins (UPs) and cytokeratin 20 (CK20), which are markers of highly differentiated urothelial cells [21, 22, 23, 24], in murine embryonic stem cells via the GATA4/6 signaling mechanism [19]. Since VitA regulates the adult stationary urothelium [25] and studies show that retinoic acid can induce embryonic stem cells to differentiate into urothelial cells [19], we sought to investigate whether the VitA diet facilitates urothelial regeneration.

Urothelial regeneration involves three key processes: proliferation, apoptosis, and differentiation. The urothelium has a rapid regenerative capacity after injury caused by cyclophosphamide (CP), although the timeline varies across species [26, 27, 28]. The intense proliferation of urothelial cells after injury is crucial for the formation of blood–urine barrier and is usually followed by differentiation in the early stages of urothelial regeneration (Days 1–3 after CP injection) [26, 29, 30]. Conversely, apoptosis is more important in the later stages of regeneration [31, 32, 33]. Although the role of VitA in urothelial regeneration has been recognized, its exact effect on each stage of urothelial regeneration is not fully understood. Therefore, the present study aimed to establish a link between VitA‐enriched diet and early urothelial regeneration after injury, focusing on cell proliferation, apoptosis, and differentiation. In addition, our study provides an RNA‐seq reference database and the basis for future studies investigating the relationship between VitA and urothelial regeneration.

2. Methods

2.1. Animals and Treatment

A total of 72 adult female BALB/c mice (8 weeks old) weighing between 16.0 and 22.8 g were used for the experiment. The animals were housed in groups of three to five animals in polyacrylamide cages at constant humidity (55%) and temperature (22°C) with water and food ad libitum and 12 h/12 h light–dark cycle. All animals were allowed an acclimatization period of 7 days. The animals were purchased from Charles River Laboratories Italia (Calco, Italy). The experiment was conducted in three time‐independent experiments. The animals were randomly divided into eight groups. Four groups were treated with CP (n = 10 for each group). The other four groups were treated with sterile saline (S; n = 8 for each group) and served as control groups. The experimental design is shown in Figure 1. CP (Sigma–Aldrich, Merck, Darmstadt, Germany, Cat. No. C0768) was diluted in sterile saline and injected once intraperitoneally (i.p.) at a concentration of 150 mg CP/kg body weight. For the control groups, sterile saline was i.p. injected in the same volume as for the CP injections. One week before the CP or saline injections, half of the mice had their normal diet (N) replaced with a VitA‐enriched diet (VitA). A VitA‐enriched diet contained 566081UI retinyl‐acetate per kilogram, 36 times more VitA than in a normal diet [3, 34]. After the change of diet, mice consumed the same diet (normal or VitA‐enriched) until the end of the experiment. The diets were weighed and changed daily (Figure S1). The animals were weighed every 3 days and examined daily for signs of pain or discomfort. Animals were euthanized by CO₂‐asphyxia, which was followed by urinary bladder dissection on Day 1 (N S1d, N CP1d, VitA CP1d, and VitA S1d) or Day 3 (N S3d, N CP3d, VitA CP3d, and VitA S3d) after injection of CP or saline (Figure 1). The obtained urinary bladders were dissected and processed for RNA isolation, paraffin embedding, and scanning electron microscopy (SEM). All animal experiments were performed in accordance with the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector, and Plant Protection, permit number U34401‐1/2022/18. Experiments were conducted according to ARRIVE guidelines [35].

FIGURE 1.

Schematic representation of the animal experiment. Groups of animals are labeled as VitA S1d (1 day after i.p. injection of S with VitA‐enriched diet), VitA S3d (3 days after i.p. injection of S with VitA‐enriched diet), VitA CP1d (1 day after i.p. injection of CP with VitA‐enriched diet), VitA CP3d (3 days after i.p. injection of CP with VitA‐enriched diet), N S1d (1 day after i.p. injection of S with normal diet), N S3d (3 days after i.p. injection of S with normal diet), N CP1d (1 day after i.p. injection of CP with normal diet), N CP3d (3 days after i.p. injection of CP with normal diet). CP, cyclophosphamide; i.p., intraperitoneally; N, normal diet; S, saline; VitA, vitamin A.

2.2. Dosage Information

A VitA‐enriched diet consumed by mice contained 566081UI retinyl‐acetate per kilogram (194 µg/g diet), 36 times more VitA than in a normal mouse diet [3, 34]. The current recommended daily intake of VitA for adult humans is 750 and 650 µg/day for men and women, respectively [36].

2.3. Total RNA Isolation

After dissection, urinary bladder tissue from 72 mice was quickly frozen in liquid nitrogen and stored at −80°C until RNA isolation. Frozen samples were homogenized using a tissue pulverizer (Products 59012N, Cole‐Parmer, BioSpec) cooled with liquid nitrogen. Then, RNA was isolated from the samples using TRI Reagent (RNA/DNA/Protein Isolation Reagent, Research Centre, MSPR‐TR118, VWR, Vienna, Austria) and PeqGOLD Total RNA Kit (Cat. No. 13‐6834‐01P, VWR, Vienna, Austria) according to the manufacturer's instructions. The purity and quantity of the isolated RNA were determined by reading the absorbance at 230, 260, and 280 nm using Cytation 1 (Agilent, Santa Clara, CA, USA).

