Renin mRNA Levels Research Articles

Abstract P434: Piezo2 Channels in JG cells do not Regulate Renin Expression or Renin Release to the Circulation

Kidneys are central to blood pressure regulation through the renin-angiotensin system. Renin-producing juxtaglomerular (JG) cells act as renal baroreceptors by controlling renin release according to the changes in perfusion pressure. Recent studies suggest that Piezo2 calcium channels may regulate the baroreceptor mechanism of renin release. However, our single-cell RNAseq and RNAScope analysis on adult kidneys showed a very low expression for Piezo2 in the JG cells. Since it is unclear if Piezo2 in the JG cells regulates renin expression and release, we defined the in vivo role of Piezo2 in the current study using Piezo2 conditional mutant mice. For this we generated Piezo2 fl/fl ;Ren1 d Cre, Piezo2 fl/del ;Ren1 d Cre and Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice. Though we detected an efficient deletion of Piezo2 by RNAScope in the JG cells of the mutant kidneys, we did not observe a significant difference in the renin signals between the control and the mutant groups by renin immunostaining. q-RT-PCR of the kidney cortices to measure renin mRNA levels and renin-specific ELISA to quantify plasma renin levels also indicated no significant differences (NS) between the control and the mutant groups ( Piezo2 fl/fl ;Ren1 d Cre - Relative renin mRNA levels: Control (n=11) 1.05±0.113 Mutants (n=6) 0.90±0.20, NS; Plasma renin levels (pg/ml): Control (n=11) 17472±3712 Mutants (n=6) 14934±3547, NS; Piezo2 fl/del ;Ren1 d Cre - Relative renin mRNA levels: Control (n=3) 0.98±0.13 Mutants (n=4) 1.04±0.19, NS; Plasma renin levels (pg/ml): Control (n=3) 20221±4303 Mutants (n=4) 15014±1901, NS). Furthermore, subjecting the mice to hypotension and volume depletion with captopril and a low salt diet for one week did not affect the renin expression due to Piezo2 deletion in the renin cells. Finally, our results from the aortic coarctation surgery model showed that Piezo2 deletion in the adult JG cells of Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice did not affect the changes in renin expression between the left and right kidneys in response to perfusion pressure (Relative renin mRNA levels: Control (n=11) LK-1.102 ±0.173 RK-0.316±0.046, p=0.002 Mutants (n=9) LK-1.191±0.266 RK-0.524±0.212, p=0.05). Collectively, our in vivo data shows that renin expression is unaffected during normal conditions and when homeostasis is threatened by hypotension, sodium depletion, or changes in blood pressure, indicating that Piezo2 channels are not necessary for renin synthesis and release to circulation in JG cells.

Abstract 084: Smooth Muscle Cullin-3 Regulates The Renal Baroreceptor Mechanism Via Lamin AC

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. Patients carrying mutations in CUL3 exhibit early onset of hypertension (HTN). We demonstrated that conditional ablation of CUL3 in vascular smooth muscle cells (VSMCs) results in vascular dysfunction, arterial stiffness, and severe HTN in mice. We hypothesized that 1) conditional deletion of CUL3 in VSMCs (S-CUL3KO) results in severe HTN via the renin-angiotensin system (RAS)-dependent mechanism, and 2) S-CUL3KO mice exhibit an impaired baroreceptor mechanism regulating renin. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited elevated systolic blood pressure (SBP) 3 weeks after tamoxifen administration (S-CUL3KO: 164±1 vs. ISM-CRE: 124±4 mmHg, p<0.05). To test the first hypothesis captopril or candesartan (10 mg/kg/day and 100 mg/kg/day in drinking water, respectively) were administered to S-CUL3KO once severe HTN was already established. Captopril or candesartan treatment for 1 week reversed elevated SBP to 117±4 mmHg (p<0.05) and 127±2 mmHg (p<0.05), respectively, in S-CUL3KO. Despite the severity of HTN in S-CUL3KO, the level of renin mRNA in the kidney was surprisingly unaltered (p>0.05) when compared to the normotensive control mice. The protein expression of lamin AC, a crucial component of the nuclear mechanotransducer controlling renin expression, was significantly increased in S-CUL3KO kidneys (0.11±0.02 vs. 0.05±0.01 normalized RFU, p<0.05). This suggests lamin AC may be a target protein of CUL3-ubiquitin ligase complex. Supporting this concept, treatment of A10 smooth muscle cells with MLN4914, an inhibitor of the Cullin pathway, results in a 2-fold increase in lamin AC expression (0.007±0.001 vs. 0.0154±0.0004, normalized RFU, p<0.05). Furthermore, co-immunoprecipitation of CUL3 in HEK293 cells immunoprecipitated lamin AC. Our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor mechanism and 2) lamin AC is a potential target of the CRL3-mediated regulation. Understanding CUL3 target proteins might help the development of new strategies to mitigate HTN.

