Mouse Renin Research Articles

An efficient inducible model for the control of gene expression in renin cells.

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/-;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells.NEW & NOTEWORTHY Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy.

Abstract P104: Kidney arteriolar responses to liver-targeted small interference RNA (siRNA) targeting angiotensinogen in diabetic rats: comparison with other renin-angiotensin system blockers

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin’s fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan + captopril group only (P<0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 (14%) diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

O52 KIDNEY ARTERIOLAR RESPONSES TO LIVER-TARGETED SMALL INTERFERENCE RNA (SIRNA) TARGETING ANGIOTENSINOGEN IN DIABETIC RATS: COMPARISON WITH OTHER RENIN-ANGIOTENSIN SYSTEM BLOCKERS

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril+valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin's fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan+captopril group only (P<0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

Angiotensin II Directly Increases Endothelial Calcium and Nitric Oxide in Kidney and Brain Microvessels InVivo With Reduced Efficacy in Hypertension.

The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) invivo and the mechanisms how VECs may mitigate angiotensin II-mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels invivo. Changes in VEC intracellular calcium ([Ca2+]i) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5-Salsa6f mice or the endothelial uptake of NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready-to-use adeno-associated virus expressing Mouse Renin 1 gene (Ren1-AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4-fold increases in VEC [Ca2+]i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5-fold increase in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II-induced VEC [Ca2+]i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation. The present study directly visualized angiotensin II-induced increases in VEC [Ca2+]i and NO production that serve to counterbalance agonist-induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium-specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Abstract P224: The Role Of Gata3 In Renin Cell Identity

Renin cells act as precursors cells of the kidney vasculature and show high plasticity in postnatal life in response to challenges to homeostasis. Our scRNA-seq studies revealed that the dual-zinc finger transcription factor Gata3, important for cell lineage commitment and differentiation inseveral tissues, is expressed in mouse renin cells. We found an enhancer associated with Gata3 in renin cells leading us to hypothesize that GATA3 is important for renin cell identity. We studied 60 and 120-day-old mice carrying a conditional deletion of Gata3 in renin cells: Gata3 fl/fl ;Ren1 dCre/+ ( Gata3-cKO ) and control Gata3 fl/fl ;Ren1 d+/+ . BUN and plasma renin levels were measured from blood samples collected by cardiac puncture. Ren1, Acta2, and Gata3 were visualized in kidney sections by immunostaining. Histological analysis was performed with H&E, PAS, or Masson’s Trichrome chemical reactions. The juxtaglomerular area (JGA) index was calculated as a ratio of renin-positive JGAs per the total number of glomeruli. Gata3-cKO mice have: 1) a marked reduction in Gata3 staining; 2) a marked reduction of Ren1 (1.78 ± 0.51 (n=5) vs. 12.96 ± 2.29 (n=6), p=.0006) and Akr1b7 (2.12 ± 0.35 (n=5) vs. 19.75 ± 5.43 (n=7), p<0.0001) mRNAs compared to controls under threat; 3) a significant decrease in circulating plasma renin compared to controls under basal conditions (15609 ± 14281 pg/ml (n=5) vs. 78778 ± 33925 pg/ml (n=7), p=.0039) and physiological threat (265981 ± 79431 vs. 62347 ± 75631 pg/ml (n=5), p=.0032); 4) a significantly reduced JGA index (28.47 ± 2.32 (n=5) vs. 44.42 ± 2.72 (n=5), p=.0001); 5) flattened JG cells with misplaced renin staining in the Bowman’s capsule; 6) renal abnormalities with evidence of glomerular sclerosis/fibrosis, hemorrhage with aneurysms, and evidence of inflammatory cell infiltration; 7) misplaced Acta2 staining in the intraglomerular mesangium, interstitium, and Bowman’s capsule. Our results suggest a role of Gata3 in the identity of cells of the renin lineage.

The role of Gata3 in renin cell identity.