2.4. RNAseq and Gene Enrichment Analysis

Isolated RNA from three independent experiments, in total 18 samples (groups VitA S1d, N S1d, VitA CP1d, N CP1d, VitA CP3d, and N CP3d; n = 3 per group) were sent to Novogene (Sacramento, CA, USA), where RNAseq was performed. The RNAseq and data analysis were similar to those described in Peskar et al. [37]. Before library preparation, RNA integrity was assessed using the RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). As input material for the RNA sample preparation, a total amount of 0.5 µg RNA per sample was used. Quantified libraries were sequenced to a depth of 26 million reads/sample on an Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA), generating 150 bp paired‐end fastq files [37]. Raw and processed data are available in the NCBI Gene Expression Omnibus database with the accession number GSE282487.

Raw data in fastq format was processed for quality control using Novogene Co., Ltd. in‐house perl scripts (Novogene, Sacramento, CA, USA). High‐quality reads were mapped to the mouse genome (Ensembl GRCm39) using Hisat2 (v2.0.5). The mapped reads to genes were quantified by FeatureCounts (v1.5.0‐p3) and expressed as Fragments Per Kilobase of transcript sequence per Million base pairs (FPKM). Differential expression analysis using the DESeq2 R package (1.20.0) was performed on all groups. The p values for differentially expressed genes (DEGs) were obtained using the negative binomial distribution, followed by Benjamini–Hochberg's procedure for adjustment. The adjusted p values < 0.05 were considered statistically significant. Enrichment analysis of DEGs was conducted using clusterProfiler (v.3.8.1). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed [37]. Pathways with adjusted p values of <0.05 were considered significantly enriched.

2.5. Reverse Transcription and qPCR

As an additional validation method for RNAseq‐obtained results, we performed qPCR on the same samples we used for RNAseq and added five to eight samples per group (n = 8 samples per S groups, n = 10 samples per CP groups; n = 72). According to the manufacturer's instructions, reverse transcription of total RNA (1 µg of total RNA) to cDNA was conducted with FastGene Scriptase II cDNA 5× ReadyMix (Cat. No. LS64, Nippon Genetics Europe, Germany). The qPCR analysis was performed using 5× HOT FIREPol EvaGreen qPCR Mix Plus (Cat. No. 08‐24‐0000S, Solis Biodyne, Tartu, Estonia) and self‐designed primers (Integrated DNA Technologies, Coralville, IA, USA) on the QuantStudio5 (ThermoFisher Scientific, Waltham, Massachusetts, USA). Samples were analyzed in triplicates in one run of 40 cycles, composed of denaturation (15 s at 95°C), annealing (30 s at 60°C), and elongation (30 s at 72°C). The melting curves generated at the end of the run resulted in a single peak. Data were analyzed with a comparative Ct method relative to endogenous control (L32) expression and presented as log2^−ΔΔCt. First ΔCt was calculated as the difference between the average Ct of the gene of interest and the average Ct of endogenous control, and then the ΔΔCt was calculated as the difference between the ΔCt of the gene of interest and the average ΔCt of the control group (N S1d). The primer sequences are listed in Table S1.

2.6. Immunohistochemistry

For immunohistochemistry (IHC), paraffin‐embedded tissue (parts of the urinary bladders of 72 mice; n = 8 samples per S groups, n = 10 samples per CP groups) was used and cut into 6 µm thick sections. IHC was used to label the proteins Ki‐67, CK20, and UPs. The tissue sections were first deparaffinized in xylene and hydrated in decreasing ethanol concentrations. Antigen retrieval was performed by microwave heating in Tris‐EDTA buffer (pH 9.00), and the endogenous peroxidase was inactivated with 3% H₂O₂ in methanol. Nonspecific labeling was blocked with 5% normal goat serum (X0907, Aglient Dako, Santa Clara, CA, USA) in 1% bovine serum albumin (BSA) diluted in phosphate‐buffered solution (PBS) at room temperature for 2 h. The sections were then incubated overnight at 4°C with primary listed in Table S2. For the negative controls, the primary antibodies were replaced with 1% BSA/PBS. The next day, the sections were incubated with secondary antibodies (Table S2) diluted in 1% BSA in PBS for 2 h at room temperature. The sections were washed with PBS and incubated with the ABC Peroxidase Staining Kit (Cat. No.32020, Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. After washing in PBS, a standard DAB development procedure with a DAB Substrate Kit (Cat. No. 34002, Thermo Scientific, Waltham, Massachusetts, USA) was performed. Finally, the sections were counterstained with hematoxylin and examined using an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan). Images were analyzed by ImageJ version 1.53t [38] by calculating the proliferation rate (counting ki‐67 positive and negative cells in urothelium) and measuring the length of UPs and CK20 positive luminal surface of urothelial cells.

After hematoxylin–eosin staining of paraffin sections, we measured the area of edema in the lamina propria of the urinary bladder wall. Results are shown as a percentage of the edema area versus the entire urinary bladder wall area.

