Renin Gene Expression Research Articles

Evaluation Of Possible Antifibrotic Effect Of Cholecalciferol, Calci-Potriol, Aliskiren, Ramipril And Eprosartan On Carbon Tetrachloride Induced Liver Fibrosis In Albino Rats

the present study aims to evaluate the possible anti-fibrotic effect of calci-potriol, cholecalciferol, aliskiren, eprosartan and ramipril on carbon tetra-chloride (CCl4) induced liver fibrosis in rats. Methods: Fifty six Spague Dawely rats were randomly allocated into seven groups. All rats were ex-posed to induction of hepatic fibrosis by intraperitoneal (ip) injection of CCl4 twice/week for 8 successive weeks except control healthy group. The rats were treated with either calcipotriol (20 ug/Kg), Cholecalciferol (500 IU/kg), aliskiren (10mg/kg), eprosartan (60mg/kg) and ramipril (10mg/kg) simulta-neously with CCl4 treatment. Results: all the tested drugs have significant improvement in liver function tests (liver enzymes, bilirubin and albumin), oxidative stress markers (reduced glutathione, malondialdehyde and nitric oxide), transforming growth factor-b1 (TGF-b1) and fibrotic score. Only cal-cipotriol and cholecalciferol are significantly decrease renin gene expres-sion in liver and kidney tissues while cholecalciferol alone has significant in-crease in serum calcium and 25 hydroxy vitamin D [25(OH)D] levels. Conclusion: these results suggest that calcipotriol, cholecalciferol, aliskiren, eprosartan and ramipril have antifibrotic effect. Vitamin D receptor (VDR) agonists (Calcipotriol and cholecalciferol) inhibit renin gene expression and have better antifibrotic effect than renin angiotensin blockers (aliskiren, ep-rosartan and ramipril). Eprosartan has better antifibrotic effect than aliskiren and ramipri

Topiroxostat Showed Renoprotection via Various Mechanisms Including Suppression of Xanthine Oxidoreductase-Induced-Oxidative Stress and Inflammation in db/db Mice

Background: Xanthine oxidoreductase (XOR) produces superoxide along with uric acid. This reactive oxygen species induces organ damage. Therefore, we aimed to investigate whether XOR-induced superoxide is associated with renal abnormality in diabetic mice. Methods: Db/db mice (9-week-old) were fed standard diets with or without topiroxostat (1, 3, and 10 mg/kg/day) or febuxostat (3, 10, 30, and 45 mg/kg/day) for 4 weeks. Furthermore, topiroxostat (1 and 3 mg/kg/day) and febuxostat (3 mg/kg/day) were administered for 7 days or 8 weeks. The levels of urinary albumin excretion (UAE), monocyte chemoattractant protein-1 (MCP-1), and angiotensin II were determined. For renal pathological or mechanistic analysis, periodic acid-Schiff (PAS), nitrotyrosine, and MCP-1 staining or renal gene expression, XOR activity, and purine body levels were evaluated. Results: Both topiroxostat and febuxostat suppressed UAE : topiroxostat 3 mg/kg/day, 11.5 ± 33 μg/day versus vehicle 253.7 ± 69 μg/day, p<0.05; febuxostat 3 mg/kg/day, 255 ± 92 μg/day versus vehicle 634 ± 101 μg/day, p=0.05, for 4 weeks, respectively, and reduced immunohistological staining of enhanced oxidative stress and inflammation markers without affecting glycated hemoglobin. Topiroxostat but not febuxostat decreased urinary MCP-1 and angiotensin II levels and attenuated morphological hypertrophy in the glomerulus and epithelial cell enlargement (diameter and height) in the proximal tubules. In the early stage, topiroxostat and febuxostat decreased renal XOR activity in response to increased purine body levels (topiroxostat 3 mg/kg/day, 2.25 ± 0.19 mg/g tissue, p=0.07; febuxostat 3 mg/kg/day, 2.34 ± 0.09 mg/g tissue, p<0.05 to vehicle 2.01 ± 0.12 mg/g tissue, for 7 days) relative to the vehicle. Moreover, febuxostat upregulated the gene expression of renin 1 (Ren1) and downregulated that of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HPRT) compared with that of the diabetic control. Conclusion: Topiroxostat and febuxostat exhibited renoprotective effects by suppressing oxidative stress and inflammation, and enhancing the salvage pathway via XOR inhibition.

