Izražanje mehanoreceptorjev PIEZO v uroteliju med obnovo po kemijsko izzvanem cistitisu

Deutsch, Maja (author), Romih, Rok (mentor)

Urotelij ima pomembno vlogo pri zaznavanju in posredovanju mehanskih dražljajev, ki nastopijo pri raztezanju sečnega mehurja med polnjenjem z urinom. Pri tem so ključni mehanoreceptorji v uroteliju, med njimi ionska kanala Piezo1 in Piezo2. Pri intersticijskem cistitisu, kjer je urotelij spremenjen, je zaznavanje mehanskih dražljajev moteno. Kako spremembe urotelija med cistitisom vplivajo na izražanje Piezo1 in 2, ni znano. Cistitis smo izzvali pri podganah z enkratno peritonealno injekcijo ciklofosfamida. Prisotnost mehanoreceptorjev Piezo smo preverjali s prenosom western in imunoofluorescenco. Primerjali smo različne protokole ter ustreznost metod in protiteles za imunolokalizacijo mehanoreceptorjev Piezo. Uporabili smo dve različni protitelesi proti Piezo1 in protitelo proti Piezo2 ter različna sekundarna protitelesa. S prenosom western smo prisotnost Piezo1 dokazali z enim protitelesom, medtem ko za Piezo2 protitelo ni pokazalo lis pri ustrezni velikosti. Na zamrznjenih rezinah smo z imunofluorescenco s protitelesom proti Piezo1 dokazali močnejšo reakcijo pri vzorcih, tretiranih s ciklofosfamidom v primerjavi z rezinami netretiranih podgan. Reakcija je bila prisotna na apikalni plazmalemi dežnikastih celic ter tudi na bazalnih in intermediarnih celicah. Reakcija za Piezo2 je bila šibkejša, vidna je bila v vseh treh skladih urotelija, lamini proprii in detruzorju. Pri obeh mehanoreceptorjih Piezo je bila vidna reakcija tako v vrhu in telesu sečnega mehurja, med tema predeloma razlik ni bilo. V bazi sečnega mehurja je bila v primeru Piezo1 reakcija šibka, pri Piezo2 pa le-te ni bilo. Z imunofluorescenco smo tako dokazali močnejšo reakcijo Piezo1 in Piezo2 po tretiranju s ciklofosfamidom in dokazali njihovo prisotnost v vseh skladih urotelija. Ovrednotili smo nove postopke priprave vzorcev, tako omogočili študije sečnega mehurja podgan in s tem postavili izhodišče za nadaljne študije pri ugotavljanju vloge mehanoreceptorjev Piezo med cistitisom.

• Language: Slovenian
• Keywords: urotelij, Piezo, ciklofosfamid, prenos western, imunofluorescenca
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-149315
• COBISS.SI-ID: 168136707
• Publication date in RUL: 06.09.2023

Secondary language

• Language: English
• Title: The expression of PIEZO mechanoreceptors in the urothelium during regeneration after a chemically induced cystitis

Abstract

The urothelium has an important role in sensing and communicating mechanical stimuli, that occur during the stretching of the urinary bladder filling. The key component to this are the mechanoreceptors, including the ion channels Piezo1 and Piezo2. In interstitial cystitis, where the urothelium is changed, the sensing of mechanical stimuli is interrupted. How the changes of the urothelium during cystitis affect the expression of Piezo1 and Piezo2 is not yet known. Cystitis was induced with a single peritoneal cyclophosphamide injection. The presence of Piezo mechanoreceptors in the urinary bladder of rats was checked with western blotting and immunofluorescence. We compared different protocols and antibodies for immunolocalizations of mechanoreceptors Piezo. We used two different antibodies for Piezo1 and an antibody for Piezo2 and different secondary antibodies. The presence of Piezo1 was demonstrated by a single antibody while western blotting of Piezo2 did not show marks on the correct size. On cryosections with immunofluorescence, Piezo1 showed a stronger reaction in samples injected with cyclophosphamide in comparison to sections of untreated rats. The reaction was mainly present on the apical plasmalemma of umbrella cells and also on basal and intermediate cells. In Piezo2 the reaction was weaker but was also seen in all three urothelium layers, the lamina propria, and the detrusor. With both Piezo mechanoreceptors a reaction was visible on the top and body of the urinary bladder and there were no differences between these two regions. The reaction was weaker in Piezo1 or non-existent in the case of Piezo2 in the basal region. With immunofluorescence we thus demonstrated a stronger reaction of Piezo protein in the urothelium after being injected with cyclophosphamide and showed their presence in all layers of the urothelium. We evaluated new protocols of sample preparation, thus enabling new studies of urinary bladder of rats and setting the stage for further studies of determining the role of Piezo mechanoreceptors during cystitis.

• Keywords: urothelium, Piezo, cyclophosphamide, western blot, immunofluorescence