Renin cells are precursors for other cell types in the kidney and show high plasticity in postnatal life in response to challenges to homeostasis. Our previous single-cell RNA-sequencing studies revealed that the dual zinc-finger transcription factor Gata3, which is important for cell lineage commitment and differentiation, is expressed in mouse renin cells under normal conditions and homeostatic threats. We identified a potential Gata3-binding site upstream of the renin gene leading us to hypothesize that Gata3 is essential for renin cell identity. We studied adult mice with conditional deletion of Gata3 in renin cells: Gata3fl/fl;Ren1dCre/+ (Gata3-cKO) and control Gata3fl/fl;Ren1d+/+ counterparts. Gata3 immunostaining revealed that Gata3-cKO mice had significantly reduced Gata3 expression in juxtaglomerular, mesangial, and smooth muscle cells, indicating a high degree of deletion of Gata3 in renin lineage cells. Gata3-cKO mice exhibited a significant increase in blood urea nitrogen, suggesting hypovolemia and/or compromised renal function. By immunostaining, renin-expressing cells appeared very thin compared with their normal plump shape in control mice. Renin cells were ectopically localized to Bowman's capsule in some glomeruli, and there was aberrant expression of actin-α2 signals in the mesangium, interstitium, and Bowman's capsule in Gata3-cKO mice. Distal tubules showed dilated morphology with visible intraluminal casts. Under physiological threat, Gata3-cKO mice exhibited a lower increase in mRNA levels than controls. Hematoxylin-eosin, periodic acid-Schiff, and Masson's trichrome staining showed increased glomerular fusion, absent cubical epithelial cells in Bowman's capsule, intraglomerular aneurysms, and tubular dilation. In conclusion, our results indicate that Gata3 is crucial to the identity of cells of the renin lineage.NEW & NOTEWORTHY Gata3, a dual zinc-finger transcription factor, is responsible for the identity and localization of renin cells in the kidney. Mice with a conditional deletion of Gata3 in renin lineage cells have abnormal kidneys with juxtaglomerular cells that lose their characteristic location and are misplaced outside and around arterioles and glomeruli. The fundamental role of Gata3 in renin cell development offers a new model to understand how transcription factors control cell location, function, and pathology.

Abstract P091: Role Of Gata3 In Renin Cell Identity

Renin cells act as precursors for other cell types in the kidney vasculature and show high plasticity in postnatal life in response to challenges to homeostasis. Our scRNA-seq studies revealed that the dual-zinc finger transcription factor (TF) Gata3, important for cell lineage commitment and differentiation in several tissues, is expressed in mouse renin cells under normal conditions and homeostatic threat. In addition, we found an enhancer associated with Gata3 in renin cells leading us to hypothesize that GATA3 is important for renin cell identity. We studied 60 and 76 day-old mice carrying a conditional deletion of Gata3 in renin cells: Gata3fl/fl;Ren1dCre/+ (Gata3 cKO), and control Gata3fl/fl;Ren1+/+ counterparts. Gata3 cKO mice exhibited increased BUN (43±4.6mg/dl) vs. control mice (BUN: 21±2mg/dl), suggestive of hypovolemia and/or compromised renal function. By immunostaining, renin expressing cells appeared very thin compared to the normal plump shape in control mice. Renin signal was abnormally localized to the Bowman’s capsule in some glomeruli, and there was an atypical strong Acta2 signal in the mesangium and Bowman’s capsule in Gata3 cKO mice. Distal tubules showed dilated morphology without visible intraluminal casts whereas proximal tubules appeared normal. Gata3 cKO recruited mice exhibited marked reductions in Ren1+ cells compared to controls by an 87% reduction of Ren1 mRNA levels by RT-qPCR (1.68 ± 0.11 vs 12.98 ± 0.98, p=0.0006). H&E, PAS, and Mason’s Trichrome reactions demonstrated increased glomerular fusion, absent cubical epithelial cells in Bowman’s capsule, and intraglomerular aneurysms. In summary, we found abnormal expression and localization of Ren1 and Acta2 and abnormal morphology of kidneys in Gata3 cKO mice. Our results suggest a role of Gata3 in the identity of cells of the renin lineage. Future studies will investigate the role of Gata3 during development and in older animals.