Abstract P221: Soluble (pro)renin Receptor Contributes To 2-kidney, 1-clip-induced Renovascular Hypertension And Ischemic Nephropathy In Mice

The Goldblatt two-kidney, one-clip (2K1C) model features renovascular hypertension and ischemic nephropathy due to overactivated renin-angiotensin system (RAS). Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease (S1P) and implicated as a potential regulator of the RAS and renal function. Here, we examined the function and underlying mechanism of sPRR in 2K1C model by pharmacological inhibition of S1P and genetic mutagenesis of S1P cleavage site. Male 2-mo-old C57BL/6 mice were subjected to the 2K1C procedure or sham operation for 1 month, followed by a 14-day infusion of vehicle or a specific S1P inhibitor PF429242 (PF) (30 mg/kg/day via minipump). The 2K1C procedure induced a 2-fold increase in plasma sPRR, which was reduced by 50% following PF treatment. This was paralleled by improvement of renovascular hypertension (MAP:142.1±1.3 vs. 129.9±2.8mmHg, n=7, p < 0.05) and cardiac hypertrophy (HW/BW:6.3±0.3 vs. 5.0 ± 0.3mg/g, n=7, p < 0.01) accompanied with suppressed cortical and medullary renin activity, active renin content, prorenin content and total renin content in clipped kidney. Meanwhile, cortical and medullary renin mRNA level in clipped kidney was increased 3.8-fold and 5.3-fold, which was reduced by 41% and 55% respectively, following PF treatment. The level of urinary albumin, KIM-1 and NGAL was elevated 5.1-fold, 4.0-fold and 5.7-fold after 2K1C surgery, which was blunted 35%, 49% and 59%, respectively, following PF treatment. Secondly, we employed a novel mouse model with mutagenesis of the cleavage site in PRR (termed as Mutant mice) to verify the role of endogenous sPRR in 2K1C model. Similarly, the hypertensive response and ischemic nephropathy of Mutant mice to 2K1C procedure was blunted (24-day MAP: 2K1C/WT 142.5±2.1 vs. 2K1C/Mutant 128.2±3.2mmHg; 24-day urinary albumin/creatinine: 2K1C/WT 64.9±7.2 vs. 2K1C/Mutant 33.0±1.9mg/g, n=6, p < 0.05), in parallel with attenuated response of systemic and intrarenal RAS. Together, consistent results with the different approaches have established an important role of S1P-derived sPRR in regulation of the RAS and thus the pathogenesis of 2K1C-induced renovascular hypertension and ischemic nephropathy.

Abstract P250: Targeting The Thrombospondin-CD47 Axis Attenuates Blood Pressure And Renal Injury In Female Hypertensive (mRen2)27 Rats

Alterations in the extracellular matrix (ECM) can influence vascular stiffness and compromise organ function contributing to hypertension. A key component of the ECM is the type I integral membrane receptor CD47 that regulates the ECM by binding to the matricellular protein thrombospondin-1(TSP1). Increased circulating levels of TSP1 and CD47 activation are associated with cardiac and renal pathologies. Thus, the objective of the current study determined the efficacy of targeting CD47 with an anti-sense morpholinos to reduce blood pressure and renal injury in 20-week old hypertensive (mRen2)27 female rats. The CD47 morpholino (Morph) or the missense probe (Control) were dissolved in sterile PBS and administered by tail vein injection twice weekly for 6 weeks. Systolic blood pressures (SBPs) were monitored in conscious rats by a tail cuff auscultation and expression of RAS components assessed by RT-qPCR; all data are means ± SEM with n=8-10 per group. Prior to treatment, the baseline SBPs did not differ between the Control and Morph groups [196 ± 5 vs.197 ± 4 mmHg; p=0.87]. However, as early as one week of treatment, the CD47 Morph group exhibited a significant reduction in SBPs compared to the Control transgenics [185 ± 5 vs. 204 ± 3 mmHg; p<0.01]. At the end of the 6 week treatment period, SBPs remained significantly lower in the CD47 Morph group compared to the Controls [185 ± 4 vs.199 ± 3 mmHg; p<0.02]. In addition, CD47 blockade was associated with reduced proteinuria in the CD47 Morph compared to Controls [7 ± 1 vs. 26 ± 7 mg protein/24 hr excretion; p<0.05]. Regarding the mechanism of CD47 blockade to reduce blood pressure and proteinuria, mRNA analysis revealed lower renal renin mRNA levels in the CD47 Morph versus the Control group [0.54 ± 0.11 vs.1.04 ± 0.11; p<0.01]. Moreover, CD47 Morph treatment significantly reduced the mRNA levels of renal ACE [0.78 ± 0.13 vs.1.05 ± 0.14; p<0.05] and the renal AT1A receptor [0.74 ± 0.07 vs.1.05 ± 0.12; p<0.05], however, renal angiotensinogen mRNA levels were not different among the CD47 Morph and Controls [1.04.± 0.10 vs. 1.16 ± 0.20, p=0.83]. We conclude that silencing CD47 attenuates hypertension and renal injury in the female (mRen2)27 potentially by reducing expression of the renin-ACE-AT1 receptor axis within the kidney.

