Renin Protein Research Articles

Abstract P433: Impaired Renin Activity in a Bicistronic Ren1 c -T2A-tdTomato Reporter Mouse

The study of renin cell identity and function often requires the isolation of this rare cell type –0.01% of the kidney cells. Multicistronic constructs are commonly used to express reporter genes in mice. They consist of two or more genes linked by 2A peptide sequences, which produce separate proteins through a ribosomal skipping mechanism. Here, we report on the generation of a mouse model to label renin-expressing cells with a bright fluorescent reporter for the tracking and isolation of renin cells. Ren1 ctdTomato mice were generated by inserting a bicistronic T2A-tdTomato knock-in cassette upstream of the TGA stop codon of the Ren1 c gene. Kidneys from adult heterozygous (het) Ren1 ctdTomat o mice showed tdTomato signal confined to the JG area under basal conditions, and extending along the afferent arterioles and in the intraglomerular mesangium upon treatment with captopril + low-salt diet to induce the endocrine transformation of renin cells. Unexpectedly, homozygous (homo) mice exhibited increased tdTomato signal that extended in the afferent arterioles and the mesangium even under normal physiological conditions, and progressive thickening of the kidney arterioles with age. Despite reduced Renin immunostaining in the renal cortex, homo mice exhibited significantly higher kidney Ren1 mRNA (7.9 ± 1.2 vs 1.2 ± 0.6, p< 0.0001) and circulating Renin levels (121.7 ± 44.0 vs 66.7 ± 29.4 ng/ml, p< 0.003) when compared to het controls. Moreover, homo mice showed significantly lower blood pressure measured under anesthesia (62.3 ± 5.9 vs 90.0 ± 5.7 mmHg, p< 0.0001), and Ang I plasma levels (159.3 ± 34.8 vs 500.0 ± 184.1 pmol/L, p< 0.05), indicating compromised Renin activity. The concentric arteriolar hypertrophy phenotype observed in these mice is identical to that described when RAS is genetically or pharmacologically inhibited, including the presence of mutations in the renin gene. Unlike mice with global deletion of renin, these animals did not require neonatal saline injections to survive and did not develop other kidney abnormalities, indicating that the bicistronic approach rendered a renin hypomorphic mouse. Ren1 ctdTomato mice constitute an excellent model for the labeling of renin-expressing cells and for the study of the mechanisms involved in the development of concentric vascular hypertrophy under RAS inhibition. In addition, this model may provide a better understanding of factors controlling renin protein folding, stability, packaging, and release.

Abstract P429: Stronger Association of Angiotensinogen with Mortality than Renin or Lactate in Critical Illness

Introduction: Sepsis and septic shock remain global healthcare issues associated with high mortality rates affecting millions every year despite best care efforts. Activation of the renin-angiotensin-system (RAS) is an early event in sepsis, and renin levels may more strongly associate with mortality than serum lactate, a widely accepted index of patient status. We postulated that reduced levels of Angiotensinogen (AGT) may reflect a dysfunctional RAS driven by high renin levels but an attenuated expression of Ang II to adequately support tissue perfusion and blood pressure. This study compared the association with mortality of AGT to serum lactate and active renin in a subset of the VICTAS (Vitamin C, Thiamine, and Steroids in Sepsis) cohort (N=103) at baseline (day 0) prior to treatment. Methods: Serum levels of intact AGT and active renin protein were quantified by separate ELISAs. Biomarker discrimination for all-cause 30-day mortality was compared by receiver operating characteristic (ROC) curves generated by logistic regression models adjusted for age, sex, sequential organ failure assessment (SOFA) score, systolic blood pressure, and in-hospital vasopressor support. Association of serum AGT, renin, and lactate with mortality was assessed by Kaplan-Meier curves with log rank test and hazard ratios derived from Cox regression. Results: Median [interquartile range] serum levels for AGT were: 114 [78.2-205.4] nM; renin: 4.9 [2.2-13.6] pM; and lactate: 2.6 [1.7-3.8] mM. ROC curves revealed better discrimination of AGT for mortality than either active renin or lactate. The area under the curve (AUC) of AGT (0.82, 95% CI: 0.70, 0.93) was greater than lactate (AUC=0.68, 95% CI: 0.57, 0.80; p=0.022). Kaplan-Meier curves and log rank test also revealed that AGT levels below 114 nM associated with reduced survival. Renin levels greater than 4.9 pM were also associated with patient mortality albeit less than that for AGT while higher lactate levels (>2.6 mM) were not associated with mortality in this cohort. Conclusion: AGT levels more strongly associated with 30-day mortality than renin or lactate in the VICTAS cohort. Reduced AGT may reflect increased consumption by high renin coupled with reduced expression by the liver ultimately impairing Ang II-AT 1 R tone in critically ill patients. The prospective assessment of circulating AGT may facilitate more precise therapeutic approaches treatment to restore a dysfunctional RAS and improve overall mortality in sepsis.