Renal‐derived human sPRR increases blood pressure in female but not male mice

Previous studies showed that soluble prorenin receptor (sPRR) plays an important role in blood pressure regulation. In rodent models, sPRR contributes to AngII production by increasing renin activity and systolic blood pressure (SBP). Moreover, reducing sPRR release into plasma decreased blood pressure and attenuated angiotensin II (AngII) induced hypertension. However, there is a gap of knowledge concerning the functional role of locally produced sPRR from the kidney in blood pressure regulation. Additionally, the role of human sPRR in blood pressure regulation has not been delineated. Therefore, we evaluated the renal derived human sPRR role in SBP control.Human sPRR‐Myc‐tag transgenic mice were bred with mice expressing Hoxb7/Cre to selectively express human sPRR in the collecting duct (RHsPRR). RHsPRR and control (CTL) male and female mice were fed a standard diet for 10 months (n=8‐11/group). Body weight and urine flow rate were examined and SBP measured by radiotelemetry. Western blot analysis depicted the presence of human sPRR‐Myc‐tag (28 KDa) in the cortex and medulla of RHsPRR male mice which validated the humanized mouse model.Male mice have higher body weights than female mice but renal derived human sPRR did not influence body weights (M: 34.1±0.8 and 32.9±0.8g, F: 28.4±0.8 and 29.6±0.5g, P<0.05). Circulating sPRR was unchanged between male and female mice (M: 3995±643 and 4342±500pg/ml, F: 3479±194 and 3948±238pg/ml). SBP increased significantly in female RHsPRR mice compared to CTL (F: 118.7±2 and 127.2±3 mmHg, P<0.05) but not in male mice (M: 123.8±2 and 119.9±6 mmHg). The SBP response to losartan, an AT1R antagonist, was unchanged in females but was impaired in males (M: ‐7.6±1.2 and ‐5.5±0.8 mmHg, F: ‐6.5±1.2 and ‐7.9±1.5 mmHg, P<0.05). Renal derived human sPRR significantly increased AT1R gene expression in females but not in males (M: 1.8±0.6 and 1.2±0.3 2‐ΔΔCT, F: 1.6±0.5 and 3.8±0.9 2‐ΔΔCT, P<0.05). Moreover, renal derived human sPRR decreased the gene expression of renin and angiotensinogen in female mice (Renin: 1.5±0.5 and 0.4±0.1 2‐ΔΔCT, Angiotensinogen: F: 0.9±0.1 and 0.6±0.1 2‐ΔΔCT, P<0.05) suggesting a negative feedback loop involving Angiotensin II‐AT1R pathway. These effects were not observed in males (Renin: 3.3±1.2 and 4.1±1.2 2‐ΔΔCT; Angiotensinogen: M: 1.4±0.4 and 1.4±0.4 2‐ΔΔCT).Overall, our data suggest that renal‐derived human sPRR increases blood pressure in female mice likely via the activation of renal AT1R expression. In contrast, renal‐derived human sPRR expression impaired the blood pressure response to Losartan in male mice. In conclusion, our data support renal derived human sPRR as a promising therapeutic target in hypertension in humans.

DOCA-SALT DIMINISHES BRAIN RAS ACTIVITY IN PARALLEL WITH PLASMA AND RENAL RAS ACTIVITY – NO EVIDENCE FOR SELECTIVE BRAIN RAS ACTIVATION

Objective: Deoxycorticosterone acetate (DOCA)-salt is suggested to promote hypertension by selectively upregulating the brain renin-angiotensin system (RAS), while suppressing the circulating RAS. Yet, we previously observed parallel downregulation of plasma and brain renin in mice. Here, we quantified angiotensinogen (AGT) and angiotensin (Ang) in rat brainstems. Design and method: For 7 weeks, rats received DOCA (200 mg; 60-day release pellet) and saline as drinking water. The contribution of AGT (liver vs. kidney/brain), Ang II type 1 receptors (AT1R) and blood pressure to tissue Ang content was determined by treatment with vehicle, liver-targeted AGT siRNA (30 mg/kg fortnightly; s.c.), valsartan (31 mg/kg/day; s.c.) or spironolactone (80 mg/kg/day; s.c.) during the final 3 weeks (n = 7–8/group). Plasma renin and AGT were quantified by enzyme-kinetic assay, tissue AGT by western blotting, and Ang I and II by LC-MS/MS. Results: Plasma renin, AGT, Ang I and II in rats not exposed to DOCA-salt were 20 ± 4 ng Ang I/mL/h, 730 ± 37 nM, 140 ± 24 pM and 108 ± 24 pM. AGT was present in liver, kidney and brainstem. Kidney Ang I and II were 540 ± 135 and 510 ± 97 fmol/g, while brainstem Ang I and II were undetectable in all and 30% of the rats, respectively (LLOQ <6 fmol/g). DOCA-salt increased blood pressure by 19 ± 1 mmHg and lowered plasma and renal Ang I and II by ∼90%, while brainstem Ang II became undetectable in 70% of the rats. AGT siRNA lowered plasma AGT by 97 ± 0.3%, with similar reductions in the liver and kidney, while brain AGT was unaltered. Whereas spironolactone normalized blood pressure, it increased plasma renin, and plasma, renal and brain Ang I and II. siRNA and valsartan tended to further lower renal Ang, without affecting blood pressure, plasma renin or plasma and brain Ang I and II. Conclusions: DOCA-salt suppresses plasma, renal and brain Ang II in parallel. Renal Ang II depends on hepatic AGT and AT1R-mediated internalization. Brain AGT is liver-independent, but unaltered by DOCA-salt, thus not permitting selective brain RAS upregulation. The RAS upregulation during spironolactone, combined with its antihypertensive effect, argues against the (brain) RAS as a major blood pressure determinant in this model.