Vitamin D improves diabetic nephropathy in rats by inhibiting renin and relieving oxidative stress

It is well established that the activation of the renin-angiotensin system (RAS) and the oxidative stress caused by hyperglycemia are major mediators of the development and progression of diabetic nephropathy (DN). Vitamin D may be important in maintaining podocyte health, preventing epithelial-to-mesenchymal transformation, and suppressing renin gene expression and inflammation, but its mechanism requires clarification. This study evaluated the specific mechanism of vitamin D to DN improvement. We induced a rat model of diabetes with an intraperitoneal injection of streptozotocin (60mg/kg). The streptozotocin-induced diabetic rats were fed normal chow for about 2months to induce the DN model. The DN rats were then treated with irbesartan and/or calcitriol, administered intragastrically about 1month. The rats displayed the early characteristics of DN, including increased microalbuminuria, obvious hypertrophic kidney, and a markedly increased kidney weight/bodyweight ratio. Vitamin D inhibited the compensatory increase in renin expression. Malondialdehyde, which reflects oxidative stress levels, was elevated in the DN group rats and their antioxidant capacity was significantly reduced. The irbesartan and calcitriol interventions markedly improved the renal pathology and pathophysiological changes. Irbesartan combined with vitamin D (calcitriol) more effectively ameliorated DN than irbesartan alone. Vitamin D combined with angiotensin II type 1 receptor blockers exerts a synergistic effect on the treatment of DN, not only by inhibiting renin but also by reducing oxidative stress and increasing the renal antioxidant capacity.

Antihypertensive and cardioprotective effects of the dipeptide isoleucine-tryptophan and whey protein hydrolysate.

Angiotensin-converting enzyme inhibitors are treatment of choice in hypertensive patients. Clinically used inhibitors exhibit a structural similarity to naturally occurring peptides. This study evaluated antihypertensive and cardioprotective effects of ACE-inhibiting peptides derived from food proteins in spontaneously hypertensive rats. Isoleucine-tryptophan (in vitro IC50 for ACE = 0.7 μm), a whey protein hydrolysate containing an augmented fraction of isoleucine-tryptophan, or captopril was given to spontaneously hypertensive rats (n = 60) over 14 weeks. Two further groups, receiving either no supplement (Placebo) or intact whey protein, served as controls. Systolic blood pressure age-dependently increased in the Placebo group, whereas the blood pressure rise was effectively blunted by isoleucine-tryptophan, whey protein hydrolysate and captopril (-42 ± 3, -38 ± 5, -55 ± 4 mm Hg vs. Placebo). At study end, myocardial mass was lower in isoleucine-tryptophan and captopril groups but only partially in the hydrolysate group. Coronary flow reserve (1 μm adenosine) was improved in isoleucine-tryptophan and captopril groups. Plasma ACE activity was significantly decreased in isoleucine-tryptophan, hydrolysate and captopril groups, but in aortic tissue only after isoleucine-tryptophan or captopril treatment. This was associated with lowered expression and activity of matrix metalloproteinase-2. Following isoleucine-tryptophan and captopril treatments, gene expression of renin was significantly increased indicating an active feedback within renin-angiotensin system. Whey protein hydrolysate and isoleucine-tryptophan powerfully inhibit plasma ACE resulting in antihypertensive effects. Moreover, isoleucine-tryptophan blunts tissue ACE activity, reduces matrix metalloproteinase-2 activity and improves coronary flow reserve. Thus, whey protein hydrolysate and particularly isoleucine-tryptophan may serve as innovative food additives with the goal of attenuating hypertension.

A Genetic Variant in the Distal Enhancer Region of the Human Renin Gene Affects Renin Expression.