Beneficial effects of Panax notoginseng (Burkill) F. H. Chen flower saponins in rats with metabolic hypertension by inhibiting the activation of the renin–angiotensin–aldosterone system through complement 3

BackgroundMetabolic hypertension (MH) has become the most common type of hypertension in recent years due to unhealthy eating habits and lifestyles of people, such as over-eating alcohol, high fat, and sugar diets (ACHFSDs). Therefore, effective means to combat MH are needed. Previous studies have shown that Panax notoginseng (Burkill) F. H. Chen flower saponins (PNFS) can lower blood pressure in spontaneously hypertensive rats (SHR). However, whether it acts on MH and its mechanism of action remain unclear. MethodsThe pharmacodynamic effects of PNFS were evaluated in rats with ACHFSDs-induced MH. The blood pressure, blood biochemical, grip strength, face temperature, vertigo time, and liver index were estimated. The histological changes in the liver and aorta were observed using hematoxylin and eosin staining. The levels of ET-1, TXB2, NO, PGI2, Renin, ACE, Ang II, and ALD in plasma were detected using ELISA. The levels of C3, KLF5, LXRα, and Renin in kidney tissues were measured using qRT-PCR.The expression levels of C3, KLF5, LXRα, and Renin in kidney tissues were examined using Western blotting.ResultsIn the present study, PNFS was found to reduce blood pressure, face temperature, and vertigo time, increase grip strength and improve dyslipidemia in rats with MH. In addition, PNFS decreased the plasma levels of ET-1 and TXB2, elevated the levels of NO and PGI2, and improved pathological aortic injury. Meanwhile, PNFS decreased the plasma levels of Renin, ACE, Ang II, and ALD. QRT-PCR and Western bolt showed that PNFS downregulated C3, KLF5, LXRα, and Renin protein and mRNA expression in the kidneys of rats with MH.ConclusionThe finding of the present study suggested that PNFS could downregulate C3 and KLF-5 expression in rats with MH, thereby inhibiting the overactivation of the renin–angiotensin–aldosterone system, while improving vascular endothelial function and ultimately reducing blood pressure in rats with MH.

Structural and dynamic investigation of non-synonymous variations in Renin-AGT complex revealed altered binding via hydrogen-bonding network reprogramming to accelerate the hypertension pathway.

Hypertension is one of the major issues worldwide and one of the main factors involved in heart and kidney failure. Angiotensinogen and renin are key components of the renin-angiotensin-aldosterone system, which plays an indispensable role in hypertension. The aim of this study was to find out the non-synonymous mutations and structure-based mutation-function correlation in the renin-AGT complex and reveal the most deleterious mutations to accelerated hypertension. In the current study, we employed computational modeling and molecular simulation approaches to demonstrate the impact of specific mutations in the REN-AGT interface in hypertension. Computational algorithms, that is, PhD-SNP, PolyPhen-1, MAPP, Sorting Intolerant from Tolerant, Screening of non-acceptable polymorphism, PredictSNP, PolyPhen-2, and Protein Analysis Through Evolutionary Relationships predicted 20 mutations as deleterious in AGT while only five mutations were confirmed as deleterious in the renin protein. Investigation of the bonding analysis revealed that two mutations S107L and V193F in renin altered the hydrogen-bonding paradigm at the interface site. Furthermore, exploration of structural-dynamic behaviors demonstrated by that these mutations also increases the structural stability to regulate the expression of disease pathway. The flexibility index of each residues and structural compactness analysis further validated the findings by portraying the difference in the dynamic behavior in contrast to the wild type. Binding energy calculations based on molecular mechanics/generalized Born surface area methods were used which further established the binding differences between the wild type, S107L, and V193F mutant variants. The total binding energy for wild type, S107L, and V193F was reported to be -27.79, -47.72, and -38.25, respectively. In conclusion, these two mutations increase the binding free energy alongside the docking score to enhance the binding between renin and AGT to overexpress this pathway in a hypertension disease condition. Patients with these mutations may be screened for potential therapeutic intervention.