The role of mineralocorticoid receptor in the potassium switch: effect on NCC regulation+

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

Uncovering a sex‐specific role for olfactory receptor 558 (Olfr558) in blood pressure regulation

Olfactory receptors are primarily studied in the nose; but are also expressed and play pivotal roles in non‐olfactory tissues as ecnomotopic receptors. We find that olfactory receptor 558 (Olfr558) is expressed in vascular smooth muscle cells in the kidney, including the renal afferent and efferent arterioles. We developed Olfr558 whole‐body knockout (KO) mice to test the hypothesis that Olfr558 plays a role in blood pressure (BP) regulation. Olfr558 wild‐type (WT) vs. KO (males and females) exhibited no genotypic differences in body weight, kidney weight/body weight, heart weight/body weight, glomerular filtration rate, or blood values (electrolytes, glucose, blood urea nitrogen, creatinine, and hemoglobin). However, Olfr558 KO males had lower diastolic blood pressure (DBP) (81.1±1.6 mmHg, n=9, p=0.009) than WT males (89.1±2.2 mmHg, n=7) during the light cycle (telemetry) without a change in mean arterial (MAP) or systolic blood pressure (SBP). In contrast, Olfr558 KO females had increased MAP (KO: 95.0±1.7 mmHg, WT: 89.0±0.9 mmHg, p=0.003), SBP (KO: 108.0±1.7 mmHg, WT: 102.1±1.4 mmHg, p=0.02), and DBP (KO: 81.6±1.6 mmHg, WT: 75.0±0.9 mmHg, p=0.003) during the light cycle (n=8 for WT and KO). A similar trend was observed during the dark cycle. These data suggest that Olfr558 regulates BP in a sex‐specific manner.To determine the mechanisms underlying the sexually dimorphic effect of Olfr558 on BP regulation, we are examining renin signaling and vascular tone. In males, kidney renin mRNA levels were significantly decreased in Olfr558 KO (0.43±0.04, n=12, p=0.001 vs WT 1±0.14, n=11). Akr1b7 (renin cell marker) mRNA was also reduced in male KO (0.5±0.06, n=12, p=0.002, vs WT 1±0.09, n=10). We then evaluated the % of renin positive to total glomeruli and found that it was reduced in male KO (29.0±0.95, p=0.004, vs WT 43.2±1.88; n=8). We also found that plasma renin activity (PRA) was dramatically decreased in male KO (200.8±20.5, n=11, p=0.001) than WT (401.9±15.2, n=10). These results suggest that Olfr558 regulates BP in males at least in part via renin. In females, there were no differences in renin or Akr1b7 mRNA, nor in the % of renin positive glomeruli, nor PRA. These results suggest that, in females, Olfr558 modulates BP independent of renin. Studies of ex vivo vascular reactivity in males show that aortic rings exhibited less constriction to Phenylephrine (PE) in KO vs WT, which could contribute to the hypotension seen in these mice. In contrast, constriction to KCl and relaxation to Acetylcholine (ACh) and sodium nitroprusside (SNP) showed no genotypic difference. KO mesentery exhibited more relaxation to SNP, but not ACh, and similar constriction to KCl and PE as WT. In females, KO aortic rings exhibited more constriction to KCl, but not PE, and similar relaxation to ACh and SNP as WT. However, mesentery showed no genotypic difference in constriction to KCl and PE, relaxation to ACh and SNP. Thus, our findings suggest that the hypotension seen in Olfr558 KO males is likely contributed to both by decreased renin and by changes in vascular reactivity, whereas the origin of hypertension seen in Olfr558 KO females is still an area of active investigation.

Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and it is a known risk factor for hypertension. The upregulation of the renin-angiotensin system has been reported in IH, and the correlation between renin and CD38 has been noted. We exposed human HEK293 and mouse As4.1 renal cells to experimental IH or normoxia for 24 h and then measured the mRNA levels using a real-time reverse transcription polymerase chain reaction. The mRNA levels of Renin (Ren) and Cd38 were significantly increased by IH, indicating that they could be involved in the CD38-cyclic ADP-ribose signaling pathway. We next investigated the promotor activities of both genes, which were not increased by IH. Yet, a target mRNA search of the microRNA (miRNA) revealed both mRNAs to have a potential target sequence for miR-203. The miR-203 level of the IH-treated cells was significantly decreased when compared with the normoxia-treated cells. The IH-induced upregulation of the genes was abolished by the introduction of the miR-203 mimic, but not the miR-203 mimic NC negative control. These results indicate that IH stress downregulates the miR-203 in renin-producing cells, thereby resulting in increased mRNA levels of Ren and Cd38, which leads to hypertension.

Angiotensin II up-regulates sodium-glucose co-transporter 2 expression and SGLT2 inhibitor attenuates Ang II-induced hypertensive renal injury in mice.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P<0.001). In vitro, angiotensin II (Ang II) dose-dependently stimulated SGLT2 expression in HK-2, human immortalized renal proximal tubular cells (RPTCs); losartan and antioxidants inhibited it. Sglt2 expression was increased in transgenic (Tg) mice specifically overexpressing Agt in their RPTCs, as well as in WT mice with a single subcutaneous injection of Ang II (1.44 mg/kg). Moreover, Ang II (1000 ng/kg/min) infusion via osmotic mini-pump in WT mice for 4 weeks increased systolic blood pressure (SBP), glomerulosclerosis, tubulointerstitial fibrosis, and albuminuria; canaglifozin (Cana, 15 mg/kg/day) reversed these changes, with the exception of SBP. Fractional glucose excretion (FeGlu) was higher in Ang II+Cana than WT+Cana, whereas Sglt2 expression was similar. Our data demonstrate a link between intrarenal RAS and SGLT2 expression and that SGLT2i ameliorates Ang II-induced renal injury independent of SBP.

Anabolic steroid excess promotes hydroelectrolytic and autonomic imbalance in adult male rats: Is it enough to alter blood pressure?

AimThe present study investigated the effects of anabolic steroid (AS) excess on blood pressure regulation. MethodsMale Wistar rats were treated with nandrolone decanoate (AS) or vehicle (CTL) for 8 or 10 weeks. Saline (1.8%) and water intake were measured in metabolic cages. Urinary volume, osmolarity, Na+ and K+ concentrations, and plasma osmolarity were measured. The autonomic balance was estimated by heart rate variability at baseline or after icv injection of losartan. Cardiac function was assessed by echocardiography and ex vivo recordings. Myocardial collagen deposition was evaluated by Picrosirius-Red staining. Vascular reactivity and wall thickness were investigated in aortic sections. Blood pressure (BP) was assessed by tail-cuff plethysmography. Angiotensin II type I receptor (AT1R), renin, and mineralocorticoid receptor (MR) mRNA expression was measured in the kidneys and whole hypothalamus. ResultsAS group exhibited decreased urinary volume and Na+ concentration, while urinary K+ concentration, plasma osmolarity, and renal AT1R and renin mRNA levels were increased compared to CTL (p < 0.05). Water intake was increased, and saline intake was decreased in the AS group (p < 0.01). AS group exhibited increased low-frequency/high-frequency-ratio, while it was decreased by icv injection of losartan (p < 0.05) compared to baseline. Neither cardiac function nor vascular reactivity/morphology was affected by AS excess (p > 0.05). Ultimately, BP levels were not altered by AS excess (p > 0.05). ConclusionAS excess promoted hydroelectrolytic and autonomic imbalance but did not alter vascular or cardiac function/morphology.

Dietary Potassium Downregulates Angiotensin-I Converting Enzyme, Renin, and Angiotensin Converting Enzyme 2.

BackgroundThe importance of dietary potassium in health and disease has been underestimated compared with that placed on dietary sodium. Larger effort has been made on reduction of sodium intake and less on the adequate dietary potassium intake, although natural food contains much more potassium than sodium. The benefits of a potassium-rich diet are known, however, the mechanism by which it exerts its preventive action, remains to be elucidated. With the hypothesis that dietary potassium reduces renal vasoconstrictor components of the renin-angiotensin system in the long-term, we studied the effect of high potassium diet on angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2.MethodsSprague Dawley male rats on a normal sodium diet received normal potassium (0.9%, NK) or high potassium diet (3%, HK) for 4 weeks. Urine was collected in metabolic cages for electrolytes and urinary volume measurement. Renal tissue was used to analyze angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2 expression. Protein abundance analysis was done by Western blot; gene expression by mRNA levels by RT-qPCR. Renal distribution of angiotensin-I converting enzyme and renin was done by immunohistochemistry and morphometric analysis in coded samples.ResultsHigh potassium diet (4 weeks) reduced the levels of renin, angiotensin-I converting enzyme, and angiotensin converting enzyme 2. Angiotensin-I converting enzyme was located in the brush border of proximal tubules and with HK diet decreased the immunostaining intensity (P < 0.05), decreased the mRNA (P < 0.01) and the protein levels (P < 0.01). Renin localization was restricted to granular cells of the afferent arteriole and HK diet decreased the number of renin positive cells (P < 0.01) and renin mRNA levels (P < 0.01). High potassium intake decreased angiotensin converting enzyme 2 gene expression and protein levels (P < 0.01).No morphological abnormalities were observed in renal tissue during high potassium diet.The reduced expression of angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2 during potassium supplementation suggest that high dietary potassium intake could modulate these vasoactive enzymes and this effects can contribute to the preventive and antihypertensive effect of potassium.

