Control Of Renin Gene Expression Research Articles

Long-Range Control of Renin Gene Expression in Tsukuba Hypertensive Mice.

Renin, a rate-limiting enzyme in the renin–angiotensin system, is regulated to maintain blood pressure homeostasis: renin gene expression in the kidney is suppressed in a hypertensive environment. We found that expression of a 15-kb human RENIN (hREN) transgene was aberrantly upregulated (>4.2-fold), while the endogenous mouse renin (mRen) gene was suppressed (>1.7-fold) in Tsukuba hypertensive mice (THM), a model for genetically induced hypertension. We then generated transgenic mice using a 13-kb mRen gene fragment that was homologous to the 15-kb hREN transgene and found that its expression was also upregulated (>3.1-fold) in THM, suggesting that putative silencing elements of the renin genes were distally located in the loci. We next examined the possible role of a previously identified mouse distal enhancer (mdE) located outside of the 13-kb mRen gene fragment. Deletion of the mdE in the context of a 156-kb mRen transgene did not affect its transcriptional repression in THM, implying that although the silencing element of the mRen gene is located within the 156-kb fragment tested, it is distinct from the mdE. Consistent with these results, deletion of the 63-kb region upstream of the mdE from the endogenous mRen gene locus abrogated its transcriptional repression in THM. We finally tested whether dysregulation of the short renin transgenes also occurred in the fetal or neonatal kidneys of THM and found that their expression was not aberrantly upregulated, demonstrating that aberrant regulation of short renin transgenes commences sometime between neonate and adult periods.

Renin gene expression is regulated by Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP‐TF II)

We aimed to investigate the involvement of the nuclear receptor COUP‐TFII in the regulation of renin gene expression.Protein‐DNA binding studies demonstrated that COUP‐TFII binds to the proxDR sequence in the proximal renin promoter. Knockdown of COUP‐TFII in the renin‐producing cell lines As4.1 and Calu‐6 reduced the stimulation of renin mRNA expression by cAMP agonists. The mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was essential for the function of a renin promoter cAMP‐response element. Knockdown of COUP‐TFII or cAMP‐binding protein (CREB) decreased the basal renin gene expression. When CREB was knocked down, the deficiency of COUP‐TFII did not further diminish the renin expression. Mice deficient in COUP‐TFII have lower renin expression than their control strain. Surprisingly, there was no significant difference in both plasma renin concentration and blood pressure between wildtype and COUP‐TFII‐deficient mice. Altogether our data show that COUP‐TFII is involved in the control of renin gene expression. To better characterize the role of COUP‐TFII for the renin production in vivo we are generating mice with selective COUP‐TFII deletion in the renin‐producing cells of the kidney.

Chicken Ovalbumin Upstream Promoter Transcription Factor II Regulates Renin Gene Expression

This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.

Replacement of connexin 40 by connexin 45 causes ectopic localization of renin-producing cells in the kidney but maintains in vivo control of renin gene expression

Deletion of connexin 40 (Cx40) leads to ectopic hyperplasia of renin-producing cells in the kidney, which is associated with dysregulated hyperreninemia and hypertension. The aim of this study was to determine whether Cx45 is able to substitute the function of Cx40 with regard to the localization of renin-producing cells. For this purpose, we have studied the distribution of renin-expressing cells under both normal conditions and during a stimulatory challenge of the renin system by inducing salt deprivation in mice, achieved by replacing the coding sequence of the Cx40 gene with that of Cx45 (Cx40ki45). In both wild-type (WT) mice and Cx40ki45 mice under normal conditions, renin-expressing cells were located at the juxtaglomerular position, whereas in Cx40-deficient mice they were located in the periglomerular interstitium. Upon challenge of the renin system, renin mRNA and the number of renin-expressing cells increased in WT mice in the media layer of afferent arterioles, while neither parameter changed significantly in Cx40-deficient mice. In Cx40ki45 mice, challenge of the renin system markedly increased both renin mRNA and the number of renin-expressing cells. However, the newly recruited renin-expressing cells were localized mainly outside the afferent vessels in the periglomerular interstitium. We found no evidence of cell divisions in renin-expressing cells in any of the genotypes investigated in this study, suggesting that the ectopically localized, renin-expressing cells in Cx40ki45 mice were already preexisting but were not renin-expressing under normal conditions. In summary, we infer from our findings that the function of Cx40 for the localization of potential renin-producing cells cannot be substituted by that of Cx45, although the regulability of renin gene expression can.