BackgroundThe high heritability of plasma renin activity was confirmed in recent investigations. A variation located near the strong enhancer of the human renin gene (REN), C-5312T, has been shown to have different transcription activity levels depending on its allele: the 5312T allele shows transcription levels that are 45% greater than those of the 5312C allele. The purpose of this study was to confirm the hypothesis that variations in the enhancer region of the REN gene are involved in regulating renal expression of renin.MethodsSixty-four subjects with biopsy-proven renal diseases were included in this study (male/female: 35/29, age 41.9 ± 20.9 years, SBP/DBP 123.1 ± 23.7/73.4 ± 14.8 mmHg, s-Cr 0.93 ± 0.63 mg/dl). A genetic variant of REN, C-5312T, was assayed by PCR-RFLP and the TaqMan method. Total RNAs from a small part of the renal cortex were reverse-transcribed and amplified for REN and GAPDH with a real-time PCR system.ResultsLogarithmically transformed expression values of the relative ratio of REN to GAPDH (10−3) were as follows (mean ± SE): CC (26 cases), 0.016 ± 0.005; CT (33 cases), 0.047 ± 0.021 (p = 0.41 vs. CC); TT (5 cases), 0.198 ± 0.194 (p = 0.011 vs. CC, p < 0.031 vs. CT). Thus, significant differences in REN expression were observed among the genetic variants.ConclusionThe results suggest that variants in the enhancer region of the human renin gene have an effect on the expression levels of renin in renal tissue; this observation is in good accordance with the results of the transcriptional assay.

Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P&lt;0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P&lt;0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P&lt;0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P&lt;0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.

Calcitriol ameliorates renal damage in a pre-established proteinuria model.

Proteinuria is critical in the tubulointerstitial changes that ultimately lead to renal insufficiency. Increased protein filtration has direct toxic effects on tubular epithelial cells, leading to epithelial mesenchymal transition (EMT) to a myofibroblast phenotype. Angiotensin II and transforming growth factor (TGF)-β1 are the main mediators of EMT. Calcitriol may exert a potential renoprotective effect by reducing the activity of the renin angiotensin system by suppressing renin gene expression and also by inhibiting the proinflammatory nuclear factor-κB pathway. The present study investigated the benefits of calcitriol treatment in a puromycin-induced protein-uric nephropathy model. Uninephrectomized adult male Wistar rats received intraperitoneal administration of a single dose of puromycin (100 mg/kg) or vehicle. After eight weeks, the animals were divided into two groups and received vehicle or calcitriol (0.5 μg/kg) for four weeks. The vehicle-treated, proteinuric rats developed progressive proteinuria and tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and fibrosis were significantly ameliorated by calcitriol treatment. Calcitriol was effective in preventing an increase in the EMT markers, α-smooth muscle actin and fibroblast-specific protein 1, reducing macrophage infiltration as evidenced by levels of ED-1. In addition, calcitriol increased the anti-inflammatory cytokine interleukin-10 and reduced the pro-oxidant p47 phox enzyme. These effects were paralleled by a reduction in TGF-β/Smad3 expression. Calcitriol may have therapeutic potential in the proteinuric nephropathy model used in the present study by inhibiting the TGF-β1 axis.

Genetic Variant of C-5434T REN Enhancer on Serum Renin Levels and Binding Pattern of Signal Transducers and Activators Transcription 3.

The human renin gene has been widely known to be involved in essential hypertension (EH) pathogenesis. Genetic variant C-5434T of REN enhancer contributed to renin gene transcription and serum renin regulation. However, the mechanism associated with the transcription level changes remains unknown, and only a few reports exist that discussed serum renin levels on C-5434T of REN. Thus, this study aims to investigate the relationship between genetic variant C-5434T of REN enhancer and serum renin levels in Indonesian hypertensive patients. SNP of C-5434T was genotyped in 56 hypertensive patients by using RFLP. The data showed that serum renin is slightly higher in hypertensive patients with the TT genotype (39 ± 10.3) than patients with the CC genotype (33 ± 10.6) but the difference was not statistically significant (p = 0.35). Here, we also present a docking approach for predicting interaction between genetic variant -5434C/T and STAT3 (Signal Transducers and Activators Transcription 3), the predicted transcription factor that regulates renin gene enhancer. The results showed that STAT3-DNA allele T more favorably binds to DNA than STAT3-DNA allele C. These data suggest that the presence of genetic variant C-5434T has changed the binding pattern of STAT3 to REN enhancer. This is likely to influence STAT3 activity to stimulate the expression of renin gene in producing renin.