Neural suppression of miRNA-181a in the kidney elevates renin expression and exacerbates hypertension in Schlager mice.

BPH/2J mice are a genetic model of hypertension with overactivity of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). BPH/2J display higher renal renin mRNA and low levels of its negative regulator microRNA-181a (miR-181a). We hypothesise that high renal SNS activity may reduce miR-181a expression, which contributes to elevated RAS activity and hypertension in BPH/2J. Our aim was to determine whether in vivo administration of a renal-specific miR-181a mimic or whether renal denervation could increase renal miR-181a abundance to reduce renal renin mRNA, RAS activity and hypertension in BPH/2J mice. Blood pressure (BP) in BPH/2J and normotensive BPN/3J mice was measured via radiotelemetry probes. Mice were administered miR-181a mimic or a negative control (1-25 nmol, i.v., n = 6-10) with BP measured for 48 h after each dose or they underwent renal denervation or sham surgery (n = 7-9). Injection of 5-25 nmol miR-181a mimic reduced BP in BPH/2J mice after 36-48 h (-5.3 ± 1.8, -6.1 ± 1.9 mmHg, respectively, P < 0.016). Treatment resulted in lower renal renin and inflammatory marker (TLR4) mRNA levels in BPH/2J. The mimic abolished the hypotensive effect of blocking the RAS with enalaprilat (P < 0.01). No differences between mimic or vehicle were observed in BPN/3J mice except for a higher level of renal angiotensinogen in the mimic-treated mice. Renal miR-181a levels that were lower in sham BPH/2J mice were greater following renal denervation and were thus similar to those of BPN/3J. Our findings suggest that the reduced renal miR-181a may partially contribute to the elevated BP in BPH/2J mice, through an interaction between the renal sympathetic nerves and miR-181a regulation of the RAS.

Susceptibility of Mice Lacking Renin‐b to Chronic Angiotensin II Infusion

In addition to the classical endocrine renin angiotensin system (RAS), local activity of the RAS has been detected in many tissues including the brain. The brain renin angiotensin system is known for its importance in blood pressure regulation and water homeostasis. The brain specific isoform of renin, termed renin‐b, has been proposed as a negative regulator of the brain renin‐angiotensin system (RAS) since the selective ablation of ren‐b (Ren‐b KO) in mice results in brain RAS disinhibition leading to blood pressure (BP) elevation. New cohorts of Ren‐b KO mice, however, do not exhibit elevated BP and the retrospective analysis of five previous cohorts indicated that Ren‐b KO model exhibits a high degree of variability in BP, which may be attributed to the susceptibility to different stressors. Importantly, Ren‐b KO (including recent cohorts) exhibit suppressed plasma renin and angiotensin II (Ang II) implying that suppression of the endocrine RAS might compensate for the elevation of the brain RAS activity in those Ren‐b KO mice exhibiting normal BP. Therefore, we hypothesized that Ren‐b KO are sensitized to exogenous systemic infusion of Ang II. Wildtype (WT) or Ren‐b KO were infused with either vehicle or Ang II (400 ng/kg/min; sc.) via osmotic minipumps for 4 weeks. Ang II significantly increased BP in both WT and Ren‐b KO to the same degree (WT+veh: 103±4 vs WT+Ang: 120±4 mmHg; p = 0.01 (n=10–12) and KO+veh: 104±3 vs KO+Ang: 118±5 mmHg; p = 0.03 (n=10–14)). Although the BP between Ren‐b KO and WT mice was elevated equally, Ren‐b KO exhibited enhanced Ang II‐induced dipsogenic response (WT+Ang: 3.7±0.3 vs KO+Ang: 4.6±0.4 ml/day; p = 0. 05; n=10–12) and exhibited a trend toward increase in levels of urinary aldosterone (WT+Ang: 14.6±1.4 vs KO+Ang: 17.8±1.7 mmHg) and copeptin, a marker of arginine vasopressin (WT+Ang: 11.5±2.6 vs KO+Ang: 22.2±5.2 mmHg). BP, drinking, aldosterone, and copeptin data in Ang II‐treated Ren‐b KO exhibits a bi‐modal distribution indicating that some mice are, whereas others, are not sensitized to Ang II. Ang II‐treated Ren‐b KO exhibited elevated heart weight (WT+Ang: 4.8±0.2 vs KO±Ang: 5.2±0.1 mg/g of body weight; p = 0.03; n=10–12) concomitant with an increased cardiac collagen type I alpha‐2 mRNA expression (p = 0.03; n=7–9) and trend toward increase in tumor necrosis factor‐α. Ang II induced an exaggerated suppression of plasma renin in Ren‐b KO compared to WT (WT+Ang: 14.8±2.1 vs KO+Ang: 7.2±2.6 ng/ml; p = 0. 03; n=7–9). However, there was no difference in the renin mRNA and protein levels of renin in the renal cortex. We used in situ hybridization technique to localize renin expression in the renin cortex. We did not observe a shift from glomerular to tubular renin expression in Ren‐b KO. We conclude Ren‐b KO exhibits higher sensitivity to Ang II‐induced aldosterone secretion and drinking responses as well as elevated susceptibility to Ang II‐evoked cardiac remodeling. The mechanism causing suppressed plasma renin in Ren‐b KO remains unclear. Studies of a conditional Ren‐b KO model will be used to elucidate the physiological consequences of downregulating renin‐b in specific neuroanatomical regions.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