The endocrine kidney: tampering with oxygen sensors may change your character

The endocrine kidney: tampering with oxygen sensors may change your character

Interaction of the renin inhibitor aliskiren with the SARS-CoV-2 main protease: a molecular docking study

The renin protein is an upstream enzymatic regulator of the renin-aldosterone-angiotensin system (RAAS) essential for the maintenance of blood pressure. The angiotensin-converting enzyme-2 (ACE2) is a major component of the RAAS and a cell surface receptor exploited by the SARS-CoV-2 virus to enter host cells. A recent molecular modeling study has revealed that the direct renin peptide inhibitor remikiren can bind to the catalytic site of SARS-CoV-2 main protease (Mpro). By analogy, we postulated that the non-peptidic drug aliskiren, a more potent renin inhibitor than remikiren and a drug routinely used to treat hypertension, may also be able to interact with Mpro. An in silico comparison of the binding of the two compounds to Mpro indicates that aliskiren (ΔE = −75.9 kcal/mol) can form stable complexes with the main viral protease, binding to the active site, as remikiren (ΔE = −83.2 kcal/mol). The comparison with a panoply of 30 references compounds (mainly antiviral drugs) indicated that remikiren is a potent Mpro binder comparable to drugs like glecaprevir and pibrentasvir (ΔE = −96.5 kcal/mol). The energy of interaction (ΔE) of aliskiren with Mpro is about 10% lower than with remikiren, comparable to that calculated with drugs like velpatasvir and sofosbuvir. A model is proposed to define the drug binding site, with the best binders (including remikiren) penetrating deeply into the site, whereas the less potent binders (including aliskiren) interact more superficially with the protein. Communicated by Ramaswamy H. Sarma

Abstract P145: Renal Proximal Tubule-specific Renin Deficiency Has No Effect On Atherosclerosis In Hypercholesterolemic Mice

Objective: Renin cleaving angiotensinogen to release angiotensin I is the rate-limiting step to produce angiotensin II. Although juxtaglomerular cells are the predominant source of renin in circulation, renin protein is also present in proximal tubules. In this study, we investigated the effects of renin in proximal tubule cells (PTC) on development of atherosclerosis in hypercholesterolemic mice Approach and Result: Inducible PTC-specific renin deficient (PTC-renin -/-) mice were developed by breeding renin floxed mice with mice expressing Cre under the control of the N-myc downstream regulated gene 1 (Ndrg1-Cre ERT2). All study mice were in a low-density lipoprotein receptor (LDLR) deficient background, and both male and female littermates were studied. Littermates of renin floxed mice with or without Ndrg1-Cre ERT2 transgene were injected intraperitoneally with tamoxifen (150 mg/kg/day) for 5 consecutive days when they were 4-6 weeks old. Cre genotypes were determined after weaning (~ 3-4 weeks old) and deletion of renin gene in kidney was verified by PCR of DNA extracted from kidney after termination (~18-20 weeks old). Two weeks after the last injection of tamoxifen, mice were fed a Western diet for 12 weeks, which led to plasma total cholesterol concentrations above 1000 mg/dl (P = 0.893 in male and P = 0.192 in female between PTC-renin +/+ and -/- mice). PTC-specific renin deficiency did not affect blood pressure (PTC-renin+/+ vs -/-: 110 ± 1 mmHg vs 112 ± 1 mmHg in male, P = 0.452; and 105 ± 3mmHg vs 106 ± 2 mmHg in female, P = 0.875), as measured by a tail-cuff system. Atherosclerosis in the ascending and aortic arch regions was measured with an en face method. No differences on percent lesion area between the two genotypes in both male and female mice (PTC-renin+/+ vs -/-: 18.1 ± 1.4% vs 18.9 ± 1.5% in male, P = 0.315; and 16.2 ± 1.5% vs 16.2 ± 1.2% in female, P = 0.648). Conclusion: Renal proximal tubule-specific renin deficiency does not affect blood pressure and atherosclerosis in hypercholesterolemic mice.