Abstract 080: Selective Knockout of the Intracellular Isoform of Renin in the Brain Contributes to Metabolic and Cardiovascular Control

Renin gene expression is regulated by two distinct promoter-first exon combinations that target renin for either secretion (exon 1a initiating secreted renin, sREN) or for cytoplasmic retention (exon 1b initiating intracellular renin, icREN). The icREN isoform is expressed predominantly in the brain and its expression is downregulated whereas sREN expression is upregulated by deoxycorticosterone (DOCA)-salt suggesting each isoform may be differentially regulated. We generated mice that lack icREN, but preserve sREN, by flanking exon-1b and its surrounding sequences (including the promoter) with loxP sites and breeding successively with mice expressing flipase and cre-recombinase. Real time quantitative RT-PCR analysis revealed a loss of icREN mRNA in the brain, but a preservation of sREN mRNA in the kidney. Total body weight was normal in male icREN-KO mice (26.3±0.7 g, n=8, 12 wk) compared to controls (25.3±0.7 g, n=12, 12 wk), but total (2.14±0.09 g vs 2.46±0.11 g) and relative (9.0±0.5 % vs 10.7±0.6%) fat mass as measured by NMR were reduced in 8-week icREN-KO mice (n=14-16, P&lt;0.05). Whereas food intake was normal, there was an increase in resting metabolic rate as measured by respirometry in icREN-KO (0.1484±0.0036 kcal/hr, n=41, P=0.02 by ANCOVA) vs controls (0.1389±0.0038 kcal/hr, n=38). There was also a trend toward decreased digestive efficiency (81.1±1.0%, n=6, P=0.06) vs controls (84.9±1.2%, n=3) at 22 wk of age. Systolic blood pressure was decreased in icREN-KO mice (113±1 mmHg, n=12, 17.0±1.2 wk vs 120±3 mmHg, n=5, 15.9±1.9 wk, P&lt;0.05). We previously reported a functional link between the synthesis and action of angiotensin-II and the action of leptin. We tested if this was impaired in icREN-KO mice by measuring the renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular (ICV) administration of leptin. The decrease in arterial pressure was confirmed in this cohort of icREN-KO mice. Surprisingly, the RSNA response to leptin was greater in icREN-KO mice compared to controls (257.9±16.0%, n=3 vs 157.0±13.9%, n=4, 4 hr after leptin, P=0.01). Captopril attenuated the sympathetic response to ICV leptin. Together these data suggest that this novel icREN isoform contributes to metabolic and cardiovascular control.

Parallel changes in the urinary excretion of ANG II and angiotensinogen and kidney ANG II levels in slowly progressive ANG II‐dependent hypertension (1136.20)

Previous studies have demonstrated that the urinary excretion of ANG II and AGT, and kidney ANG II levels are markedly elevated in ANG II‐dependent malignant hypertension associated with marked renal injury. The present study was performed to determine the temporal changes in urinary ANG II and AGT excretion and kidney ANG II levels in a model of slowly progressive ANG II‐dependent hypertension in Cyp1a1‐Ren2 transgenic rats with inducible expression of the Ren2 renin gene. Male Cyp1a1‐Ren2 rats (n=6/group) were induced to develop slowly progressive ANG II‐dependent hypertension by dietary administration of indole‐3‐carbinol (I3C, 0.15% wt/wt) for 14 days. Conscious systolic blood pressures (SBP) increased from 140±8 to 176±9 mmHg (P&lt;0.05) by day 4 of I3C induction, whereas the urinary excretion of ANG II and AGT both increased by day 3 of I3C induction (32.1±3 to 64.8±6 fmol/hr and from 123±25 to 437±75 ng/day, P&lt;0.05 in both cases). Both urinary ANG II and AGT excretion continued to increase (P&lt;0.01) and averaged 120±5 fmol/hr and 1384±514 ng/day, respectively, by day 13 of induction. Similarly, kidney ANG II levels were increased by day 13 of induction (1042±219 vs. 349±96 fmol/g, P&lt;0.05). SBP, urinary ANG II and AGT excretion and kidney ANG II levels promptly decreased to control levels following termination of dietary I3C administration (to 127±2 mmHg, 60±5 fmol/hr, 83±34 ng/day, and 473±185 fmol/g, respectively, P&lt;0.01 in all cases). The present findings demonstrate that the urinary excretion of ANG II and AGT increases progressively, and that total kidney ANG II levels are increased, following induction of ANG II‐dependent hypertension and decrease within 3 days to control levels following termination of dietary I3C administration. The data also indicate that the intratubular generation of ANG II increases progressively following induction of ANG II‐dependent hypertension and decreases promptly to control levels following termination of induction of ANG II‐dependent hypertension.Grant Funding Source: 5P30GM103337