2.7. Scanning Electron Microscopy

Urinary bladder samples (n = 56) (n = 6 mice per S groups and n = 8 mice per CP groups) were fixed in 4% formaldehyde and 2% glutaraldehyde for 2 h and 45 min at room temperature. After washing in 0.1 M cacodylate buffer overnight at 4°C, samples were post‐fixed in osmium tetroxide for 1 h at room temperature in the dark. After rapid washing in distilled water, the samples were dehydrated in increasing concentrations of acetone. Drying was then performed at the critical point with liquid CO₂. The dry samples were attached to specific holders and underwent gold sputter‐coating. The samples were analyzed using a Tescan Vega3 scanning electron microscope (Tescan, Brno, Czech Republic). Images were analyzed by ImageJ version 1.53t [38]. We measured the area of individual structures on the luminal site of the urothelium, visible under SEM (microvilli, ropy ridges, microridges). We calculated the percentage of each regarding the complete measured area.

2.8. Statistical Analysis

Statistical analysis of the data was conducted using GraphPad Prism version 8.01 (Dotmatics, Boston, MA, USA). The normality of data distributions was investigated by the Shapiro–Wilk test. Summary statistics are expressed as mean and standard deviation (SD). Statistical differences between groups were calculated using Ordinary one‐way analysis of variance (ANOVA) or the Kruskal–Wallis test, depending on the normality of the data distribution. All tests were two‐tailed and a p value <0.05 was considered statistically significant.

3. Results

3.1. Vitamin A‐Enriched Diet Modifies the Transcriptome of the Mouse Urinary Bladder One Day After CP Injection

We first conducted complete transcriptome profiling of mouse urinary bladders using RNAseq to evaluate the overall effect of a VitA‐enriched diet on the early stages of urothelial regeneration, that is, 1 day after CP‐induced injury. To assess the inflammatory response in the bladder wall after CP injection‐induced urothelial injury, we compare the expression of TNF and IL17 signaling pathway genes in the N CP1d and N S1d groups. The heatmap and PCA showed that the expression of the analyzed genes changed 1 day after CP injection compared to S injection (Figure S2). In addition, paraffin sections were stained with hematoxylin–eosin, and the edema area was measured. This showed that edema increased after CP injection regardless of diet (Figure S3).

Differential expression analysis comparing the VitA CP1d and N CP1d groups revealed that 1303 genes were upregulated, and 916 genes were downregulated in the VitA CP1d group (|log2FC| ≥ 0; p ≤ 0.05) (Figure 2a). To further understand the function of DEGs and their involvement in biological pathways, we performed a KEGG enrichment analysis. A total of 63 significantly enriched KEGG pathways were identified, with most of them (n = 42) being upregulated. Next, we focused only on the upregulated KEGG signaling pathways, specifically on the cell cycle and PI3K‐Akt signaling pathways, which were at the top of the list and are associated with cell proliferation, a potential target of VitA activity [2, 39] (Figure 2b).

FIGURE 2.

The effects of VitA‐enriched diet on the transcriptional profile of mouse urinary bladder 1 day after CP injection (comparison between VitA CP1d and N CP1d groups of mice). (a) Volcano plot shows 916 downregulated (green dots) and 1303 upregulated (red dots) genes, while the expression of 34 363 (gray dots) is not significantly altered (log2FC ≥ 0; p ≤ 0.05). (b) Bubble plot of 20 most significantly upregulated KEGG enrichment signaling pathways in VitA CP1d group in comparison to N CP1d group. The top two upregulated KEGG pathways are highlighted, because of their connection to proliferation, which is a potential target of VitA activity. n = 3 mice (per group). CP, cyclophosphamide; KEGG, Kyoto Encyclopedia of Genes and Genomes; N, normal diet; VitA, vitamin A.

We also acknowledge that a VitA‐enriched diet alone influences gene expression. Differential gene expression analysis comparing the VitA S1d group and N S1d group showed that there were 1009 upregulated genes and 1054 downregulated genes in the VitA S1d group (|log2FC| ≥ 0; p ≤ 0.05) (Figure S4a).

The same analysis was also performed 3 days after CP injection comparing the VitA CP3d group to the N CP3d group (Figure S4b). The number of DEGs on Day 3 was substantially lower than those on Day 1, indicating that the effects of a VitA‐enriched diet are most pronounced on Day 1 and diminish on Day 3 after CP‐induced urothelial injury.

3.2. Vitamin A‐enriched Diet Modulates the Expression of Cell Cycle Genes One Day After CP‐Induced Injury

To visualize the expression of genes within the KEGG cell cycle pathway, we generated a volcano plot using the RNAseq data from this pathway to compare the VitA CP1 and N CP1d groups (Figure 3a). Subsequently, the expression of six significantly upregulated genes detected with RNAseq (Cdk1, Cdc25b, Ccna2, Bub1, Bub1b, and Knl1) was evaluated by qPCR in a larger sample size (n = 10 for the CP‐treated groups and n = 8 for the S‐treated groups). The analysis revealed that Cdk1, Ccna2, Bub1, and Bub1b genes were significantly upregulated in the CP‐treated groups compared to the S‐treated groups, regardless of the diet (Figure 3b), which can be attributed to the effects of CP. We also observed a slightly higher mean expression of Cdc25b and Knl1 in the VitA CP1d group compared to the N CP1d group, but this was not significant (Figure 3b), presumably due to the high biological variability within the groups. Similar results were also obtained on Day 3 after CP or S injection (Figure S5). These results suggest that increased VitA intake had no significant effect on the expression of genes involved in the cell cycle pathway in the early stage of urothelial regeneration. Nevertheless, the expression of the genes Cdk1, Ccna2, Bub1, and Bub1b was increased due to CP treatment (1 day after CP‐induced injury).