Upregulation of Cortical Renin and Downregulation of Medullary (Pro)Renin Receptor in Unilateral Ureteral Obstruction.

Chronic kidney disease (CKD) is characterized by renal dysfunction, which is a common feature of other major diseases, such as hypertension and diabetes. Unilateral ureteral obstruction (UUO) has been used as a model of CKD in experimental animals and consists of total obstruction of one kidney ureter. The UUO decreases renal blood flow, which promotes the synthesis of renin in the juxtaglomerular apparatus, the first step in renin–angiotensin system (RAS) cascade. RAS induces inflammation and remodeling, along with reduced renal function. However, it remains unknown whether intrarenal RAS (iRAS) is activated in early stages of CKD. Our objective was to characterize different iRAS components in the renal cortex and in the medulla in an early phase of UUO. Male C57BL/6 mice (8–12 weeks old) were subjected to UUO in the left kidney, or to sham surgery, and were euthanized after 7 days (n = 5/group). Renal function, renal inflammatory/remodeling processes, and iRAS expression were evaluated. UUO increased plasma creatinine, right renal hypertrophy (9.08 ± 0.31, P < 0.05 vs. Sham), and tubular dilatation in the left kidney cortex (42.42 ± 8.19µm, P < 0.05 vs. Sham). This correlated with the increased mRNA of IL-1β (1.73 ± 0.14, P < 0.01 vs. Sham, a pro-inflammatory cytokine) and TGF-β1 (1.76 ± 0.10, P < 0.001 vs. Sham, a pro-fibrotic marker). In the renal cortex of the left kidney, UUO increased the mRNA and protein levels of renin (in 35% and 28%, respectively, P < 0.05 vs. Sham). UUO decreased mRNA and protein levels for the (pro)renin receptor in the renal medulla (0.67 ± 0.036 and 0.88 ± 0.028, respectively, P < 0.05 vs. Sham). Our results suggest that modulation of iRAS components depends on renal localization and occurs in parallel with remodeling and pro-inflammatory/pro-fibrotic mechanisms.

Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein-dependent, salt-sensitive hypertension.

New Findings • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension.• What is the main finding and its importance? The main finding is that PVN‐specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension in Sprague–Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. The central mechanisms underlying salt‐sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt‐resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein‐dependent, salt‐sensitive hypertension. Male Sprague–Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal‐salt (NS; 0.6% NaCl) or high‐salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co‐infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro‐inflammatory cytokines TNFα, IL‐1β and IL‐6 and the anti‐inflammatory cytokine IL‐10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt‐sensitive hypertension, increased plasma noradrenaline, PVN pro‐inflammatory cytokine mRNA upregulation, anti‐inflammatory cytokine mRNA downregulation and PVN‐specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co‐infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN‐infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial‐mediated PVN inflammation and the development of salt‐sensitive hypertension.