Abstract P159: Induction Of Human Angiotensinogen In Hepatocytes And Human Renin In Renal Proximal Tubule Cells Does Not Accelerate Atherosclerosis In Mice

Objective: Hepatocyte-produced angiotensinogen (AGT) can be filtered by glomeruli and retained in renal proximal tubules. Renin protein has also been detected in proximal tubules (PTC) of kidney. However, it is unknown whether AGT and renin in proximal tubules contribute to atherosclerosis. This study aimed to determine whether hepatocyte-derived AGT interacts with renin in proximal tubules to promote atherosclerosis. Approach and Results: Transgenic mice expressing human renin driven by a kidney androgen-related protein promoter (KAP-hREN) in an LDLR -/- background were administered subcutaneously with testosterone (15 mg/mouse, constant release for 60 days) to activate the human renin transgene in PTCs. To induce synthesis of human AGT in hepatocytes, adeno-associated viral (AAV; 3 x 10 10 genomic copies/mouse) vector containing human AGT with a liver-specific promoter was injected intraperitoneally. Three groups of male littermates were administered testosterone: (1) wild type mice administered null AAV (n=11), (2) KAP-hREN transgenic mice administered null AAV (n=7), and (3) KAP-hREN transgenic mice (n=8) administered AAV containing human AGT. Two weeks after administration of testosterone and AAVs (either null or human AGT), all mice were fed a Western diet for 6 weeks. Induction of human renin was confirmed by mRNA abundance of human renin in kidney of KAP-hREN transgenic mice. In mice administered human AGT AAV, presence of human AGT was detected in plasma with a human AGT ELISA kit. Induction of both human AGT in liver and human renin in proximal tubules did not affect plasma cholesterol concentrations or systolic blood pressure. Atherosclerotic lesion sizes, as quantified by percent lesion area using an en face method, were not different among the 3 groups (Group 1 vs 2 vs 3: 3.7 ± 0.4% vs 3.3 ± 0.9% vs 2.3 ± 0.3%; P = 0.19). Conclusions: Induction of human AGT in hepatocytes and human renin in proximal tubules does not augment atherosclerosis in hypercholesterolemic mice.

Olfactory receptor 78 modulates renin but not baseline blood pressure.

Olfactory receptor 78 (Olfr78) is a G protein‐coupled receptor (GPCR) that is expressed in the juxtaglomerular apparatus (JGA) of the kidney as well as the peripheral vasculature, and is activated by gut microbial metabolites. We previously reported that Olfr78 plays a role in renin secretion in isolated glomeruli, and that Olfr78 knockout (KO) mice have lower plasma renin activity. We also noted that in anesthetized mice, Olfr78KO appeared to be hypotensive. In this study, we used radiotelemetry to determine the role of Olfr78 in chronic blood pressure regulation. We found that the blood pressure of Olfr78KO mice is not significantly different than that of their WT counterparts at baseline, or on high‐ or low‐salt diets. However, Olfr78KO mice have depressed heart rates on high‐salt diets. We also report that Olfr78KO mice have lower renin protein levels associated with glomeruli. Finally, we developed a mouse where Olfr78 was selectively knocked out in the JGA, which phenocopied the lower renin association findings. In sum, these experiments suggest that Olfr78 modulates renin, but does not play an active role in blood pressure regulation at baseline, and is more likely activated by high levels of short chain fatty acids or hypotensive events. This study provides important context to our knowledge of Olfr78 in BP regulation.