English

Renin distal enhancer plays a pivotal role in renin gene expression, and the genetic variants C-5312T of renin enhancer can affect renin gene transcription level. However, the mechanism associated with the transcription level changes remains unknown. Therefore, it is of interest to investigate the possible role of distal enhancer in regulating the expression of renin gene. Single nucleotide polymorphism in renin distal enhancer was identified in 34 hypertensive patients by automatic sequencing. The data showed that the renin enhancer from the patients have genetic variants C-5312T or C-5312T SNP. Hence, the functionality of the renin enhancer and influence of the genetic variants C-5312T on binding to Sp1 is studied. These results from the binding study suggested that Sp1 binds to the DNA in GC rich region. Thus, the genetic variant C-5312T has changed the binding pattern of Sp1 to renin enhancer. This is likely to influence Sp1 activity to stimulate the expression of renin gene. The binding of Sp1 to the cis-element will enhance transcription of renin gene. Thus, polymorphism within C-5312T might contribute to the reduction of renin transcription.

Abstract 191: Transcriptional Regulation of Renin by Nuclear Receptors Co-regulated With Renin

Expression of the renin gene is required to maintain normal morphological and physiological identity of renal juxtaglomerular (JG) cells, yet the mechanisms regulating renin gene transcription remain elusive. We re-examined data from Brunskill et. al (JASN 22:2213, 2011), investigating genome-wide gene expression in JG and other renal cell types. Based on our previous data implicating nuclear receptors (RAR, RXR, VDR, PPARG, Nr2f2 and Nr2f6) in the regulation of mouse and human renin gene expression, we focused our analysis on the expression of genes encoding the 48 nuclear hormone receptors and their co-regulation with renin. Several nuclear receptors have an expression pattern emulating that of renin, that is, they were similarly enriched in JG cells but not in other cell types. These include Esr1, Nr1h4, Ppara, VDR, Nr1i2, Ppard, Hnf4g, Nr1h3, Thrb, Hnf4a, Esrrg, Nr4a3, Nr3c2, and Ar. We tested the hypothesis that a nuclear receptor that is co-regulated with renin may participate in renin gene regulation. To accomplish this, endogenous renin expression was evaluated in renin-expressing As4.1 cells after siRNA-mediated knock down of selected nuclear receptors. Each experiment included a negative control siRNA duplex (NC) that does not target any known genes. By way of example, siRNA-mediated inhibition of estrogen receptor alpha (Esr1) by 70-80% resulted in a 2-fold decrease in renin mRNA (fold change ± SEM: siEsr1: 0.4±0.2, p&lt;0.001 vs NC). Similar results were obtained with a different siRNA targeting Esr1. Interestingly, loss of Esr1 also caused up-regulation of vitamin D receptor (VDR, 2.8±0.7 fold, p&lt;0.001 vs NC) and Nr2f6 (2.0±0.2 fold, p&lt;0.05 vs NC), both of which are known to be negative regulators of renin. Similarly, both renin (0.1±0.02, p&lt;0.001 vs untreated) and Esr1 (0.3±0.1, p&lt;0.05 vs untreated) mRNA were reduced in the kidney from mice treated with deoxycorticosterone acetate (50mg) and receiving 0.15 M NaCl in drinking water for 21 days (DOCA-salt). These data suggest Esr1 may regulate renin expression. Studies are in progress to assess if Esr1 stimulates renin expression on its own or acts by affecting the level of other nuclear receptors; and to determine if other co-regulated nuclear receptors also regulate expression of the renin gene.