Alteration of Cx37, Cx40, Cx43, Cx45, Panx1, and Renin Expression Patterns in Postnatal Kidneys of Dab1-/- (yotari) Mice.

Numerous evidence corroborates roles of gap junctions/hemichannels in proper kidney development. We analyzed how Dab1 gene functional silencing influences expression and localization of Cx37, Cx40, Cx43, Cx45, Panx1 and renin in postnatal kidneys of yotari mice, by using immunohistochemistry and electron microscopy. Dab1 Δ102/221 might lead to the activation of c-Src tyrosine kinase, causing the upregulation of Cx43 in the medulla of yotari mice. The expression of renin was more prominent in yotari mice (p < 0.001). Renin granules were unusually present inside the vascular walls of glomeruli capillaries, in proximal and distal convoluted tubules and in the medulla. Disfunction of Cx40 is likely responsible for increased atypically positioned renin cells which release renin in an uncontrolled fashion, but this doesn’t rule out simultaneous involvement of other Cxs, such as Cx45 which was significantly increased in the yotari cortex. The decreased Cx37 expression in yotari medulla might contribute to hypertension reduction provoked by high renin expression. These findings imply the relevance of Cxs/Panx1 as markers of impaired kidney function (high renin) in yotari mice and that they have a role in the preservation of intercellular signaling and implicate connexopathies as the cause of premature death of yotari mice.

Short-Term Western Diet Aggravates Non-Alcoholic Fatty Liver Disease (NAFLD) With Portal Hypertension in TGR(mREN2)27 Rats.

Non-alcoholic fatty liver disease (NAFLD) is gaining in importance and is linked to obesity. Especially, the development of fibrosis and portal hypertension in NAFLD patients requires treatment. Transgenic TGR(mREN2)27 rats overexpressing mouse renin spontaneously develop NAFLD with portal hypertension but without obesity. This study investigated the additional role of obesity in this model on the development of portal hypertension and fibrosis. Obesity was induced in twelve-week old TGR(mREN2)27 rats after receiving Western diet (WD) for two or four weeks. Liver fibrosis was assessed using standard techniques. Hepatic expression of transforming growth factor-β1 (TGF-β1), collagen type Iα1, α-smooth muscle actin, and the macrophage markers Emr1, as well as the chemoattractant Ccl2, interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) were analyzed. Assessment of portal and systemic hemodynamics was performed using the colored microsphere technique. As expected, WD induced obesity and liver fibrosis as confirmed by Sirius Red and Oil Red O staining. The expression of the monocyte-macrophage markers, Emr1, Ccl2, IL1β and TNFα were increased during feeding of WD, indicating infiltration of macrophages into the liver, even though this increase was statistically not significant for the EGF module-containing mucin-like receptor (Emr1) mRNA expression levels. Of note, portal pressure increased with the duration of WD compared to animals that received a normal chow. Besides obesity, WD feeding increased systemic vascular resistance reflecting systemic endothelial and splanchnic vascular dysfunction. We conclude that transgenic TGR(mREN2)27 rats are a suitable model to investigate NAFLD development with liver fibrosis and portal hypertension. Tendency towards elevated expression of Emr1 is associated with macrophage activity point to a significant role of macrophages in NAFLD pathogenesis, probably due to a shift of the renin–angiotensin system towards a higher activation of the classical pathway. The hepatic injury induced by WD in TGR(mREN2)27 rats is suitable to evaluate different stages of fibrosis and portal hypertension in NAFLD with obesity.

