Angiotensinogen and AT(1) antisense inhibition of osteopontin translation in rat proximal tubular cells

Antisense oligonucleotide inhibition of angiotensinogen and ANG II type 1 r eceptor (AT(1)) mRNA translation in rat proximal tubules (PT) was examined to provide direct evidence for a role of the renin-angiotensin system (RAS) in upregulated osteopontin expression observed following mechanical cell s tretch. Male Sprague-Dawley rats underwent unilateral ureteral obstruction (UUO) under Brevital anesthesia. In situ hybridization and Western blot ana lysis demonstrated angiotensinogen mRNA and angiotensin converting enzyme ( ACE) protein localized to PTs and upregulated in obstructed kidneys, respec tively, confirming an increased expression of renal RAS in vivo. In vitro s tudies were performed to provide mechanistic insight into ANG II-dependent osteopontin expression following mechanical cell stretch, which putatively mimics the increased PT luminal pressure post-UUO. A cationic transfection method was used to introduce either angiotensinogen or AT(1) antisense olig onucleotide into cultured rat PT cells prior to 1 h of cyclic mechanical ce ll stretch. Northern blot analysis revealed that PT cells subjected to cycl ic mechanical stretch with/without prior transfection with a sense oligonuc leotide exhibited increased osteopontin mRNA expression compared with unstr etched cells. Blockade of either angiotensinogen or AT1 mRNA translation by antisense oligonucleotide inhibition prior to cell stretch was found to si gnificantly decrease osteopontin mRNA levels 2.4-fold (P < 0.004) and 1.6-f old (P < 0.001), respectively, compared with values observed in control uns tretched cells. This study provides evidence that stretch-induced upregulat ion of osteopontin mRNA expression is mediated, in part, via production of ANG II. These results lend insight into upregulation of osteopontin via a l ocal PT RAS leading to macrophage infiltration in the tubulointerstitium in experimental hydronephrosis.