FIGURE 3.

Expression profile of the genes involved in the cell cycle pathway in the mouse urinary bladder 1 day after CP or S injection. (a) Volcano plot showing expression of genes involved in cell cycle pathway in the VitA CP1d and N CP1d groups, determined by RNAseq. Significantly upregulated genes in the VitA CP1d group are pointed out. (b) Expression of Cdk1, Cdc25b, Ccna2, Bub1, Bub1b, and Knl1 in the mouse urinary bladders 1 day after CP or S injection, determined by qPCR. Shown is mean ± SD for each group; n = 10 mice per group (CP‐injected groups) and n = 8 mice per group (S‐injected groups); p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Statistical analysis of Cdc25b, Bub1, Bub1b, and Ccna2 data was conducted using Ordinary one‐way ANOVA with Tukey's post‐test, and analysis of Cdk1 and Knl1 data was conducted using Kruskal–Wallis test with Dunn's post‐test. N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, and VitA S1d–1 day after S‐injection with VitA‐enriched diet. ANOVA, analysis of variance; CP, cyclophosphamide; N, normal diet; S, saline; VitA, vitamin A.

3.3. Genes Itga3 and Areg, Involved in the PI3K‐Akt Signaling Pathway, Are Upregulated One Day After CP‐Induced Injury Due to a Vitamin A‐Enriched Diet

The second KEGG‐enriched pathway that we investigated was the PI3K‐Akt signaling pathway, which is also involved in cell proliferation. We generated a volcano plot of genes involved in this pathway based on RNAseq data comparing the VitA CP1d and N CP1d groups (Figure 4a) and selected significantly upregulated genes for analysis by qPCR on a larger sample (n = 10 for CP‐treated groups and n = 8 for S‐treated groups). We focused on the genes Itga3, Areg, Epha2, Spp1, and Ereg (Figure 4b, Figure S6), while we excluded Il6 due to its low expression in the RNAseq data (the count for this gene was below 100 in all groups). We confirmed that the VitA‐enriched diet caused a statistically significant upregulation of Itga3 and Areg compared with a normal diet 1 day after CP injection (Figure 4b). A trend toward increased expression of the Epha2, Spp1, and Ereg genes was also observed in the VitA CP1d group compared to the N CP1d group, but there was no statistical significance (Figure 4b). Our results showed that Itga3 gene expression was also significantly higher in the VitA S1d group compared to the N S1d group (Figure 4b). This suggests that increased VitA intake affects Itga3 gene expression in the urinary bladder, independent of CP‐induced injury. On Day 3 after CP injection, no significant effect of a VitA‐enriched diet was observed on the expression of these genes (Figure S6), showing that the effects of VitA on the PI3K‐Akt signaling pathway are more pronounced at early stages of urothelial regeneration (1 day after CP‐induced injury).

FIGURE 4.

Expression profile of the genes involved in the cell cycle pathway in the mouse urinary bladder 1 day after CP or S injection. (a) Volcano plot showing expression of genes involved in PI3k‐Akt signaling pathway in the VitA CP1d and N CP1d groups, determined by RNAseq. Significantly upregulated genes in the VitA CP1d group are pointed out. (b) Expression of Itga3, Areg, Ereg, Spp1, and Epha2 in the mouse urinary bladders 1 day after CP or S injection, determined by qPCR. Shown is mean ± SD for each group; n = 10 mice per group (CP‐injected groups) and n = 8 mice per group (S‐injected groups); p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Statistical analysis of Itga3, Spp1, Ereg, and Areg data was conducted using Ordinary one‐way ANOVA with Tukey's post‐test, and analysis of Epha2 data was conducted using the Kruskal–Wallis test with Dunn's post‐test. N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, and VitA S1d–1 day after S‐injection with VitA‐enriched diet. ANOVA, analysis of variance; CP, cyclophosphamide; N, normal diet; S, saline; VitA, vitamin A.

