Prorenin Receptor Deletion Research Articles

Collecting Duct Pro(Renin) Receptor Contributes to Unilateral Ureteral Obstruction-Induced Kidney Injury via Activation of the Intrarenal RAS.

Although the concept of the intrarenal renin-angiotensin system (RAS) in renal disease is well-described in the literature, the precise pathogenic role and mechanism of this local system have not been directly assessed in the absence of confounding influence from the systemic RAS. The present study used novel mouse models of collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin together with pharmacological inhibition of soluble PRR production to unravel the precise contribution of the intrarenal RAS to renal injury induced by unilateral ureteral obstruction. We examined the impact of CD-specific deletion of PRR, CD-specific deletion of renin, and S1P (site-1 protease) inhibitor PF429242 treatment on renal fibrosis and inflammation and the indices of the intrarenal RAS in a mouse model of unilateral ureteral obstruction. After 3 days of unilateral ureteral obstruction, the indices of the intrarenal RAS including the renal medullary renin content, activity and mRNA expression, and Ang (angiotensin) II content in obstructed kidneys of floxed mice were all increased. That effect was reversed with CD-specific deletion of PRR, CD-specific deletion of renin, and PF429242 treatment, accompanied by consistent improvement in renal fibrosis and inflammation. On the other hand, renal cortical renin levels were unaffected by unilateral ureteral obstruction, irrespective of the genotype. Similar results were obtained via pharmacological inhibition of S1P, the key protease for the generation of soluble PRR. Our results reveal that PRR-dependent/soluble PRR-dependent activation of CD renin represents a key determinant of the intrarenal RAS and, thus, obstruction-induced renal inflammation and fibrosis.

Abstract 47: Deletion of PRR in AgRP Neurons of the Arcuate Nucleus Contributes to the Development of DOCA-Salt Hypertension

Agouti-related protein (AgRP) neurons, a key neuronal population within the arcuate nucleus (ARC), are known for their pivotal roles in regulating energy- and glucose homeostasis. However, their involvement in cardiovascular regulation remains poorly understood. Using RNAscope in situ hybridization, we found that 95.4% of the ARC AgRP neurons express the (pro)renin receptor (PRR), a component of the brain renin-angiotensin system that is crucial for blood pressure (BP) regulation. We therefore hypothesized that PRR signaling in the ARC AgRP neurons plays a regulatory role in the development of hypertension. To test this hypothesis, we selectively deleted PRR in ARC AgRP neurons by crossing AgRP-IRES-Cre mice with PRR fl/fl mice. Using RNAscope, as expected, AgRP Cre+ PRR fl/y mice displayed a significantly lower PRR mRNA in the ARC AgRP neurons than WT mice (76.3 ± 6.1 vs. 127.7 ± 2.9 AFU, p < 0.05), indicating efficient ablation of PRR in ARC AgRP neurons. To examine the impact of PRR deletion in the ARC AgRP neurons on BP, the AgRP Cre+ PRR fl/y and control mice were implanted with telemetry probes to monitor BP continuously. After baseline recording, mice received DOCA-salt treatment (50 mg DOCA + saline as drinking water) for two weeks. BP variability and standard time domain heart rate variability (HRV) parameters, including overall HRV (SDNN) and short-term HRV (rMSSD), were analyzed from beat-to-beat intervals (n = 2-7 mice/group). At baseline, there was no difference between the two groups in BP, BP variability, and HRV. Two weeks of DOCA-salt induced a significantly higher BP in AgRP Cre+ PRR fl/y mice than the controls (156.2 ± 6 vs. 119.4 ± 3 mmHg, p < 0.05), while BP variability did not differ between the two groups. Noteworthy, AgRP Cre+ PRR fl/y mice exhibited lower SDNN than the controls (13.3 ± 4 vs. 19.4 ± 4 ms), indicating increased sympathetic and/or reduced parasympathetic nervous activity. This lower SDNN suggests impaired cardiac autonomic function and adaptability to physiological demands in AgRP Cre+ PRR fl/y mice. Concurrently, AgRP Cre+ PRR fl/y mice showed a lower rMSSD (4.7 ± 0.5 vs. 10.8 ± 3.5 ms), implying reduced parasympathetic activity. In conclusion, deletion of PRR in the ARC AgRP neurons results in compromised cardiac autonomic function associated with the development of hypertension. Our findings underscore a novel protective role of PRR signaling in the ARC AgRP neurons in BP regulation.

