MP68-06 FORMATION OF DOUBLE-STRANDED RNA ACTIVATES STRESS KINASES AND INITIATES CYTOSKELETON ORGANIZATION AND CELL DEGENERATION IN BLADDER ISCHEMIA

Abstract

INTRODUCTION AND OBJECTIVES: Double-stranded RNA (ds-RNA) is RNA with two complementary strands, which is not readily detected in normal tissues but is produced as a replicative intermediate in cellular stress conditions. By binding to and activating stress kinases and inducing mRNA degradation, ds-RNA can serve as a potent stimulus to cellular and subcellular structural modifications. We examined the formation and structural consequences of ds-RNA in chronic bladder ischemia. METHODS: A rat bladder ischemia model was developed by creating aorto-iliac arteries atherosclerosis. After 8 weeks, the animals underwent metabolic cage studies then hemodynamic measurements and cystometrograms (CMG) were obtained. Bladder tissues were processed for RT-PCR of ds-RNA precursor Alu-RNA, dot blot analysis of ds-RNA formation, western blotting of ds-RNA-dependent protein kinase (PKR), mass spectrometry, ELISA of stress markers, and transmission electron microscopy (TEM). RESULTS: Chronic bladder ischemia increased micturition frequency, decreased voided volume and led to detrusor instability. Ischemia upregulated Alu-RNA expression and led to the formation of ds-RNA and activation of the stress kinase PKR in the bladder. These changes were associated with cellular lipid and protein stress markers. Accumulation of ds-RNA and phosphorylation of PKR resulted in DNA damage, loss of proteins, and activation of degenerative pathways. Mass spectrometry and Gen Ontology analysis suggested cytoskeleton organization and degenerative activities mediated by ubiquitination and increased proteolysis, peptidase and hydrolase activities. EM confirmed cytoskeleton organization and cell degeneration characterized by swollen mitochondria with disrupted membrane and decreased granules, swollen elongated ER, collagen invasion of smooth muscle cells and nerve fibers, sporadic loss of epithelial mucosal membrane, twisted smooth muscle cells, diffused vacuolization, and loss of neural structural integrity. CONCLUSIONS: We report the formation of ds-RNA and subsequent phosphorylation of PKR in bladder ischemia. Activation of dsRNA/PKR pathway in the bladder resulted in widespread structural modifications via mechanisms involving DNA damage, mRNA degradation and loss of proteins. Our data suggest a close link between dsRNA/PKR pathway, proteolysis, cytoskeleton organization and loss of cellular structural integrity. Formation of ds-RNA and activation of PKR may precede cellular ultrastructural deterioration and play a central role in loss of smooth muscle cells and neurodegeneration in the ischemic overactive bladder.