Abstract 16: Physiological Characterization of a Selective Knockout of the Intracellular Isoform of Renin in the Brain of Mice

Renin gene expression is regulated by two distinct promoter-first exon combinations that target renin for either secretion (exon 1a initiating secreted renin, sREN) or for cytoplasmic retention (exon 1b initiating intracellular renin, icREN). The icREN isoform is expressed predominantly in the brain and its expression is downregulated whereas sREN expression is upregulated by deoxycorticosterone (DOCA)-salt suggesting each isoform may be differentially regulated. We generated mice that conditionally lack icREN, but preserve sREN, by targeting a novel renin allele in ES cells by flanking exon-1b and its surrounding sequences (including the promoter) with loxP sites. Mice carrying this allele were first bred with flippase mice to remove the neo cassette and then with cre-recombinase mice to generate the null allele lacking icREN. The structure of the final allele was confirmed by Southern blot. There was no apparent lethality in the homozygous mice when heterozygous mice were intercrossed. Real time quantitative RT-PCR analysis revealed a loss of icREN mRNA in the brain, but a preservation of sREN mRNA in the kidney. Male icREN-KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P&lt;0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN-KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Studies are ongoing to assess arterial pressure. Together these data suggest that this novel icREN isoform contributes to metabolic and possibly to cardiovascular control. Future studies using the conditional allele (icREN-flox) will provide an opportunity to dissect the neural circuits involved in these responses.

Maternal vitamin D deficiency programmes adult renal renin gene expression and renal function.

Renin is essential for renal development and in adult kidneys vitamin D deficiency increases renin gene expression. We aimed to determine whether maternal vitamin D deficiency upregulates fetal renal renin expression, and if this is sustained. We also examined growth and the long-term renal effects in offspring on a normal diet. Female Sprague-Dawley rats in UVB-free housing were fed either vitamin D deficient chow (DEF) or normal chow from 4 weeks and mated with vitamin D replete males at 10 weeks. Fetuses were collected at E20 or dams littered and the pups were weaned onto normal chow. Kidney mRNA levels for renin, (pro)renin receptor [(P)RR], transforming growth factor β 1 (TGF-β1), and nephrin were determined in E20 fetuses and in male offspring at 38 weeks. Renal function was assessed at 33 weeks (24 h, metabolic cage) in both sexes. Renal mRNA expression was upregulated for renin in fetuses (P < 0.05) and was almost doubled in adult male offspring from DEF dams (P < 0.05). Adult males had reduced creatinine clearance, solute excretion and a suppressed urinary sodium-to-potassium ratio (P < 0.05). Female adult DEF offspring drank more and excreted more urine (P < 0.05) but creatinine clearance was not impaired. We conclude that maternal vitamin D depletion upregulates fetal renal renin gene expression and this persists into adulthood where, in males only, there is evidence of sodium retention and compromised renal function. Importantly these effects occurred despite the animals being on a normal diet from the time of weaning onwards.

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.

Angiotensin II Stimulates Renin Synthesis and Secretion in Mouse Collecting Duct M‐1 cells via a PKC alpha‐mediated cAMP Stimulation Mechanism

To test the hypothesis that Ang II stimulates renin synthesis and secretion in collecting duct (CD) cells via activation of PKC and subsequent stimulation of cAMP/PKA/CREB pathway, we treated mouse cortical CD (M‐1) cells with Ang II (10−7M). Ang II treatment for 6‐h increased intracellular renin gene expression and renin content in culture media (Ang II: 31±4 vs. 19±4 ng Ang I/hr/ml; p&lt;0.05). Likewise, this treatment increased intracellular cAMP levels by 1 min (148±32 pmol/mg protein) which reached a peak at 30 min (290±27 vs. 44±0 pmol/mg protein; p&lt;0.05). The adenlyate cyclase activator, forskolin and the analogue of cAMP, dibutyryl‐cAMP both significantly increased renin gene expression. These responses were followed by increases in pCREB levels and prevented by PKA inhibition with H89 or PKI. PKC inhibition either with calphostin C or a PKCα dominant negative prevented the augmentation of intracellular levels of cAMP, pCREB and renin mRNA in Ang II‐treated M‐1 cells. Ang II stimulates renin synthesis and secretion in M‐1 cells via PKCα‐mediated cAMP accumulation and PKA/CREB activation. By this mechanism distal tubular renin may contribute to local Ang II generation, thus to activation of sodium reabsorption.

