Acute nonbacterial cystitis is an inflammation of the urinary bladder accompanied by damage to the urothelium. The causes are varied and mostly unknown. The inner (luminal) surface of the urinary bladder is covered with an epithelium, the urothelium, which forms a blood-urine barrier. In studies of acute nonbacterial cystitis, cyclophosphamide (CP)-induced cystitis, which causes haemorrhagic cystitis, is often used as an in vivo model by single intraperitoneal (i.p.) administration of CP. A high dose of CP causes acute urinary bladder inflammation and damage to the urothelium, which is followed by rapid urothelial regeneration. Urothelial regeneration begins in the early stages with an increased proliferation rate. At the same time, the non-proliferating cells undergo a differentiation process. In the late stages of urothelial regeneration, we can observe urothelial hyperplasia, which returns to normal due to a higher rate of apoptosis. The SV-HUC1 cell line, in which inflammation is triggered by tumour necrosis factor alpha (TNFα), is generally used as an in vitro model for inflammation in urothelial cells.
Retinoids are a group of isoprenoids (natural and synthetic) that have a hydrophilic polar chain and the same properties as all-trans retinol, which we also refer to as vitamin A. Vitamin A and retinoids cannot be synthesized by human cells, so we must get them from our diet. Biologically, the most active vitamin A metabolite is retinoic acid (RA). Retinoids play an important role in embryonic development and influence vision, immunity, reproductive capacity, cell differentiation, proliferation, apoptosis, and the integrity of epithelial and immune cells. Retinoids also have anti-inflammatory properties. Many studies have shown that RA has an important impact on the development and regeneration of the urothelium via the RA signalling pathway. However, the effect of vitamin A on inflammation in the urinary bladder, proliferation, apoptosis, and differentiation of urothelial cells during regeneration of the urothelium after i.p. injection of CP is still unknown. Moreover, the effects of retinoids ATRA and acitretin on inflammation in the SV-HUC1 cell line in the in vitro TNFα inflammation model are also still unknown.
Therefore, we have divided our research into two parts: in vivo and in vitro.
The first in vivo part aimed to investigate the influence of a vitamin A-enriched diet on the extent of urothelial injury and urinary bladder inflammation, proliferation, apoptosis and differentiation of urothelial cells, and the expression of genes involved in RA signalling pathway in a mouse model of acute nonbacterial cystitis that we induced with a single dose of CP. We demonstrated that CP induces inflammation and alters the expression of genes involved in interleukin 17 (IL-17), tumour necrosis factor (TNF), hypoxia-inducible factor 1 (HIF-1) signalling pathways, and T helper (Th) cell differentiation. Although a vitamin A-enriched diet does not affect bladder inflammation or the before mentioned signalling pathways, we also acknowledge that it does not alter the extent of subepithelial oedema in the lamina propria and does not increase bladder injury. We also found that increased intake of vitamin A alters the expression of genes involved in RA metabolism (LRAT, RBP4, Aldh1a1, and Aldh1a7). Vitamin A-enriched diet also increased the proliferation rate of urothelial cells in the early stages (1st day after CP injection) of urothelial regeneration by increasing the expression of Itga3 and Areg, allowing rapid and effective restoration of the urothelium and the blood-urine barrier. However, a vitamin A-enriched diet had no significant effect on urothelial cell apoptosis and differentiation in the early stages of regeneration.
In the second in vitro part, we investigated the effects of all-trans RA (ATRA) and acitretin, a synthetic retinoid, on the immune response and changes in RA signalling pathway in an in vitro model of acute nonbacterial inflammation induced with tumour necrosis factor alpha (TNFα) in the SV-HUC1 cell line. We found that treatment with ATRA or acitretin caused a slight increase in the expression of IL1b and IL8 after induction of inflammation with TNFα but decreased the expression of IL6. ATRA also caused a decrease STAT1 and NFκB1 levels in SV-HUC1 cells after treatment with TNFα. Treatment with TNFα and retinoids also increased the expression of LRAT and Aldh1a3, which are involved in RA metabolism. We find that ATRA increases the expression of Areg, Ereg, and Mki67, and acitretin increases the expression of Itga3 and Cdk1. All these genes are involved in increasing cell proliferation. Finally, we proved that short-term treatment of SV-HUC1 cells with retinoids does not affect their differentiation stage.
The results of this doctoral thesis contribute to the knowledge of the effects of vitamin A and retinoids on inflammation and regeneration of the urothelium. Our findings may contribute to further investigations that could potentially lead to a clinical application of vitamin A and retinoids in the treatment of urothelial injury and acute nonbacterial cystitis, most likely in the form of supportive therapy.
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