Peroxisome Proliferator-Activated Receptor-γ Is Involved in the Control of Renin Gene Expression

Based on the presence of a functional retinoic acid receptor/retinoid X receptor transcription factor binding sequence (hormone-responsive element) in the renin gene enhancer and on the fact that the peroxisome proliferator-activated receptors (PPARs) bind to DNA as heterodimers with retinoid X receptors, we speculated that PPARs are involved in the regulation of renin gene expression. To test this hypothesis, we used the human renin-producing cell line CaLu-6. Endogenous or pharmacological PPARgamma agonists (unsaturated fatty acids and thiazolidinediones, respectively) stimulated renin gene expression. Surprisingly, we found that PPARgamma targets a palindromic repeat with a 3-bp spacer (Pal3) in the proximal human renin promoter. Thus, renin is the first gene described with a functional Pal3 sequence. PPARgamma agonists also stimulated renin gene expression in cultured native juxtaglomerular cells, which are the main source of renin in vivo. In summary, PPARgamma was identified as a novel intracellular mediator involved in the upregulation of renin transcription.

Renin Enhancer Is Critical for Control of Renin Gene Expression and Cardiovascular Function

Renin Enhancer Is Critical for Control of Renin Gene Expression and Cardiovascular Function

Renin Enhancer Is Critical for Control of Renin Gene Expression and Cardiovascular Function

The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked beta-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 +/- 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin

Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R−/− mice by 40–50%. On the other hand, renal COX-1 levels were similar in B2R−/− and +/+ mice. Renal renin protein was 61% lower in B2R−/− compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R−/− mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R−/− mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261±16 and 266±15 fmol/g in B2R+/+ and B2R−/− mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R−/− mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression.

Angiotensin II inhibits renin gene transcription via the protein kinase C pathway.

Angiotensin II (ANGII) inhibits renin gene expression in vivo, a mechanism considered to be a physiologically important negative feedback of the renin-angiotensin-system. How this particular effect of ANGII is achieved at the cellular level is unknown. Our study therefore aimed to determine whether ANGII exerts a direct effect on renin gene expression and, if so, to characterise the molecular mechanisms involved. In the mouse renal juxtaglomerular cell line As4.1, ANGII decreased steady-state renin mRNA levels and prorenin secretion time and concentration (EC(50) 100 nmol/l) dependently. The effects of ANGII were blunted by the protein kinase C (PKC) inhibitor bisindolylmaleimide I and mimicked by the PKC activator phorbol 12-myristate-13-acetate (PMA) (EC(50) 10 nmol/l). ANGII also inhibited renin promoter activity PKC-dependently. The inhibitory sequences triggered by ANGII appear to reside within the first 2.9 kb in the 5'-flanking region of the mouse ren1c gene but are not related to the two canonical activator protein-1 (AP-1) binding sites at positions -16 to -22 and -141 to -147. In summary, our data suggest that ANGII acts directly on renal juxtaglomerular cells to inhibit renin gene transcription through the PKC pathway. Since the PKC pathway can be activated by a variety of hormones it represents a powerful and probably rather important downstream pathway in the control of renin gene expression.

The role of angiotensin II in the feedback control of renin gene expression.

This study aimed to characterize the influence of endogenous angiotensin II on renal renin gene expression during different states of a stimulated and of a suppressed renin system. To this end the renin system in male Sprague Dawley rats was stimulated by unilateral renal artery clipping (0.2 mm clip), by furosemide (60 mg/kg per diem) or isoproterenol (160 microg/kg per diem), and by ingestion of a low-salt diet (0.02%), or was suppressed by setting a contralateral renal artery clip (0.2-mm clip) or by ingestion of a high-salt diet (4%). During the last 2 days of these different treatment regimens, the animals were treated with the angiotensin II AT1 receptor antagonist losartan (40 mg/kg per diem) and renal renin mRNA levels were assayed. Renin gene expression was stimulated four- to fivefold by renal artery clipping and isoproterenol infusion, two- to three-fold by furosemide and a low-salt diet, and about four-fold by losartan. Additional treatment with losartan potentiated the stimulatory effects of a low-salt diet, of furosemide and of isoproterenol infusion on renin gene expression, whilst there was no significant additional effect of losartan on renin gene expression in clipped kidneys. Both contralateral renal artery clipping and a high-salt diet decreased renin mRNA levels to about 50% of the control value. In rts with a unilateral clip, additional losartan treatment caused renin mRNA to increase to about 350% of the control value in the contralateral kidney but to only 100% of the control value in animals on a high-salt diet. These findings suggest that the enhanced formation of angiotensin II during a low-salt intake, during tubular inhibition of salt reabsorption or during beta-adrenoreceptor activation plays a relevant negative feedback role in the activation of the renin gene. Moreover, in rats with one hypoperfused kidney, angiotensin II could be involved in the inhibition of renin gene expression in the contralateral kidney. In hypoperfused kidneys, however, and in animals on a high-salt diet, angiotensin II appears to play a only a minor feedback role in the regulation of the renin gene.