3.4. Vitamin A‐Enriched Diet Increases the Proliferation of Urothelial Cells One Day After CP‐Induced Injury

The RNAseq and qPCR data have shown that a VitA‐enriched diet alters the expression of genes involved in cell proliferation in the early stages of urothelial regeneration 1 day after CP‐induced injury. To investigate this process in more detail, we determined the expression of the gene Mki67 with qPCR and its corresponding protein, Ki‐67, a commonly used proliferation marker, with IHC (Figure 5). The expression of Mki67 was upregulated in all CP‐treated groups compared to S‐treated groups on Days 1 and 3 after injection; however, no influence of the diet was observed on RNA level (Figure 5a, b). IHC showed that the proliferation rate of urothelial cells significantly increased 1 day after CP injection in mice receiving a VitA‐enriched diet (VitA CP1d group; 34.91% ± 13.44%) compared with mice receiving a normal diet (N CP1d group; 18.40% ± 8.06%; p < 0.001) (Figure 5c). On the third day after CP administration, there was no significant difference in proliferation rates between the VitA CP3d (42.48% ± 10.61%) and N CP3d (28.65% ± 10.45%) groups (Figure 5d). These results show that urothelial cell proliferation is increased on Days 1 and 3 after CP injection. Moreover, a VitA‐enriched diet leads to an even higher proliferation rate of urothelial cells on the first day after injury, but this effect is no longer observed on the third day.

FIGURE 5.

Proliferation rate of urothelial cells 1 and 3 days after CP and S injection. Gene expression of Mki67 (a) 1 day after injections and (b) 3 days after injections. Proliferation rate (c) 1 day and (d) 3 days after injections, determined as a percentage of Ki67 positive cells. Shown is mean ± SD for each group. n = 10 mice per group (CP‐injected groups) and n = 8 mice per group (S‐injected groups). p < 0,05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Statistical analysis of data on graphs (a) and (c) was conducted using Ordinary one‐way ANOVA with Tukey's post‐test and analysis of data on graphs (b) and (d) was conducted using Kruskal–Wallis test with Dunn's post‐test. (e) Representative images of Ki67, determined by IHC (L–lumen, U–urothelium, LP–lamina propria, black arrow–positive nuclei of urothelial cells). N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, VitA S1d–1 day after S‐injection with VitA‐enriched diet, N S3d–3 days after S‐injection with a normal diet, N CP3d–3 days after CP‐injection with a normal diet, VitA CP3d–3 days after CP injection with VitA‐enriched diet, VitA S3d–3 days after S injection with VitA‐enriched diet. ANOVA, analysis of variance; CP, cyclophosphamide; IHC, immunohistochemistry; N, normal diet; S, saline; VitA, vitamin A.

3.5. Vitamin A‐Enriched Diet Inhibits Apoptotic Responses One and Three Days After CP‐Induced Injury

Since proliferation during urothelial regeneration is in a certain balance with apoptosis [31], we next retrieved the genes involved in the apoptosis KEGG pathway and checked whether their expression was significantly altered in the VitA CP1d versus N CP1d groups using RNAseq data 1 day after CP injection. This analysis demonstrated that the expression of Ptges, Ccn1, and Angptl4 was significantly upregulated, while the expression of Ttc36 was significantly downregulated in the VitA CP1d group (Figure 6a). A subsequent qPCR analysis on a larger sample size shows a trend toward higher mean expression levels of Ccn1, Ptges, and Angptl4 in the VitA CP1d group. The Ttc36 gene was excluded from the qPCR analysis due to its low expression (the count was below 100 in all samples). No significant differences in the expression of these genes were observed on Day 3 after CP injection between these two groups (Figure S7). On the other hand, we observed significantly decreased expression of the Ccn1 gene in the VitA S3d group in comparison to the N S3d group. This shows that increased VitA intake over a longer period could lower the apoptosis rate in the normal urothelium. Nevertheless, Angptl4 gene expression was significantly increased in the CP groups at 1 and 3 days compared to the S groups, regardless of diet type (Figure 6b). The VitA‐enriched diet appears to increase the expression of several genes involved in inhibition of apoptosis on the first day after CP injection, while it has no significant effect on apoptosis‐related gene expression in normal urothelium.

FIGURE 6.

Expression profile of genes involved in the KEGG apoptosis signaling pathway in the mouse urinary bladder 1 day after CP injection. (a) Volcano plot comparing gene expression between VitA CP1d and N CP1d groups, determined by RNAseq. Significantly deregulated genes are pointed out. (b) Gene expression of Ptges, Ccn1, and Angptl4, determined by qPCR. Shown is mean ± SD for each group; n = 10 mice per group (CP‐injected groups) and n = 8 mice per group (S‐injected groups); p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Statistical analysis of Ptges and Angptl4 data was conducted using Ordinary one‐way ANOVA with Tukey's post‐test, and analysis of Ccn1 data was conducted using Kruskal–Wallis test with Dunn's post‐test. N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, VitA S1d–1 day after S‐injection with VitA‐enriched diet. ANOVA, analysis of variance; CP, cyclophosphamide; KEGG, Kyoto Encyclopedia of Genes and Genomes; N, normal diet; S, saline; VitA, vitamin A.

3.6. Vitamin A‐Enriched Diet Does Not Affect the Differentiation Stage of Urothelial Cells One and Three Days After CP‐Induced Injury

In addition to proliferation and apoptosis, differentiation is a crucial aspect of urothelial regeneration, which we further investigated in our study. First, analysis of RNAseq data of VitA CP1d compared to N CP1d revealed that gene expression of Upk2, Upk3a, and Abcg1, which are associated with urothelial differentiation, was significantly downregulated (Figure S8a). Next, we performed gene expression analysis of differentiation‐related markers of highly differentiated superficial urothelial cells, that is, UPs (Upk1a, Upk1b, Upk2, and Upk3a) and CK20 (Krt20) by qPCR. The result showed no major differences in the VitA CP1d and VitA CP3d group compared to the N CP1d and N CP3d group (Figures S8b–f and S9).