Abstract P2030: DOCA-Salt Diminishes Brain RAS Activity In Parallel With Plasma And Renal RAS Activity - No Evidence For Selective Brain RAS Activation

Deoxycorticosterone acetate (DOCA)-salt is suggested to increase blood pressure via selective upregulation of the brain renin-angiotensin system (RAS), while suppressing the circulating RAS. Yet, we have observed parallel downregulation of plasma and brain renin in mice. Here, we quantified brainstem angiotensinogen (AGT) and angiotensin (Ang) levels in rats exposed to DOCA (200mg; 60-day release) and 0.9% NaCl as drinking water for 7 weeks. To determine the contribution of AGT (liver vs. kidney/brain), Ang II type 1 receptors (AT1R) and blood pressure to tissue Ang II content, rats received vehicle, liver-targeted AGT siRNA (10 mg/kg fortnightly; s.c.), valsartan (31 mg/kg/day; s.c.) or spironolactone (80 mg/kg/day; s.c.) during the final 3 weeks, the latter fully normalizing blood pressure (n=3-9 per group). Plasma renin and AGT were determined by enzyme-kinetic assay, tissue AGT by western blotting, and Ang I and II by LC-MS/MS. Plasma renin, AGT, Ang I and II in rats not exposed to DOCA-salt were 24±8ng Ang I/mL per h, 731±51nM, 123±38pM and 109±43pM. AGT was present in liver, kidney and brainstem. Kidney Ang I and II were 540±135and 510±97fmol/g, while brainstem Ang I and II were undetectable in all and 50% of the rats, respectively (&lt;6 and &lt;12±4 fmol/g). DOCA-salt lowered plasma and renal Ang I and II by ≈90%, while brainstem Ang II became undetectable in 70% of the rats. AGT siRNA reduced plasma AGT by 97±1%, with similar reductions in the liver and kidney, while brain AGT was unaltered. Neither valsartan nor spironolactone affected AGT at any site. Spironolactone increased plasma renin, and hence plasma, renal and brain Ang I and II also rose modestly. Both siRNA and valsartan tended to further lower renal Ang levels, while neither drug affected plasma renin or Ang I and II in plasma and brain. In conclusion, DOCA-salt suppresses plasma, renal and brain Ang II in parallel. Renal Ang II is determined by hepatic AGT and relies on AT1R-mediated internalization. Although brain AGT is liver-independent, it is unaltered by DOCA-salt and does not permit selective brain RAS upregulation. The RAS upregulation induced by spironolactone, combined with its blood pressure-normalizing effect, argues against the (brain) RAS as a major blood pressure determinant in this model.

TGR(mREN2)27 rats develop non-alcoholic fatty liver disease-associated portal hypertension responsive to modulations of Janus-kinase 2 and Mas receptor

Prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing. Resulting fibrosis and portal hypertension, as a possible secondary event, may necessitate treatment. Overexpression of mouse renin in the transgenic rat model, TGR(mREN2)27, leads to spontaneous development of NAFLD. Therefore, we used TGR(mREN2)27 rats as a model of NAFLD where we hypothesized increased susceptibility and investigated fibrosis and portal hypertension and associated pathways. 12-week old TGR(mREN2)27 rats received either cholestatic (BDL) or toxic injury (CCl4 inhalation). Portal and systemic hemodynamic assessments were performed using microsphere technique with and without injection of the Janus-Kinase 2 (JAK2) inhibitor AG490 or the non-peptidic Ang(1-7) agonist, AVE0991. The extent of liver fibrosis was assessed in TGR(mREN2)27 and wild-type rats using standard techniques. Protein and mRNA levels of profibrotic, renin-angiotensin system components were assessed in liver and primary hepatic stellate cells (HSC) and hepatocytes. TGR(mREN2)27 rats developed spontaneous, but mild fibrosis and portal hypertension due to the activation of the JAK2/Arhgef1/ROCK pathway. AG490 decreased migration of HSC and portal pressure in isolated liver perfusions and in vivo. Fibrosis or portal hypertension after cholestatic (BDL) or toxic injury (CCl4) was not aggravated in TGR(mREN2)27 rats, probably due to decreased mouse renin expression in hepatocytes. Interestingly, portal hypertension was even blunted in TGR(mREN2)27 rats (with or without additional injury) by AVE0991. TGR(mREN2)27 rats are a suitable model of spontaneous liver fibrosis and portal hypertension but not with increased susceptibility to liver damage. After additional injury, the animals can be used to evaluate novel therapeutic strategies targeting Mas.