GLOBAL DELETION OF MICRORNA-181A RESULTS IN INCREASED SALT SENSITIVITY IN MICE

Objective:MicroRNA-181a (miR-181a) is down-regulated in the kidney of both human hypertensive subjects and genetically hypertensive mice (BPH/2J). We have previously shown miR-181a can bind to and negatively regulate renin mRNA levels. Low miR-181a may exert post-transcriptional control over the ren

498-P: Involvement of Phosphorylation of Beta-1 Integrin in Renin Production and Blood Pressure Regulation in Diabetic Nephropathy

Diabetic nephropathy (DN) and hypertension exacerbate each other, but many aspects of the mechanisms between these two pathologies remain unclear. The juxtaglomerular cells (JGC) secrete renin and trigger the renin-angiotensin-aldosterone system involved in blood pressure regulation. Our previous immunohistochemical analysis showed JGC-specific phosphorylation of β1 integrin (Itg), a cellular matrix adhesion factor. Quantitative analysis indicated the negative correlation between the phosphorylated β1 (pβ1) Itg and renin expression in JGC. To analyze the details of this relationship, As4.1, a cell-line of JGC was used. As protein kinase C (PKC) subtype epsilon is a putative kinase of the β1 Itg in JGC, PKC activator (phorbol 12-myristate 13-acetate, PMA) and PKC inhibitor (bisindolylmaleimide I, BIM-1) as well as TGF β1 which is known to stimulate a pathway to phosphorylate β1 Itg in JGC were applied to the culture of As4.1 cells. After 24 hours, expression of the mRNA levels of renin was gained by BIM-1 but suppressed by PMA or TGF β1. In the course of the cell culture with PMA, the mRNA levels of renin was increased till 90 minutes after application. However, it decreased subsequently, ultimately to 10% of the baseline level. Additional application of PMA at 6, 12 and 24 hours after the first application of PMA did not reproduce the temporal increase of renin expression. Time course of the cell culture with TGF β1 showed continuous decrease of the mRNA levels of renin. As PMA has broad spectrum for PKC subtypes, it was estimated that some activated PKC subtypes promoted temporal increase of renin expression in the early phase, however, later changes of cell state produced by pβ1 Itg suppressed the renin expression irreversibly. Our results confirmed a negative correlation between pβ1 Itg and renin expression in JGC cell-line, suggesting a link between β1 Itg and the regulatory system that controls renin production and secretion. Disclosure K. Sawada: None. M. Toyoda: Speaker's Bureau; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kyowa Hakko Kirin Co., Ltd., Medtronic Japan Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. N. Saito: None. M. Kimura: None. H. Moriya: None. M. Fukagawa: Research Support; Self; Kyowa Hakko Kirin Co., Ltd. Speaker's Bureau; Self; Bayer Yakuhin, Ltd., Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Torii Pharmaceutical Co., Ltd.

Rapeseed protein-derived peptides, LY, RALP, and GHS, modulates key enzymes and intermediate products of renin\u2013angiotensin system pathway in spontaneously hypertensive rat

Rapeseed proteins are a rich source of bioactive peptides. LY, RALP and GHS were previously identified from rapeseed protein hydrolysates as potent ACE and renin inhibiting peptides. In this study, the rapeseed peptides were individually evaluated for their molecular mechanisms and regulatory effects on components of the renin–angiotensin system in spontaneously hypertensive rats (SHR), including the mRNA and/or protein levels of angiotensin-converting enzyme (ACE), renin, ACE2, angiotensin II and angiotensin-(1–7) in myocardial tissues. Oral administration of 30 mg peptides/kg body weight every 2 days for five weeks significantly decreased the systolic blood pressure and the myocardial mRNA and protein levels of ACE and renin in SHR. LY, RALP and GHS also increased the expression of ACE2, angiotensin-(1-7) and Mas receptor levels, which may have mediated their antihypertensive activity. Dipeptide LY also inhibited angiotensin II protein expression in the heart tissue. Taken together, the finding demonstrates the multi-target physiological effects of the rapeseed peptides, beyond ACE and renin inhibition, which enhances knowledge of the antihypertensive mechanisms of food protein-derived peptides.

(Pro)renin receptor expression in myocardial infarction in transgenic mice expressing rat tonin

The (pro)renin receptor [(P)RR] has been implicated as a renin/prorenin receptor, and plays a role in local renin angiotensin system activation. Our goal was to investigate whether a transgenic mouse that expresses rat tonin [TGM‘(rTon)] can regulate (P)RR mRNA levels. Control (C) and TGM‘(rTon) animals were subdivided into the C sham, C MI, TGM‘(rTon) sham, and TGM‘(rTon) MI groups. The levels of tonin, (P)RR, and renin were determined using RT-PCR mRNA. Tonin activity as determined by RIE was significantly increased in the TGM‘(rTon) sham group as compared to the C sham group in the atrium (AT) and right ventricle (RV), respectively. In most mice, tonin mRNA levels were significantly reduced compared to those in the TGM‘(rTon) sham group in the atria. In this structure, the (P)RR mRNA levels were statistically significantly reduced in the TGM‘(rTon) sham and TGM‘(rTon) MI groups compared to the control groups. However, the (P)RR mRNA values were significantly increased when we compared the TGM‘(rTon) MI vs TGM‘(rTon) sham groups. In the RV, the renin mRNA levels in the TGM‘(rTon) sham group were significantly reduced compared to the C sham group. Tonin overexpression may act in the regulation of (P)RR mRNA levels during MI.