We also performed IHC labeling for UPs and CK20 (Figure 7). Quantification of UPs‐positive apical surface of urothelial cells showed no significant differences in the VitA CP1d group (69.52% ± 11.08%) compared to the N CP1d group (72.76% ± 9.48%), nor in the VitA CP3d (82.56% ± 9.76%) compared to the N CP3d group (88.95% ± 5.75%) (Figure 7e). In contrast, a significantly lower percentage of UPs‐positive surfaces was found in the CP1d groups (VitA CP1d 69.52% ± 11.08%; N CP1d 72.76% ± 9.48%) compared to the S1d groups (VitA S1d 96.46% ± 4.41%; N S1d 98.46% ± 3.33%), regardless of the diet type pointing toward an effect of CP‐induced injury on lower differentiation stage of urothelial cells (Figure 7e). In the VitA CP3d group, the UPs‐positive surface was also significantly lower (82.56% ± 9.76%) than in the VitA S3d group (99.52% ± 1.11%) (Figure 7e). IHC labeling of CK20 showed positive reaction in the apical cytoplasm of superficial cells in the S‐injected groups, while the majority of superficial cells in the CP‐injected groups were negative, confirming a lower differentiation stage of urothelial cells in the CP‐injected mice (Figure 7f–i).

FIGURE 7.

Differentiation stage of urothelial cells 1 or 3 days after CP or S injection. Representative images of IHC of a–d)UPs and f–i) CK20 in the urothelium of the bladder 1 day after CP or S injection. (e) Percentage of UPs positive apical surface of urothelial cells. Shown is the mean ± SD for all groups. n = 10 mice per group (CP‐injected groups) and n = 8 mice per group (S‐injected groups) for UPs analysis. Black arrows–positive IHC reaction (brown). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Statistical analysis of data on graph (e) was conducted using Kruskal–Wallis test with Dunn's post‐test. N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, VitA S1d–1 day after S‐injection with VitA‐enriched diet, N S3d–3 days after S‐injection with a normal diet, N CP3d–3 days after CP‐injection with a normal diet, VitA CP3d–3 days after CP injection with VitA‐enriched diet, VitA S3d–3 days after S injection with VitA‐enriched diet. ANOVA, analysis of variance; CK20, keratin 20; CP, cyclophosphamide; N, normal diet; S, saline; UP, uroplakin; VitA, vitamin A.

To further assess the differentiation stage of the urothelial cells, we quantified the structures of the luminal area of the urothelium using SEM. We measured the percentage of the urothelial surface covered with microvilli (undifferentiated cells), ropy ridges (poorly‐differentiated cells), and microridges (highly differentiated cells) (Figure 8a–d). The results showed no significant differences in percentage of microvilli (36.03% ± 40.71%), ropy ridges (57.52% ± 37.64%), or microridges (6.42% ± 12.1%) in the VitA CP1d group compared to the N CP1d group (microvilli 32.47% ± 42.52%; ropy ridges 60.2% ± 38.52%; microridges 6.61% ± 12.06%) (Figure 8e). Similar results were also found for the groups 3 days after injection. The CP‐injected groups showed an increased percentage of luminal surface area covered with ropy ridges (VitA CP3d 80.56% ± 22.11%; N CP3d 84.21% ± 34.41%) and a lower percentage of luminal surface area with microvilli (VitA CP3d 19.95% ± 23.12%, N CP3d 12.76% ± 35.25%) and microridges (VitA CP3d 0.00%; N CP3d 2.76% ± 5.52%), indicating a poorly differentiated stage of urothelial cells (Figure 8f). On the other hand, the luminal surface of the S‐injected group at Days 1 and 3 was mostly covered with cells showing microridges (approximately 100%), indicating a highly differentiated state (Figure 8e, f). All data collected indicate that increased VitA intake does not promote differentiation in the early stages of urothelial regeneration (1 and 3 days after CP‐induced injury) when proliferation is the prevailing process.

FIGURE 8.

Differentiation stage of luminal urothelial cells according to SEM analysis. (a–d) Representative images of SEM, showing the apical surface of the urinary bladder covered with microvilli (undifferentiated cells), ropy ridges (poorly differentiated cells), and microridges (highly differentiated cells). White arrows–microvilli, arrowheads–ropy ridges, asterisk–microridges. Differentiation stage of urothelial cells (microvilli–undifferentiated, ropy ridges–semi‐differentiated and microridges–differentiated cells) (e) 1 or (f) 3 days after CP or S injection. n = 8 mice per group (CP‐injected groups) and n = 6 mice per group (S‐injected groups) for SEM analysis. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Statistical analysis of data on graphs (e) and (f) was conducted using two‐way ANOVA with Tukey's post‐test. SEM‐scanning electron microscope. N S1d–1 day after S‐injection with a normal diet, N CP1d–1 day after CP‐injection with a normal diet, VitA CP1d–1 day after CP‐injection with VitA‐enriched diet, VitA S1d–1 day after S‐injection with VitA‐enriched diet, N S3d–3 days after S‐injection with a normal diet, N CP3d–3 days after CP‐injection with a normal diet, VitA CP3d–3 days after CP injection with VitA‐enriched diet, VitA S3d–3 days after S injection with VitA‐enriched diet. ANOVA, analysis of variance; CP, cyclophosphamide; N, normal diet; S, saline; SEM, scanning electron microscopy; VitA, vitamin A.