Ultrastructure study of the transgenic REN2 rat aorta – part 2: media, external elastic lamina, and adventitia

Background The renin-angiotensin-aldosterone system (RAAS) plays an important role in the development and progression of vascular stiffness, hypertension and accelerated atherosclerosis, which are associated with the metabolic syndrome (MetS) and type 2 diabetes mellitus. In addition to the intima, RAAS plays an important role in vascular media and adventitial remodeling. Methods Descending thoracic aortas of young male transgenic heterozygous (mRen2) 27 (Ren2) rats were utilized for ultrastructural study. This lean model of hypertension, insulin resistance, and oxidative stress harbors the mouse renin gene and is known to have increased aortic tissue levels of angiotensin II, angiotensin type 1 receptors, and elevated plasma aldosterone levels. Results Ultrastructural observations substantiate known and novel findings in the tunica media, internal and external elastic lamina, and tunica adventitia, which includes: increased media collagen - proteoglycan matrix expansion, increased secretory and proliferative activity and migration of vascular smooth muscle cells (VSMCs) into a newly developing subendothelial neointima, increased VSMC caveolae, mitochondria degeneration, apoptosis; and lipid retention at the elastin lamellar interface. Openings in the external elastic lamina allow pericyte-to-VSMC contacts. The tunica adventitia exhibits stromal pericyte hyperplasia with actively synthetic phenotype and pericyte-pericyte connections. Conclusion While these studies only represent a single snapshot in time, they provide an evaluation of early abnormal ultrastructural vascular remodeling in Ren-2 models of the conduit-elastic thoracic aorta.

The absence of sympathoexcitation during the development of hypertension in Cyp1a1 Ren-2 transgenic rats.

The insertion of mouse renin gene (Ren-2) into the genome of normotensive rats causes a spontaneous rise of blood pressure (BP), leading to an angiotensin II (Ang II)-dependent form of hypertension in transgenic (mRen-2)27 rats (TGR). However, enhanced sympathetic BP component was demonstrated in heterozygous TGR aged 20 weeks. In the present study we used another model, i.e. Cyp1a1-Ren-2 transgenic rats (iTGR) in which hypertension can be induced by natural xenobiotic indole-3 carbinol (I3C) added to the diet. We investigated whether the development of high blood pressure (BP) in 5-month-old iTGR animals fed I3C diet for 10 days is solely due to enhanced Ang II-dependent vasoconstriction or whether enhanced sympathetic vasoconstriction also participates in BP maintenance in this form of hypertension. Using acute sequential blockade of renin-angiotensin system (RAS), sympathetic nervous system (SNS) and NO synthase (NOS) we have demonstrated that the observed gradual increase of BP in iTGR fed I3C diet was entirely due to the augmentation of Ang II-dependent BP component without significant changes of sympathetic BP component. Thus, the hypertension in iTGR resembles to that of homozygous TGR in which high BP was entirely dependent on Ang II-dependent vasoconstriction. Moreover, our measurements of acute BP response to Rho kinase inhibitor fasudil in animals subjected to a combined blockade of RAS, SNS and NOS indicated the attenuation of basal calcium sensitization in both iTGR and homozygous TGR.

