mRNA Expression Of Key Enzymes Involved In The Eicosanoid Biosynthesis Pathway In Feline Idiopathic Cystitis

Abstract

Feline idiopathic cystitis (FIC) is a common inflammatory condition of the bladder that affects young to middle aged cats with unknown cause. Because there may be different pathological mechanisms that could cause FIC, there is a critical need to identify the key pathogenic factors in FIC. Eicosanoids are potent lipid mediators involved in the initiation and resolution of inflammation and in the maintenance of the normal health of the urothelium. Recent studies suggest that FIC may be associated with alterations in bladder eicosanoid metabolism. The altered expression of genes encoding key enzymes involved in the metabolism of eicosanoids could be important in the pathogenesis of FIC. We hypothesized that expression of the mRNAs for these enzymes would differ between affected and healthy cats. In order to test this hypothesis, mRNA was isolated from healthy and FIC cat bladders (obtained by cystotomy and embedded in formalin-fixed paraffin), as well as urolith bladders as a general inflammation control. The mRNAs for COX-1, COX-2, mPGES, cPGES, 15- LOX-2 and PGDH (which all play a role in metabolism of eicosanoids) were quantified by reverse transcription quantitative PCR (RT-qPCR). Four control genes (SDHA, RPS5, RPS19, and POL2A) were developed to allow a relative analysis by the delta delta CT method. Formalin-fixed paraffin embedded (FFPE) samples of affected cats (n=8), unaffected negative control cats (n=9) and urolith positive control cats (n=8) were obtained and used for the quantification. No significant differences were found for the expression of the genes in FIC compared to urolith and normal cats, although a trend toward of p < 0.10 was found for cPGES and 15-LOX-2. The results of these experiments may lead to a better understanding of the contribution of eicosanoids in the pathogenesis of FIC, and may eventually lead to better methods of diagnosis and treatment. Furthermore primers with high efficiencies were made for the genes, which may be useful for future studies of FIC and other feline inflammatory diseases. Moreover the use of FFPE tissues for studying mRNA expression in relation with a disease or inflammatory condition is not a common practice. However due to the lack of the availability of fresh frozen tissues, this study took the initiative to use FFPE tissues to study the mRNA expression in relation with FIC. This could make further studies easier to perform due to using stored samples instead of fresh samples.