Deep neural network modeling based virtual screening and prediction of potential inhibitors for renin protein

Renin enzyme plays an essential role in the Renin-Angiotensin System (RAS), and it is involved in the pathogenesis of hypertension and several other cardiovascular diseases (CVDs). Inhibition of renin is an effective way to intervene with the pathogenesis of these diseases. Docking-based virtual screening, 3D-Quantitative Structure-Activity Relationship (3D-QSAR), and structure-based drug design are the most frequently used strategies towards discovering novel inhibitors targeting renin. In this study, we have developed a 2D fingerprint-based Deep Neural Network (DNN) classifier for virtual screening and a DNN-QSAR model for biological activity prediction. The resulting hits from the DNN-QSAR model were then subjected to the molecular docking to identify further top hits. Molecular Dynamics (MD) simulation was conducted to get a better insight into the binding mode of identified hits. We have discovered six compounds from the Maybridge chemical database with the predicted IC50 values ranging from 24.2 nM to 83.6 nM. To the best of our knowledge, this is the first study that used a cascaded DNN model to identify potential lead compounds for the inhibition of renin target. Through the results presented in this study, we provide evidence of the DNN method being a useful approach to identify new chemical entities/novel lead compounds that may overcome the limitation of existing conventional strategies used in drug discovery research. Communicated by Ramaswamy H. Sarma

Low Nitric Oxide Bioavailability Increases Renin Production in the Collecting Duct.

In the kidney, the stimulation of renin production by the collecting duct (CD-renin) contributes to the development of hypertension. The CD is a major nephron segment for the synthesis of nitric oxide (NO), and low NO bioavailability in the renal medulla is associated with hypertension. However, it is unknown whether NO regulates renin production in the CD. To test the hypothesis that low intrarenal NO levels stimulate the production of CD-renin, we first examined renin expression in the distal nephron segments of CD-eNOS deficient mice. In these mice, specific CD-renin immunoreactivity was increased compared to wild-type littermates; however, juxtaglomerular (JG) renin was not altered. To further assess the intracellular mechanisms involved, we then treated M-1 cells with either 1 mM L-NAME (L-arginine analog), an inhibitor of NO synthase activity, or 1 mM NONOate, a NO donor. Both treatments increased intracellular renin protein levels in M-1 cells. However, only the inhibition of NOS with L-NAME stimulated renin synthesis and secretion as reflected by the increase in Ren1C transcript and renin protein levels in the extracellular media, respectively. In addition, NONOate induced a fast mobilization of cGMP and intracellular renin accumulation. These response was partially prevented by guanylyl cyclase inhibition with ODQ (1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1]. Accumulation of intracellular renin was blocked by protein kinase G (PKG) and protein kinase C (PKC) inhibitors. Our data indicate that low NO bioavailability increases CD-renin synthesis and secretion, which may contribute to the activation of intrarenal renin angiotensin system.

Expressions of Renin, angiotensin converting enzyme, angiotensin receptor 1, and angiotensin receptor 2 in synovial tissue of osteoarthritis at different stages

To study the expressions of Renin, angiotensin converting enzyme (ACE), angiotensin receptor 1 (AT1R), and AT2R in synovial tissue of osteoarthritis (OA) at different stages. The patients who were treated with upper knee amputation because of trauma or total knee arthroplasty for OA between January 2018 and December 2018 were enrolled. Among them, 32 patients who met the selection criteria were included in the study. According to the Kellgren-Lawrence (K-L) X-ray classification, they were allocated to normal synovial group (group A, n=9), moderate OA synovial group (group B, n=11, K-L level 3), and advanced OA synovial group (group C, n=12, K-L level 4). The relative expressions of Renin, ACE, AT1R, and AT2R mRNAs and proteins were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot. The relative expressions of Renin, ACE, and AT1R mRNAs and proteins were significantly higher in group B and group C than in group A ( P<0.05). The relative expressions of ACE and AT1R mRNAs and proteins and Renin protein were significantly higher in group C than in group B ( P<0.05). However, the relative expressions of AT2R mRNA and protein were lower in group B and group C than in group A ( P<0.05), and in group C than in group B ( P<0.05). The expressions of Renin, ACE, and AT1R in synovial tissue of osteoarthritis significantly increase as the K-L level increased, and the expression of AT2R decreases. Renin, ACE, AT1R, and AT2R have a certain degree of correlation with the development of OA.

