The Kidney Androgen-regulated Protein Promoter Confers Renal Proximal Tubule Cell-specific and Highly Androgen-responsive Expression on the Human Angiotensinogen Gene in Transgenic Mice

Abstract

Transgenic mice were generated containing a 1542-base pair fragment of the kidney androgen-regulated protein (KAP) promoter fused to the human angiotensinogen (HAGT) gene with the goal of specifically targeting inducible expression of renin-angiotensin system components to the kidney. High level expression of both KAP-HAGT and endogenous KAP mRNA was evident in the kidney of male mice from two independent transgenic lines. Renal expression of the transgene in female mice was undetectable under basal conditions but could be strongly induced by administration of testosterone. Testosterone treatment did not cause a transcriptional induction in any other tissues examined. However, an analysis of six androgen target tissues in males revealed that the transgene was expressed in epididymis. No other extra-renal expression of the transgene was detected. In situ hybridization demonstrated that expression of HAGT (and KAP) mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable human angiotensinogen protein in plasma, it was evident in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen. These results demonstrate that 1542 base pairs of the KAP promoter is sufficient to drive expression of a heterologous reporter gene in a tissue-specific, cell-specific, and androgen-regulated fashion in transgenic mice.The renin-angiotensin system (RAS) 1 is a classical endocrine system activated by the release of renin from the kidney and angiotensinogen (AGT) from the liver. In blood, renin proteolytically cleaves AGT to form angiotensin I (Ang-I) which is further processed by angiotensin converting enzyme to form Ang-II, a potent vasoconstrictor and antinatriuretic peptide. The RAS has been implicated in the genetic basis of hypertension and pre-eclampsia (1-4). Our understanding of the RAS in normal and pathophysiological regulation of blood pressure has been complicated by the fact that in addition to its actions as an endocrine system, certain individual tissues, such as the kidney (5-7), heart (8, 9), brain (10), and vasculature (11), contain all the components of the RAS cascade and therefore have the potential for local synthesis and action of Ang-II. In the kidney, for example, renin, AGT and ACE mRNAs, and proteins are synthesized in juxtaglomerular cells, proximal convoluted tubule (PCT) cells, and endothelial and tubular cells, respectively, and Ang-II type-1 (AT-1) and type-2 (AT-2) receptors are localized in glomeruli, collecting ducts, tubules, and vasa recta (12-18). The intrarenal RAS has been postulated to regulate various aspects of renal function including blood flow, natriuresis, and tubular-glomerular feedback, and may therefore participate in the pathogenesis of hypertension (19 -21). Our current understanding of the relative importance of the intrarenal versus systemic RAS comes largely from pharmacological studies (22) which have been limited by the specificity...

Liver Angiotensinogen Is the Primary Source of Renal Angiotensin II

“…However, our liver Agt KO study revealed that the Agt protein synthesized from the renal Agt mRNA is too low to be appreciated by either immunostaining or Western blot analysis. However, kidney Ag KO led to a decrease in urinary Agt protein, suggesting that most Agt protein synthesized in the S3 segment is immediately secreted into the urine; thus, our findings support the notion previously offered in the work by Ding et al 25 Our study also showed that, whereas disruption of kidney Agt gene has no effect on renal AII content, disruption of liver Agt gene markedly reduces renal AII. In fact, although liver Agt KO led to a moderate increase in renal Agt mRNA in a compensatory fashion, the increase was not translated into an increase in renal AII content.…”

Renal Renin-Angiotensin System

“…In situ hybridization demonstrated that expression of hAGT mRNA in males and testosterone-treated females was restricted to proximal tubule epithelial cells in the renal cortex. Although there was no detectable hAGT protein in plasma, it was shown in the urine, consistent with a pathway of synthesis in proximal tubule cells and release into the tubular lumen [75]. Mouse angiotensinogen and hAGT have a similar structure; however, mouse renin is species-specific and cannot cleave hAGT [77].…”

“…Sigmund and colleagues [74][75][76] developed a series of interesting models of inducible hypertension. Transgenic mice were generated in which a fragment of the kidney-specific androgen-regulated protein (KAP) promoter was fused to the human angiotensinogen (hAGT) gene.…”

Efficient Liver-specific Deletion of a Floxed Human Angiotensinogen Transgene by Adenoviral Delivery of Cre Recombinasein Vivo

“…The specimens were then immediately centrifuged at 12,000 rpm for 2 min at 2°C, and a 50-l plasma sample was obtained and immediately frozen at Ϫ80°C. Plasma samples were treated as described previously (18,31). Radioimmunoassays were then performed using the RIANEN angiotensin I 125 I-labeled radioimmunoassay kit (Dupont) using the directions and reagents supplied by the manufacturer.…”