Urothelial cells are epithelial cells that line the urogenital tract from renal pelvis to proximal urethra. During differentiation, the superficial urothelial cells in vivo (called umbrella cells) synthesize transmembrane proteins uroplakins, which are organised in urothelial plaques and, together with the tight junctions, form the molecular basis of the blood-urine barrier of the urinary bladder. It is known that the endoplasmic reticulum and the Golgi apparatus are involved in the biosynthetic pathway of urothelial plaques, however the role of mitochondria in urothelial cells is poorly understood. Studies suggest that mitochondria are altered by pathological conditions and are also involved in the alternative degradation of uroplakins. Mitochondria in differentiated normal and altered urothelial cells have not yet been investigated. The aim of this study was to analyse the expression and distribution of mitochondrial proteins in urothelial cells in vivo and in vitro at different stages of differentiation. Using cell-biology (western blotting, immunolabelling) and microscopic methods, we found that expression and distribution of the mitochondrial protein Tom20 differs between different cultured urothelial cells in vitro and mouse urothelial cells in vivo. In cell cultures, we observed that mitochondria were distributed in three characteristic patterns, called fragmented, tubular and network-like distribution. In the culture of non-invasive urothelial papilloma cells (RT4) we observed a predominantly fragmented mitochondrial distribution, in the culture of muscle-invasive urothelial cancer cells (T24) a predominantly network-like distribution, in the culture of partially differentiated normal porcine urothelial cells (PD NPU) predominantly a network-like and partially tubular distribution of mitochondria and in the culture of highly differentiated normal porcine urothelial cells
(HD NPU) predominantly a network-like distribution of mitochondria. We also compared mitochondrial distribution with markers of urothelial cell differentiation and found that mitochondrial distribution correlates with the degree of urothelial cell differentiation. Low differentiated cells (RT4) had a fragmented mitochondrial distribution, whereas partially differentiated cells showed a network-like distribution (PD NPU, HD NPU). We also analysed the presence and distribution of proteins involved in mitochondrial dynamics (mitofusion – MFN2, mitofission – DRP1) and mitophagy (PARK2, PINK, LC3). In low differentiated RT4 cells with fragmented mitochondrial distribution, there was more DRP1 compared to MFN2, whereas the opposite was true in partially differentiated NPU cell cultures with network-like distribution of mitochondria. The mitophagy proteins occasionally overlapped with the distribution of mitochondrial proteins. Results of this study present the first steps in the research of mitochondria in urothelial cells in vitro and in vivo and suggest that mitochondrial dynamics and mitophagy proteins are involved in the distribution of mitochondria at different stages of urothelial cell differentiation. The study also raises new questions about the significance of mitochondrial distribution in urothelial cells and their possible urothelium-specific role.
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