Protein Kinase G Is Involved in Acute but Not in Long-Term Regulation of Renin Secretion.

Pharmacological inhibition of the renin–angiotensin–aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10–20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment via activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.

Angiotensinogen and Megalin Interactions Contribute to Atherosclerosis-Brief Report.

Objective- AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results- AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions- These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.

A1166 Use of proteomics to investigate blood pressure progress in the elderly

Objectives: Hypertension increases cardiovascular risks. Apart from established clinical risk factors for hypertension, biomarkers were identified as potential risk factors for hypertension but not well-established. We therefore investigated association between cardiovascular proteins and blood pressure (BP) change in prospective cohorts. Methods: We used a proteomic profiling approach in two independent cohorts, Prospective Investigation of the Vasculature in Uppsala Seniors study (PIVUS; n = 566, mean age of 70.2 years, 50.4% female) and Uppsala Longitudinal Study of Adult Men (ULSAM; n = 238, mean age of 77.6 years) of elderly individuals at baseline and follow-up data on BP for 5 years. Proteomic chips, which used two antibodies for each protein and a PCR step to achieve a high-specific binding, was measured 92 plasma proteins simultaneously. Malmö Diet and Cancer Study (MDC) was validated the finding. Results: In PIVUS and ULSAM, renin protein significantly predicted BP progression. The odds ratio of renin and BP progression was 1.09 (95% CI, 1.02–1.15; p-value, 0.006) after adjusted for baseline characteristics using mixed models on PIVUS, ULSAM, and MDC cohort. Using linear regression models, the baseline renin level was associated with MAP (mean arterial pressure) change in composite ULSAM and PIVUS cohort (β, 1.21; 95% CI, 0.41 to 2.01), and MDC cohort (β, 0.80; 95% CI, 0.07 to 1.53). However, meta-analysis of three cohorts showed insignificantly associated between renin and MAP change (β, 0.11; 95% CI, −0.10 to 0.33). Conclusion: Using proteomics approach, we discovered and replicated the association between renin protein and BP change but weak associated with BP progression. Further studies are needed to confirm the findings.

Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin–angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin–angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d−/− mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d−/− non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin–angiotensin system.

Abstract 014: B1 Integrin is Essential for Kidney Structure, Function and Vascular Development

Integrins are the largest family of cell adhesion molecules. β1-Integrin (Itgb1) is the most abundantly expressed β subunit and is present in almost every cell type. Previous studies showed that Itgb1 is required for normal development of the ureteric bud and podocytes and for the function of the proximal tubule. However, its role in the kidney vasculature has not been explored. Renin cells are crucial for blood pressure homeostasis and for normal nephrovascular development. The mechanisms involved in their morphogenetic functions are not well understood. We found that Itgb1 is highly expressed in renin expressing cells throughout development. Therefore, to study the role of Itgb1 in renin cells we generated a conditional deletion (cKO) of Itgb1 in cells of the renin lineage by crossing floxed Itgb1 mice with mice expressing cre recombinase driven by the renin locus. Itgb1 cKO mice were smaller in size (20.32 ± 4.35 g vs 29.55 ± 7.39 g, p=0.016 ), had smaller kidney to body weight ratio (0.92 ± 0.30 vs 1.31 ± 0.30, p=0.017 ), hypotension (MABP 82.33 ± 4.00 mmHg vs 92.55 ± 7.72 mmHg, p=0.02 ), anemia (hemoglobin 11.26 ± 1.48 g/dL vs 15.07 ± 1.31 d/dL, p=0.003 ; hematocrit 40.39 ± 5.48% vs 53.8 ± 4.21%, p=0.002 ), renal failure (BUN 71.44 ± 32.81 mg/dL vs 29.4 ± 7.67mg/dL, p=0.004), and lower plasma renin levels (6664.58 ± 3251.92 vs 43357.09 ± 17032.63 pg/ml, p =0.001 ). Mutants also developed hyposthenuria (urine osmolality 452 ± 138.98 mOsm/kg vs 1460.5 ± 482.09 mOsm/kg, p&lt;0.02 ). Histological analysis revealed excessive collagen deposition in the interstitium and peri-glomerular areas; fibrocystic glomeruli; tubular dilatation and protein casts in the tubules. Immunostaining for renin and α-smooth muscle actin showed a marked decrease in renin protein expression and abundant α-smooth muscle actin expression in the interstitium. Microdissection of the renal arterial tree combined with renin immunostaining confirmed the marked decrease in renin and evidenced the overall vascular abnormalities including fewer and shorter arterial and arteriolar branches. Overall, this study shows that β1-Integrin in cells of the renin lineage is crucial for renin expression, morphogenesis of the renal vasculature and maintenance of the normal kidney architecture and function.