Selective knockout of novel intracellular renin isoform results in reduced adiposity

Renin gene expression is regulated by two distinct promoter / first exon combinations that target renin for either secretion (exon 1a) or for cytoplasmic retention (exon 1b). Renin‐1b is expressed predominantly in the brain and is differentially regulated from renin‐1a in response to stimuli such as deoxycorticosterone (DOCA)‐salt. Mice were generated that lack exon 1b (and thereby intracellular renin, icREN‐KO) but retain exon 1a. Male icREN‐KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P&lt;0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN‐KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Together these data suggest that the novel icREN isoform contributes to metabolic control.

Myocardial AT1 gene expression is increased in a model of left ventricular hypertrophy induced by high salt intake.

ObjectiveTo evaluate gene expression of the renin‐angiotensin system components in a model of left ventricular hypertrophy induced by high salt intake demonstrated in a previous study.MethodsWistar rats were fed normal (NS1.3% NaCl) or high (HS 8%) salt diet since weaning. From the 7th week of age, two HS subgroups received hydralazine or losartan (HZ 15 and LOS 20mg/kg/day). Angiotensinogen, renin, ACE, ACE2, AT1 and AT2 gene expression in left ventricle were measured.Results (n=8/group) NS HS HS+HZ HS+LOS Angiotensinogen/GAPDH (IDV) 0.21±0.017a 0.19±0.017a 0.21±0.013a 0.19±0.024a Renin/GAPDH (IDV) 0.71±0.02a 0.68±0.02a 0.67±0.02a 0.67±0.02a ACE/GAPDH (IDV) 0.87±0.03a 0.83±0.05a 0.97±0.06a 097±0.05 a ACE2/GAPDH (IDV) 0.41±0.02a 0.43±0.02a 0.37±0.03a 0.34±0.02a AT1/GAPDH (IDV) 0.23±0.00a 0.46±0.06b 0.45±0.06b 0.22±0.00a AT2/GAPDH (IDV) 0.52±0.04a 0.51±0.03a 0.45±0.03a 0.44±0.02a Data are reported as mean±SEM. Means in row with superscripts without a common letter differ, P&lt;0.05 ConclusionHigh salt intake increases AT1 gene expression, however it is prevented by losartan treatment. Supported by FAPESP and CNPq.

Aberrant Activation of the Intrarenal Renin-Angiotensin System in the Developing Kidneys of Type 2 Diabetic Rats

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.

Nitric Oxide Can Directly Mediate Renin Cell Recruitment

See related article, pp 400–407 In this issue of Hypertension , Neubauer et al1 fit missing pieces into the puzzle of how renin production is controlled, when the demand for renin is high. Our understanding of the acute release of renin is much more complete than what we know about the long-term adaptations to stimuli of renin. Our scarce knowledge of the mechanisms mediating long-term renin stimulation is remarkable with regard to the clinical importance of renin. Hypertension, fluid and electrolyte homeostasis, as well as inflammation and fibrosis all may involve chronic renin stimulation.2 More than 400 million years ago, renin first appeared in organisms,3 and renin is often seen as the first hormone discovered. Shortly before the 20th century, Tigerstedt and Bergman showed that kidney extracts can elevate blood pressure, when intravenously infused.4 Today, we recognize that the actions of renin go far beyond that of controlling blood pressure, and we have detailed insight into how renin is released into the circulation.5 The major stimuli of short-term renin release are blood pressure decreases, β1-adrenergic stimulation, and low salt, whereas …