Abstract P201: Macula Densa (Pro)Renin Receptor Contributes to 2-Kidney, 1-Clip-Induced Renovascular Hypertension but not Ischemic Nephropathy in Mice

Our previous presentation at this meeting showed that collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin attenuated hypertension and kidney damage induced by the 2-kidney, 1 clip (2K1C) procedure, highlighting an essential role of the intrarenal renin-angiotensin system (RAS) in the pathogenesis of renovascular hypertension and ischemic nephropathy. Besides the CD, PRR is also expressed in the macula densa (MD), mediating renin release from the juxtaglomerular (JG) cells. Here we examined the phenotype of NKCC2-Cre-mediated MD/TAL-specific deletion of PRR (MD/TAL PRR KO) following the 2K1C procedure. Clipping-induced hypertension was partially attenuated in MD/TAL PRR KO mice compared to floxed controls (MAP on day 24: Floxed/2K1C 148.3±1.8 mm Hg vs. MD/TAL PRR KO/2K1C 138.8±2.9 mm Hg, n=6, p < 0.05), to a similar degree in mice lacking PRR or renin in the CD. However, MD/TAL PRR KO mice did not exhibit protective effects against ischemic nephropathy compared to floxed controls following the 2K1C procedure (urinary albumin/creatinine: Floxed/2K1C 65.9 ± 4.6 mg/g vs. MD/TAL PRR KO/2K1C 59.5 ± 2.7 mg/g, n=6). Furthermore, there were no significant differences in clipping-induced protein abundance of renal fibronectin, α-SMA, or proinflammatory markers such as IL-1β, TNF-α, MCP-1. The protective phenotype against hypertension observed in KO mice was paralleled with suppressed plasma renin activity, active renin content, and total prorenin/renin levels. Additionally, while clipping upregulated renin mRNA expression in both renal cortex and medulla of floxed mice, only cortical renin expression was reduced by 39% in MD/TAL PRR KO mice. The 2K1C-induced increase in circulating sPRR was attenuated by 31% in MD/TAL PRR KO mice. Overall, our results suggest that renovascular hypertension in the 2K1C model is jointly determined by the systemic and intrarenal RAS whereas ischemic nephropathy in this model solely depends on the intrarenal RAS.

Abstract P216: Distal Convoluted Tubule-Specific Deletion of Specific Deletion of (Pro)Renin Receptor in Mice Increases Energy Expenditure and Enhances Aggressiveness

Our previous study suggested a potential role of (pro)renin receptor (PRR) in the regulation of Na + -Cl - cotransporter (NCC) activity in the distal convoluted tubule (DCT). Here, we employed Slc12a3 -CreERT2 to generate inducible DCT-specific deletion of PRR in mice (KO). As expected, the KO mice exhibited a 1.8-fold increase in renal NCC activity as reflected by HCTZ-induced natriuresis. Surprisingly, the KO mice developed metabolic and behavioral phenotypes. By calorimetry, 4-month-old male KO mice exhibited an enhanced energy expenditure as compared with floxed controls (oxygen consumption: 4172.5 ± 118.0 ml/kg/hr vs. 3303.5 ± 69.8 ml/kg/hr; CO 2 consumption: 3617.3 ± 87.8 ml/kg/hr vs. 2784.2 ± 58.5 ml/kg/hr; heat production: 20.4 ± 0.6 Kcal/kg/h vs. 16.2 ± 0.8 Kcal/kg/h, n = 5-9, p < 0.01), accompanied by enhanced locomotor activity (by 41.3%) and reduced body weight (30.2 ± 0.5 g vs. 28.0 ± 0.3 g, n = 5-9, p < 0.05). The changes in locomotor activity were confirmed by using radiotelemetry. Additionally, resident-intruder aggression test demonstrated that under basal condition, the KO mice exhibited a higher aggression rate as compared with floxed controls (80% in KO group vs . 30% in floxed group, n = 10). The KO mice also exhibited shorter clinch attack latencies, higher number of attacks and attack duration (attack latencies: 79.0 ± 17.6 s in KO group vs . 172.5 ± 5.3 s in floxed group; attack frequency: 3.4 ± 0.8 in KO group vs . 0.7 ± 0.4 in floxed group; attack duration: 10.1 ± 2.0 s in KO group vs . 1.1 ± 0.6 s in floxed group, n = 10, p < 0.01). Furthermore, the KO mice exhibited elevated plasma level of Klotho (321.4 ± 16.3 pg/ml in KO group vs. 254.1 ± 9.8 pg/ml in floxed group, n = 11-12, p < 0.01) that is known to promote energy expenditure in addition to its cardiorenal protective actions. In cultured renal HK2 cells, recombinant soluble PRR (sPRR-His) downregulated Klotho mRNA and protein expression by 29.8% and 37.9%, respectively. Overall, the present study uncovered a novel role of DCT PRR in the regulation of energy metabolism and behavior, part of which may be mediated by antagonism of Klotho action.

