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. 2012 Jan 27;17(2):196–205. doi: 10.2478/s11658-012-0002-y

The effect of hyperosmolar stimuli and cyclophosphamide on the culture of normal rat urothelial cells in vitro

Kajetan Juszczak 1,2,✉, Jolanta Kaszuba-Zwoińska 1, Paulina Chorobik 3, Agata Ziomber 1, Piotr Jan Thor 1
PMCID: PMC6275770  PMID: 22287017

Abstract

Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells (RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively. The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively, 14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%, respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l HS had the most harmful effect based on the percentage of necrotic and apoptotic cells.

Key words: Urothelial cell, Culture, Cyclophosphamide, Hyperosmolarity, Bladder, Rat, Apoptosis, Necrosis, Overactive bladder

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Abbreviations used

CYP

cyclophosphamide

CGRP

calcitonin gene-related peptide

DO

detrusor overactivity

FBS

fetal bovine serum

GAG

glycosaminoglycans

HEPES

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

HS

hyperosmolar stimuli

LUTS

lower urinary tract symptoms

MEM

minimal essential medium

RUCC

rat urothelial cultured cells

TRPV1

transient receptor potential vanilloid subtype 1

TRPV4

transient receptor potential vanilloid subtype 4

TRPM8

transient receptor potential vanilloid subfamily M member 8

TRPA1

transient receptor potential ankyrin subtype 4

SP

substance P

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