4. Discussion

VitA, particularly in its active form as RA, has long been studied for its diverse role in various cellular processes, including cell proliferation, apoptosis, and cell differentiation [1, 2, 8–10, 15]. Significant research has focused on the effects of VitA on the regeneration of various epithelial cell types [1, 8, 16, 18, 40], including the urothelial cells of the urinary bladder. For example, VitA may accelerate the regeneration of the blood–urine barrier by inducing urothelial progenitor cells to differentiate into mature superficial urothelial cells [18]. Nevertheless, the exact effect of high VitA intake on specific stages of urothelial regeneration after injury is not yet fully known, which we investigated in the present study. Therefore, the present study aimed to establish a link between VitA‐enriched diet and urothelial regeneration after severe injury, focusing on cell proliferation, apoptosis, and differentiation. We used a high‐dose CP injection to induce urinary bladder injury in female mice, a model commonly used to study urothelial regeneration [22, 41, 42]. The amount of VitA‐enriched diet used here was based on previous studies in which toxicity thresholds were defined in mice to ensure that this approach was safe and effective [34]. We used only female mice due to several reasons. First, women are much more susceptible to cystitis than men. Second, there are several studies on acute CP‐induced cystitis in female mice that have been conducted in recent years [43, 44, 45]. A study by Bon et al. showed that there are no significant differences between the behavioral responses after acute CP‐induced injury in male and female mice [46]. In addition, our group has recently shown that female mice with chronic CP‐induced cystitis (four i.p. injections of 80 mg/kg CP every other day) exhibit more pronounced urothelial hyperplasia than male mice [47].

The results of transcriptome analysis show that a VitA‐enriched diet significantly affects early‐stage urothelial regeneration, primarily through the upregulation of signaling pathways related to cell proliferation, such as cell cycle and PI3K‐Akt signaling pathways. We observed the upregulation of key genes involved in cell cycle regulation (Cdk1, Cdc25b, Ccna2, Bub1, Bub1b, and Knl1) in the VitA CP1d group compared to the N CP1d group (Figure 3). Although the biological variability within these two groups likely prevents statistical significance in qPCR validation, the upregulation of these genes is consistent, highlighting the role of VitA in modulating this pathway. Similar results were previously reported by Liu et al. who observed increased hepatocyte proliferation in the liver of mice following retinoic acid treatment. The increased hepatocyte proliferation was associated with the upregulation of cell cycle‐related genes, such as Cdk1 [48].

Further, our analysis of the PI3K‐Akt signaling pathway revealed differential gene expression 1 day after CP injection with a trend toward upregulation of several genes, including Itga3, Areg, Epha2, Spp1, and Ereg in the VitA CP1d group, indicating a more robust proliferative response. Our results after qPCR validation show that Itga3, a gene encoding integrin subunit α3, also known as integrin α3, is significantly upregulated in the groups of mice receiving VitA compared to those receiving a normal diet even under basal conditions, possibly promoting cell proliferation. This observation is consistent with the findings of Sakaguchi et al. who linked increased Itga3 expression to increased proliferation rates in urothelial cells [49]. Similarly, we confirmed that VitA increases the expression of Areg. Areg encodes amphiregulin, a member of the epidermal growth factor family and a ligand of the epidermal growth factor receptor (EGFR) [50]. Increased expression of Areg has been associated with autocrine growth regulation in urothelial cells [51], while the addition of amphiregulin, EGF, and TGFα appears to increase proliferation in human urothelial cells [52]. Suzuki et al. have shown that reduced expression of Areg and its target molecule Ki‐67 decreases urothelial cell proliferation in dimethylarsinic acid‐induced bladder carcinogenesis in rats [53], while our previous study has shown that EGFR is prominently expressed in the urothelial cells that remained viable after CP‐induced injury in rats and enables proliferation [42]. Furthermore, several other studies have shown that changes in retinoid uptake can alter cell proliferation in hepatocytes, keratinocytes, immune cells, and endothelial cells [39, 48, 52, 54, 55]. Based on these findings, we further investigated the effects of a VitA‐enriched diet on the proliferation of urothelial cells by immunolabeling the proliferation marker Ki‐67 [42, 53, 56, 57]. We confirmed higher proliferation rates of urothelial cells in mice fed with a VitA‐enriched diet 1 day after injury. However, this effect decreased on Day 3 after CP injection. These results suggest a link between VitA and increased proliferation of urothelial cells, possibly mediated by Itga3 and Areg in the early stages of urothelial regeneration after CP‐induced injury. However, this effect is transient, as proliferation rates and the expression of associated genes stabilize over time.