Abstract 015: Ctcf is Required for Renin Expression and Maintenance of the Structural Integrity of the Kidney

Renin is crucial for the regulation of blood pressure and fluid electrolyte homeostasis. The transcriptional machinery that regulates renin expression and thus determines the identity of renin cells is not completely understood. CCCTC-binding factor ( Ctcf ), is an important chromatin organizer of genes that confer cell identity and tissue-specificity. Because Ctcf binds to several sites in the neighborhood of the renin locus, we hypothesized that Ctcf may regulate renin expression. To test this hypothesis, we generated mice with conditional deletion of Ctcf in cells of the renin lineage ( Ctcf cKO). Ctcf cKO mice showed fewer renin-positive cells as shown by immunostaining, and a 70% reduction of renin mRNA levels when compared to control mice (0.292 ± 0.246 vs 1.003 ± 0.097, p&lt;0.001 ). In addition, plasma renin levels were significantly decreased in Ctcf cKO versus control mice (15276.544 ± 6778.735 pg/ml vs 62321.62 ± 21881.99 pg/ml, p&lt;0.001 ). Consistent with reduced renin levels, Ctcf cKO mice had lower mean arterial pressures (60.09 ± 3.12 mmHg vs 75.07 ± 3.06 mmHg, p&lt;0.001 ). The kidney/body weight ratio in Ctcf cKO mice was markedly reduced (1.05 ± 0.228 % vs 1.29 ± 0.074 %, p&lt;0.05 ), indicating a more pronounced effect in kidney than in somatic growth (20.09 ± 1.47 g vs 22.95 ± 2.95 g, p&lt;0.05 ). Masson’s trichrome staining revealed interstitial fibrosis coinciding with cortical depressions in Ctcf cKO kidneys. Moreover, PAS staining of Ctcf cKO kidneys showed dilated tubules with intraluminal casts, and areas with crowded sclerotic and crescent glomeruli surrounded by disorganized packed cells. Finally, Ctcf cKO mice exhibited renal failure evidenced by increased BUN (44.29 ± 17.62 mg/dL vs 26 ± 3.39 mg/dL, p&lt;0.05) , and inability to concentrate their urine (448 ± 85.57 mOsm/kg vs 1519.33 ± 382.39 mOsm/kg, p&lt;0.001 ). In summary, deletion of Ctcf in cells from the renin lineage leads to decreased endowment of renin-expressing cells accompanied by decreased circulating renin, hypotension, severe morphological abnormalities of the kidney and ultimately renal failure. We conclude that Ctcf is necessary for the appropriate expression of renin, control of renin cell number and structural integrity of the kidney.

Foxd1 is an upstream regulator of the renin-angiotensin system during metanephric kidney development.

BackgroundWe tested the hypothesis that Foxd1, a transcription factor essential for normal kidney development, is an upstream regulator of the renin-angiotensin system (RAS) during ureteric bud (UB)-branching morphogenesis.MethodsUB branching, RAS gene, and protein expression were studied in embryonic mouse kidneys. RAS mRNA expression was studied in mesenchymal MK4 cells.ResultsThe number of UB tips was reduced in Foxd1-/- compared with that in Foxd1+/+ metanephroi on embryonic day E12.5 (14±2.1 vs. 28±1.3, P<0.05). Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that renin, angiotensin I-converting enzyme (ACE), and angiotensin (Ang) II receptor type 1 (AT1R) mRNA levels were decreased in Foxd1-/- compared with those in Foxd1+/+ E14.5 metanephroi. Western blot analysis and immunohistochemistry showed decreased expression of AGT and renin proteins in Foxd1-/- metanephroi compared with that in Foxd1+/+ metanephroi. Foxd1 overexpression in mesenchymal MK4 cells in vitro increased renin, AGT, ACE, and AT1R mRNA levels. Exogenous Ang II stimulated UB branching equally in whole intact E12.5 Foxd1-/- and Foxd1+/+ metanephroi grown ex vivo (+364±21% vs. +336±18%, P=0.42).ConclusionWe conclude that Foxd1 is an upstream positive regulator of RAS during early metanephric development and propose that the cross-talk between Foxd1 and RAS is essential in UB-branching morphogenesis.