Abstract 603: Sequences Proximate to the Renin Cleavage Site in Angiotensinogen Do Not Affect Angiotensin II-mediated Functions

Objective: Angiotensinogen (AGT) cleavage by renin is the rate limiting step to produce angiotensin (Ang)II, a critical contributor to hypertension and atherosclerosis. Human AGT can not be cleaved by mouse renin as demonstrated by a transgenic mouse model expressing human AGT. Amino acids at positions 11 and 12, adjacent to the renin cleavage site in AGT, have been proposed to regulate renin cleavage between human and mouse. This study determined whether these two residues affect renin cleavage and the consequent AngII-mediated functions. Methods and Results: Hepatocyte-specific AGT deficient (hepAGT-/-) mice have low plasma AGT but high renin concentrations. This mouse model injected with adeno-associated viral vectors (AAV) encoding AGT was used to repopulate AGT. We first determined whether repopulation of human AGT using AAV recapitulate phenotypes of human AGT transgenic mice. HepAGT-/- mice were injected with AAV having a null insert or encoding human AGT, while wild type littermates (hepAGT+/+) were injected with the null AAV. Administration of AAV encoding human AGT led to high plasma human AGT concentrations without affecting plasma mouse renin concentrations, and had no effect on blood pressure and atherosclerosis. In a subsequent study, AAV encoding mutated mouse AGT with Leu11Val and Tyr12Ile that were same as the two residues in human were injected into hepAGT-/- mice. Repopulation of the mutated mouse AGT resulted in increased plasma mouse AGT concentrations and reduced renin concentrations that were comparable to their concentrations in hepAGT+/+ mice injected with the null AAV. Consequently, AAV-driven expression of mutated mouse AGT increased blood pressure and atherosclerosis in hepAGT-/- mice that were comparable to their magnitudes in hepAGT+/+ mice injected with the null AAV. Conclusion: The two amino acids proximate to renin cleavage site do not affect AngII-mediated effects.

The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Enhanced Hydrogen Peroxide and renin release in Primary cultures of Juxtaglomerular (JG) cells from diabetic mice

Diabetes mellitus and its complications represent an increasing burden for healthcare. A large fraction of patients with diabetes mellitus develops diabetic nephropathy. The Akita mice strain is one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. Previous reports have shown increased levels of reactive oxygen species in the kidneys of Akita mice. We previously showed that hydrogen peroxide is a potent stimuli for renin release from the juxtaglomerular cells, increasing plasma renin and blood pressure in mice. Thus, we hypothesized that hydrogen peroxide levels are higher in JG cells from Akita mice leading to increased renin release in this strain. Using Western Blot, we found that the NADPH oxidase isoforms NOX1 and NOX4 are expressed, but only NOX1 being upregulated in primary JG cells isolated from Akita mice, compared to JG cells from the control (C57) strain (C57=100%. Akita/NOX1= 147%±10; n=4; p&lt;0.05). Confocal imaging showed the expression of NOX1 and NOX4 in JG cells where they both co‐localized with renin immunolabeling in renin‐containing granules. In addition, the expression of Catalase, which scavenges H202, was reduced in JG cells from diabetic Akita mice (C57=100%; Akita= 72%±7.2 of C57; n=4; p&lt;0.05). We measured H202 levels by transducing JG cells with the fluorescent protein sensor Hyper under control of the renin1 promoter. We found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p&lt;0.05). Since we previously found that H202 stimulates renin release, we studied whether JG cells from Akita mice had higher total renin activity or release. For this, we measured baseline renin release to the media during 2 hours from primary JG cells cultured from Akita or control mice. JG cells were then lysed and total renin content measured. We found that in the absence of any stimuli, JG cells from Akita mice had ~2 fold higher renin release (C57= 100%, Akita=219.2±26%; p&lt;0.04, n=6). We did not observe a significant difference in total renin content between strains. Transduction of replicate plates with adenovirus encoding Catalase (AD‐Cat) decreased baseline renin release by 35±8% (p&lt;0.05) in JG cells from Akita mice whereas it did not affect renin release in control JG cells. We conclude that renin cells from diabetic Akita mice have higher endogenous H202 leading to increased renin release. The NADPH isoform NOX1 is enhanced in Akita mice and may be responsible for higher levels of H202 and renin release from JG cells. Our data suggest that direct stimulation of renin release by H202 could be potentially be involved in renal damage in diabetes mellitus.Support or Funding InformationNIH/NIDDK R03DK105300‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 082: On the Significance of Urinary Renin in Diabetic Kidney Disease: A Critical Role of Impaired Proximal Tubular Reabsorption