Treatment with CP damages the urothelium mainly by necrosis and apoptosis of the urothelial cells [42, 58]. Increased proliferation during urothelial regeneration leads to a hyperplastic urothelium, which is restored to a normoplastic state in later stages (7–14 days after CP injection) by the elimination of excess cells through apoptosis [31, 58]. In agreement with these observations, no apoptotic cells were detected by IHC in the early stages of urothelial regeneration after CP‐induced injury in our study. Nevertheless, RNAseq analysis showed significant upregulation of Ptges, Ccn1, and Angptl4 genes, which are part of the KEGG apoptosis signaling pathway, in the VitA CP1d group compared to the N CP1d group, but this could not be confirmed by subsequent qPCR analysis (Figure 6). This and our previous results [42, 58] suggest that although there may be early signals of apoptosis, its role in the regeneration process is more pronounced in the later stages (e.g., 7–14 days after injury) when the hyperplastic urothelium needs to return to the normoplastic state [42, 58].

In addition to its well‐known role in cell proliferation, VitA also has a significant influence on cell differentiation. Many studies have confirmed that VitA is one of the key substances that can drive cells in various tissue types into the early stages of the maturation process, for example, in the urothelium, endothelium, immune cells, intestinal epithelial cells, and skin [18, 19, 54, 55, 59]. To further clarify the effects of a VitA‐enriched diet on CP‐induced injury and urothelial regeneration, we analyzed in detail the most important markers of urothelial cell differentiation [60, 61, 62, 63, 64, 65] using different methods. In healthy urothelium in vivo [22, 42, 62] and urothelial models in vitro [23, 66, 67], CK20 and UPs are expressed in superficial cells, while UPs are expressed in intermediate cells as well, which is consistent with our results. In contrast, when the urothelium is injured after CP injection, we can observe a decrease in the expression of CK20 and UPs in the early stages of regeneration (up to three days after injury) [22]. Cheng et al. showed that basal cells undergo cell cycle progression rather than differentiation, and on the contrary, although intermediate cells are still highly proliferating cells, differentiation into superficial cells is more likely to be driven [62]. This is consistent with our results showing a lower percentage of UP‐ and CK20‐positive urothelial cells and a higher percentage of urothelial surface covered with microvilli and ropy ridges, characteristic of poorly and semidifferentiated cells, respectively, in the CP‐injected groups compared to the S‐injected groups (Figure 7). This indicates a lower differentiation state in these groups, which is to be expected after injury as the proliferation rate increases. On the other hand, we found no significant effect of a VitA‐enriched diet on the differentiation stage of urothelial cells 1 or 3 days after injury. Although there are no data on the influence of a VitA‐enriched diet on the differentiation process of urothelial cells, Mauney et al. have shown that the addition of a higher concentration of VitA in vitro can upregulate the expression of UP in murine embryonic cells 9 and 14 days after stimulation. However, no significant difference in UP expression was observed after a shorter period (Day 6) [19]. Thus, although retinoic acid signaling may be involved in urothelial cell differentiation [18], VitA supplementation does not significantly affect urothelial cell differentiation in the early stages of regeneration.

Our results suggest that VitA promotes urothelial cell proliferation by significantly upregulating Itga3 and Areg in the early stages (1 day) after CP‐induced injury and by increasing the expression of Cdc25b and Knl1, although not significantly. Although our RNAseq results initially indicated an effect of a VitA‐enriched diet on the expression of genes involved in apoptosis, this could not be confirmed as our results only showed an insignificant increase in the expression of Angptl4, Ccna1, and Ptges genes involved in apoptosis inhibition. Similarly, no significant effects of VitA on the differentiation stage of urothelial cells were observed in the early stages of regeneration. Our data confirm that VitA may play an important role in early urothelial regeneration, mainly by promoting urothelial cell proliferation and inhibiting apoptosis. These homeostatically regulated processes could serve as a first line of defense against the impaired blood–urine permeability barrier. Nevertheless, this study on urothelial regeneration focuses on gene expression analysis, histological and immunohistochemical evaluation, and SEM analysis, while the experiments on bladder function were not performed. Further studies, possibly with a longer duration of the VitA diet and longer follow‐up, are needed to investigate possible links between these early urothelial regeneration processes and cellular differentiation. Given the potential benefits of VitA in maintaining urothelial integrity and its possible role in improving the efficacy of chemotherapy, we suggest that further in vivo research and clinical investigations are urgently needed. In particular, additional studies should focus on the use of VitA supplements in patients with frequently injured urothelium, such as cancer patients treated with CP.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/mnfr.70045.

Supporting information

Supporting Information

Dragar B., Kranjc Brezar S., Čemažar M., Jesenko T., Romih R., Kreft M. E., Kuret T., Zupančič D., Vitamin A‐Enriched Diet Increases Urothelial Cell Proliferation by Upregulating Itga3 and Areg After Cyclophosphamide‐Induced Injury in Mice. Mol. Nutr. Food Res. 2025, 69, e70045. 10.1002/mnfr.70045

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.