Angiotensin II as a player in fibrosis

Abstract

It is generally believed that excess angiotensin is Cre–loxP system. Cre is an enzyme which catalyses a involved in the progression of glomerular and tubuloin- site-specific recombination at a unique 34 bp sequence, terstitial injury and vascular hypertrophy. In this called loxP. We separately introduced a mutation regard, the phenotype of the lack of angiotensin containing loxP sites into Ren1 and Ren2 by gene revealed by angiotensinogen gene null-mutant mice targeting, and established a mouse strain carrying each seemed to be quite paradoxical. They showed hypo- mutation. These two mutants and a transgenic mouse trophic papilla, renal vascular hypertrophy, glomerular line ubiquitously overexpressing Cre recombinase were sclerosis, tubular degeneration and interstitial fibrosis, mated to produce the mice carrying the mutant Ren1 in addition to hypotension and overexpression of renin and Ren2 genes, as well as the Cre transgene concur[1]. Null-mutant mice for the genes of AT1, i.e. AT1A rently. Initially, two mutant Ren genes were located and B [2], but not AT2 [3], showed similar phenotypes, on separate chromosomes. Analysis of the renin genoindicating that these abnormal phenotypes are caused type of the offspring revealed that 7/73 (9.6%) had by the lack of angiotensin’s function mediated by AT1 both Ren1 and Ren2 mutation, and that 6/73 (8.2%) receptors. had no mutant gene. Southern analysis confirmed that Although AT1- or angiotensinogen-deficient mice in the mice with the dual mutation, two mutant Rens did not show urinary tract obstruction, the hypotrophic were recombined at the loxP site. Thus we have papilla observed in these mice was very similar to the successfully generated Ren1 and Ren2 dual targeted phenotype of hydronephrosis generated by ureteral mice. obstruction. In AT1-deficient mice, a renal pelvis was In the resultant dual renin allele, the HA and lacZ not developed and the ureteral smooth muscle layer reporter genes, together with the Ren1 promoter, were was hypotrophic and lacked peristaltic movements [4]. incorporated. Immuno- and histochemical staining Angiotensin II induced the ureteral smooth muscles in revealed that HA and lacZ were expressed in reninorgan cultures of wild-type, but not mutant, ureteral producing cells in the kidney of heterozygotes. In tissues. These results indicate that angiotensin II homozygous mice, HA and lacZ expression were has a critical role in ureteral development and that extended into afferent arterioles, interlobular arteries the hypotrophic papilla in AT1-or angiotensinogen- and glomeruli, thereby representing highly activated deficient mice is a consequence of functional ureteral Ren1 promoter activities. No renin staining was obstruction. detected in HA-positive cells in homozygous mice. In both angiotensinogen- and AT1-deficient mice, Reverse transcriptase–polymerase chain reaction the hypertrophied vasculature is accompanied by renin (RT–PCR) amplification revealed that homozygous overproduction. We speculated that renin per se may mice express a small amount of shorter RNA, which act as a growth factor for the renal vasculature, consists of a part of Ren2, Ren1 and the reporter gene. independently of angiotensin generation. To test this Renin activity in the kidney of homozygous mice (6w, possibility, we generated renin knockout mice [5]. n=5) was undetectable, indicating that the renin genes Mouse strains commonly used for gene targeting have are functionally inactivated in homozygous mice. duplicated renin genes, Ren1 and Ren2. These two Blood pressure measured by the direct method is genes, both producing active renin in the kidney, span 85.6±6.6/53.2±6.6 mmHg in conscious homozygous >40 kb in tandem on chromosome 1. This close prox- (6w, n=5)vs 132.7±1.3/93.0±1.4 mmHg in wild-type imity makes the generation of dual gene-targeted mut- mice (6w, n=3). The kidney of homozygous mice ants by mating single gene-targeted mutants nearly showed medial hypertrophy in afferent arterioles and impossible. To solve this problem, we utilized the interlobular arteries, as well as expansion of the mesangium matrix and hypotrophic papilla, in the same