Renal tubular (pro)renin receptor deletion does not protect against kidney injury in db/db mice

Background: The (pro)renin receptor (PRR) is a multifunctional protein implicated in blood pressure regulation and kidney fibrosis. Previous studies report enhanced PRR expression in non-diabetic and diabetic kidney disease. In this study, we investigated whether deletion of renal tubular PRR attenuates kidney injury in type 2 diabetes. Methods: Floxed PRR mice were bred with mice expressing Pax8 rtTA and LC1 transgenes and db/db mice (B6.BKS) to obtain renal tubular PRR knockout (KO)-db/db mice. Age matched non-diabetic floxed controls, db/db mice and PRRKO-db/db mice were studied at 16, 20, 26 and 30 weeks of age. To induce PRR deletion, PRRKO-db/db mice were treated with 2 mg/ml doxycycline for 12 days at 8-10 weeks of age. Only male mice were included in this study. Results: All mice survived until the end of the study (N=5-7 mice/group). Compared to controls, db/db mice and PRRKO-db/db mice had higher body weights despite similar food intake throughout the study (p<0.001 by one-way ANOVA). PRRKO-db/db mice had higher urine volume and water intake compared to controls and db/db mice (p <0.01 by ANOVA). Db/db mice had elevated non-fasting blood glucose levels (average: 472 mg/dl at 16 weeks, 292 mg/dl at 30 weeks) while PRR KO-db/db mice had modestly elevated blood glucose levels (average 255 mg/dl at 20 and 30 weeks) relative to controls (average: 150 mg/dl). Urinary albumin excretion was markedly elevated in PRRKO-db/db mice relative to db/db mice and controls throughout the study (950 ug/day vs db/db:145 ug/day, controls: 50 ug/day, p<0.001). At 30 weeks, kidney histology by PAS stain showed minimal tubular injury, glomerulosclerosis or interstitial inflammation among all 3 groups. Interestingly, podocin staining by immunofluorescence was reduced in db/db mice but not PRR-KO db/db mice. Compared to control mice, db/db mice had similar KIM-1, NGAL and collagen-1 mRNA expression in kidney cortex and inner medulla. PRR KO db/db mice had increased cortical KIM-1, NGAL and collagen-I expression compared to control and db/db mice (p<0.01 by ANOVA). Plasma sPRR levels were almost two-fold higher in diabetic mice relative to controls with no difference between db/db mice and PRR KO-db/db mice. Urinary sPRR levels were undetectable in controls and mildly elevated in db/db mice (7.6 ± 0.7 pg/day) and PRR KO-db/db mice (47.6 ± 22.3 pg/ml). No differences in plasma prorenin/renin concentration was noted among the 3 groups while urinary prorenin/renin excretion was higher in PRRKO-db/db mice. Conclusion: Renal tubular deletion of PRR does not protect against kidney injury in type 2 diabetes. It is possible that loss of PRR impairs baseline tubular function which is exacerbated by type 2 diabetes. American Diabetes Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

(Pro)renin receptor signaling in hypothalamic tyrosine hydroxylase neurons is required for obesity-associated glucose metabolic impairment.