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.

Prostaglandin E2 Induces Prorenin-Dependent Activation of (Pro)renin Receptor and Upregulation of Cyclooxygenase-2 in Collecting Duct Cells

BackgroundProstaglandin E2 (PGE2) regulates renin expression in renal juxtaglomerular cells. PGE2 acts through E-prostanoid (EP) receptors in the renal collecting duct (CD) to regulate sodium and water balance. CD cells express EP1 and EP4, which are linked to protein kinase C (PKC) and PKA downstream pathways, respectively. Previous studies showed that the presence of renin in the CD, and that of PKC and PKA pathways, activate its expression. The (pro)renin receptor (PRR) is also expressed in CD cells, and its activation enhances cyclooxygenase-2 (COX-2) through extracellular signal–regulated kinase (ERK). We hypothesized that PGE2 stimulates prorenin and renin synthesis leading to subsequent activation of PRR and upregulation of COX-2. MethodsWe used a mouse M-1 CD cell line that expresses EP1, EP3 and EP4 but not EP2. ResultsPGE2 (10−6M) treatment increased prorenin and renin protein levels at 4 and 8 hours. No differences were found at 12-hour after PGE2 treatment. Phospho-ERK was significantly augmented after 12 hours. COX-2 expression was decreased after 4 hours of PGE2 treatment, but increased after 12 hours. Interestingly, the full-length form of the PRR was upregulated only at 12 hours. PGE2-mediated phospho-ERK and COX-2 upregulation was suppressed by PRR silencing. ConclusionsOur results suggest that PGE2 induces biphasic regulation of COX-2 through renin-dependent PRR activation via EP1 and EP4 receptors. PRR-mediated increases in COX-2 expression may enhance PGE2 synthesis in CD cells serving as a buffer mechanism in conditions of activated renin-angiotensin system.

Bradykinin/B2 receptor activation regulates renin in M-1 cells via protein kinase C and nitric oxide.

In the collecting duct (CD), the interactions of renin angiotensin system (RAS) and kallikrein‐kinin system (KKS) modulate Na+ reabsorption, volume homeostasis, and blood pressure. In this study, we used a mouse kidney cortical CD cell line (M‐1 cells) to test the hypothesis that in the CD, the activation of bradykinin B2 receptor (B2R) increases renin synthesis and release. Physiological concentrations of bradykinin (BK) treatment of M‐1 cells increased renin mRNA and prorenin and renin protein contents in a dose‐dependent manner and increased threefold renin content in the cell culture media. These effects were mediated by protein kinase C (PKC) independently of protein kinase A (PKA) because B2R antagonism with Icatibant and PKC inhibition with calphostin C, prevented these responses, but PKA inhibition with H89 did not modify the effects elicited by the B2R activation. BK‐dependent stimulation of renin gene expression in CD cells also involved nitric oxide (NO) pathway because increased cGMP levels and inhibition of NO synthase with L‐NAME prevented it. Complementary renin immunohistochemical studies performed in kidneys from mice with conventional B2R knockout and conditional B2R knockout in the CD, showed marked decreased renin immunoreactivity in CD, regardless of the renin presence in juxtaglomerular cells in the knockout mice. These results indicate that the activation of B2R increases renin synthesis and release by the CD cells through PKC stimulation and NO release, which support further the interactions between the RAS and KKS.