Increased expression of renin in the kidney collecting tubule of rodents made diabetic by streptozotocin (STZ) has been well demonstrated but whether this site is the main source of urinary renin is unknown. We wanted to examine the origin and significance of urinary renin in diabetic kidney disease (DKD). Total and active renin was evaluated in urines from people with longstanding type 1 diabetes of more than 25 years, with (n=36) or without DKD (n=38) (eGFR 101 vs. 39 mL/min/1.73m 2 ; p&lt;0.001). Mice given STZ (n=15) or vehicle (n=8) 20 weeks prior to study were also studied. In people with DKD, total renin was markedly increased compared to people without DKD (82 vs. 49 pg/mg Cr; p=0.023). Active renin was also significantly increased in people with DKD compared to people without DKD (3.2 vs. 1.3 pg/mg Cr; p&lt;0.001). In mice with STZ-induced DKD a significant increase in renin was found compared to controls (1093±319 vs. 64±18 pg/mg Cr; p=0.0001). To examine the role of filtration and tubular reabsorption on urinary renin, human active renin was measured in urines from non-diabetic mice infused with human recombinant renin (hrRenin) (n=8), a combination of lysine and hrRenin (n=5) and non-infused controls (n=15). Urines of mice infused with a combination of lysine (a blocker of proximal tubular protein reabsorption) and hrRenin had markedly higher urinary human active renin than those of controls (179±129 vs. 1.6±0.4 pg/mg Cr; p=0.001). The values were also markedly higher than those of mice infused with hrRenin only (4.4±1.1 pg/mg Cr; p=0.003). The effect of lysine was also evaluated in regard to endogenous mouse renin. Urinary mouse renin in mice infused with lysine (n=5) was markedly increased compared to non-infused controls (n=18) (22360±8673 vs. 346±82 pg/mg Cr; p=0.001). In conclusion, in humans with DKD, urine concentrations of both total and active renin are increased. In mice with STZ-induced DKD, urine total renin is also markedly increased. The data further demonstrate that 1) renin is both filterable and reabsorbable in normal mice and 2) the increase of urinary renin in DKD can be attributed largely to impaired reabsorption mainly in the proximal tubule.

Abstract P160: A Genome-spanning Map of Chromatin Structure Near the Renin Proximal Promoter

Chromatin conformation capture technologies (3C, 4C-seq) allow mapping of the three dimensional spatial structure of the genome. Physical proximity mapping, which captures these 3 dimensional interactions, fills an important gap in our understanding of enhancer-gene relationship by linking possible regulatory regions to specific genes that may be distant from the enhancer by thousands or millions of base pairs or even located on different chromosomes. The goal of the current study was to expand our understanding of renin regulation by identifying regions containing potential regulatory elements of renin more than a few kilobases from the renin proximal promoter, using 4C-seq technology, in isolated SS/JrHrdMcwi cardiac microvascular endothelial cells. Three replicates of ten million cells were fixed, digested with EcoRI, ligated, and then decrosslinked. Secondary digestion with NlaIII followed by a second ligation captured an unknown sequence surrounded by the sequence of the renin promoter. A sequencing library was prepared using primers specific to the renin promoter to identify the captured sequences. Sequence reads were mapped to rat genome (Rn 5) divided into fragments cut by EcoRI. Reads were verified to contain the renin proximal promoter sequence and a uniquely mapped captured sequence. The read counts were binarized and Z-scores were calculated based on a windowed average reads relative to background, and Z-scores corresponding to FDR &lt; 0.01 were considered significant. We found 62 loci spanning the genome in contact with the renin proximal promoter. Clusters of interactions were found on Chr13 at 39.6-40.5Mpb and at 53.9-56.3Mbp where the Renin gene is located. Additional contacting loci were found on all chromosomes except X. Quantitative PCR in newly isolated endothelial cells processed as 3C samples (fixed, digested, ligated, decrosslinked) were used to test 9 of the interactions, of which 8 were validated. These loci are enriched with genes whose expression is correlated with renin (53 of 268 genes, p = 7x10 -10 ) in humans, rats and mice. The present study generated a genome-wide map of segments physically interacting with the renin proximal promoter producing the first such map for a gene that is essential for cardiovascular physiology.