Control of renin gene expression in 2 kidney-1 clip rats

This study was done to investigate the mechanisms that underly the changes of renal renin gene expression upon hypoperfusion of one kidney. To this end the left renal arteries of male Sprague-Dawley rats were clipped with 0.2 mm silver clips and renal renin mRNA levels were assayed by RNase protection during the first ten days after clipping. Unilateral reduction of renal blood flow led to transient maximal fivefold increases of renin mRNA levels in the clipped kidneys and to sustained suppression of renin gene expression to 20% of the control value in the contralateral intact kidneys. Inhibition of prostaglandin (PG) formation by meclofenamate or EDRF synthesis by L-NAME markedly attenuated the increase of renin mRNA levels in response to clipping, and a combination of PG/EDRF inhibition almost abolished the increase of renin mRNA levels. Inhibition of PG/EDRF formation did not change the suppression of renin mRNA levels in the contralateral intact kidneys. Neither did renal denervation nor inhibition of macula densa function by furosemide prevent the suppression of renin gene expression in response to unilateral renal artery clipping. Only converting enzyme inhibition by ramipril and blockade of Ang II-AT1 receptors by losartan attenuated the decrease of renin mRNA levels in the contralaterals to clipped kidneys. These findings suggest that intact PG and EDRF synthesis represent stimulatory signals for renin gene expression that are required for the elevation of renin mRNA levels upon unilateral renal hypoperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium derived relaxing factor is involved in the pressure control of renin gene expression in the kidney.

To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene expression, the effects of a 2-day treatment with the NO-synthase inhibitor nitro-L-arginine-methylester (L-NAME, 40 mg/kg twice a day) on plasma renin activity (PRA) and renal and adrenal renin m-RNA levels were examined in conscious rats with and without unilateral renal clips (0.2 mm). In sham-clipped animals L-NAME led to a decrease of PRA from 7.5 to 2.5 ng angiotensin (ANGI).h-1.ml-1 and to a 35% decrease of renal renin m-RNA levels. Unilateral renal artery clipping increased PRA to 35 and to 13 ng ANGI.h-1.ml-1 in vehicle and in L-NAME-treated rats, respectively. In the clipped kidneys renin m-RNA levels increased to 450% of control values in vehicle-treated animals and to 220% of control values in L-NAME-treated animals. In the contralaterals as opposed to clipped kidneys, renin m-RNA levels decreased to 16% and 50% of the control values in vehicle- and in L-NAME-treated animals, respectively. In the adrenal glands renin m-RNA levels were not significantly changed either by clipping of one renal artery or by treatment of animals with L-NAME. The NO-donor sodium nitroprusside (100 microM) was found to increase renin secretion and renin m-RNA levels in primary cultures of renal juxtaglomerular cells. These findings suggest that EDNO is involved in the control of the renin gene by the renal perfusion pressure.

Which factor mediates reno-renal control of renin gene expression?

To obtain information about possible pathways mediating the suppression of renin gene expression in the contralateral kidneys of stenosed kidneys. The effects of unilateral renal denervation and of treatment with an angiotensin II antagonist (losartan) on renal renin gene expression were examined in a two-kidney, one-clip model. Renal renin messenger RNA levels, plasma renin activity, blood pressure and kidney weights were monitored over 10 days in adult male Sprague-Dawley rats with various unilateral reductions of renal blood flow achieved with silver clips of 0.2, 0.3 and 0.4 mm inner diameter. With all the clip sizes used, renin messenger RNA levels increased transiently in the clipped kidneys, the time course and the magnitude of the increase being dependent on the degree of flow reduction. In the contralateral kidneys clipping caused sustained decreases in renin messenger RNA to levels proportional to the clip size. The suppression of renin gene expression in the contralateral kidneys was not related to compensatory growth of the organs nor to changes in plasma renin activity or arterial pressure. Unilateral denervation of the kidney before clipping had no influence on the characteristic increase and decrease in renin messenger RNA in the stenosed and contralateral kidneys, respectively. Treatment of the rats with losartan led to fourfold increases in renal renin messenger RNA levels and to sixfold increases in plasma renin activity in control rats. A 0.3-mm clip did not further increase renin messenger RNA or plasma renin activity in losartan-treated rats but again led to suppression of renin messenger RNA in the contralateral kidney to 50% of the levels found in the clipped kidneys. The results suggest that the suppression of renin gene expression in the contralateral kidneys of stenosed kidneys is not due to compensatory renal growth nor mediated by systemic blood pressure, angiotensin II AT1 receptors or renal nerves. We therefore hypothesize that kidneys with reduced perfusion release a humoral factor that acts as a potent inhibitor of renin gene expression.