Glucose homeostasis is achieved via complex interactions between the endocrine pancreas and other peripheral tissues and glucoregulatory neurocircuits in the brain that remain incompletely defined. Within the brain, neurons in the hypothalamus appear to play a particularly important role. Consistent with this notion, we report evidence that (pro)renin receptor (PRR) signaling within a subset of tyrosine hydroxylase (TH) neurons located in the hypothalamic paraventricular nucleus (PVNTH neurons) is a physiological determinant of the defended blood glucose level. Specifically, we demonstrate that PRR deletion from PVNTH neurons restores normal glucose homeostasis in mice with diet-induced obesity (DIO). Conversely, chemogenetic inhibition of PVNTH neurons mimics the deleterious effect of DIO on glucose. Combined with our finding that PRR activation inhibits PVNTH neurons, these findings suggest that, in mice, (a) PVNTH neurons play a physiological role in glucose homeostasis, (b) PRR activation impairs glucose homeostasis by inhibiting these neurons, and (c) this mechanism plays a causal role in obesity-associated metabolic impairment.

Deletion of the prorenin receptor in the ureteric bud in mice inhibits Dot1/H3K79 pathway

BackgroundThe prorenin receptor (PRR) plays a critical role in ureteric bud (UB) branching morphogenesis. DOT1 Like (DOT1L), a histone methyltransferase specific for Histone 3 lysine 79 (H3K79), is important for differentiation of the UB-derived renal collecting duct cells. In this study, we tested whether DOT1L/H3 dimethyl K79 (H3m2K79) are regulated by PRR deletion in the UB and UB-derived collecting ducts in the embryonic mouse kidneys.MethodsMutant Hoxb7Cre+/PRRflox/flox (PRRUB-/-) and control PRRUB+/+, mice were studied on embryonic (E) day E17.5. DOT1L mRNA and protein expression in the kidney was examined by real-time qRT-PCR and immunohistochemistry, respectively. H3m2K79 protein expression was determined by immunohistochemistry and Western blot analysis.ResultsDOT1L mRNA levels were decreased in mutant compared to control mice (0.68 ± 0.06 vs. 1.0 ± 0.01, p < 0.01). DOT1L and H3m2K79 immunostaining was reduced in the mutant vs. control kidneys (Dot1: 0.62 ± 0.03 vs. 1.0 ± 0.01, p < 0.05; H3m2K79: 0.64 ± 0.04 vs.1.1 ± 0.01. p < 0.05.). Western blot analysis revealed decreased H3m2K79 protein levels in mutant compared to control kidneys (1.0 ± 0.06 vs. 1.5 ± 0.02, p < 0.05).ConclusionTargeted deletion of the PRR in the UB and UB-derived collecting ducts results in reduced DOT1L gene/protein and H3m2K79 protein expression in the embryonic mouse metanephroi in vivo.ImpactThe role of histone methylation in mediating the effect of the prorenin receptor on the ureteric bud branching (UB) morphogenesis and urine acidification during kidney development is unknown.We demonstrate that histone H3 lysine (K) 79 dimethylation by methyltransferase Dot1 is reduced in the embryonic kidney of mice that lack the prorenin receptor in the UB lineage.

Abstract P376: Collecting Duct Renin And Its Receptor Contributes To Unilateral Ureteral Obstruction-induced Kidney Injury Via Activation Of The Intrarenal Ras

The collecting duct (CD) is well known for its role in fine-tuning urinary excretion of electrolytes and water. Emerging evidence suggests a potential role played by the CD in immunity and renal injury but the underlying mechanism largely remains elusive. Within the CD, (pro)renin receptor (PRR) via site-1 protease (S1P)-derived soluble PRR interacts with renin controls the intrarenal renin-angiotensin system (RAS) to regulate Na+ and water balance and blood pressure. The present study attempted to employ mouse models of CD-specific deletion of PRR and renin (CD PRR KO and CD renin KO, respectively) and a specific S1P inhibitor PF-429242 (PF) to explore the pathogenic role of this local system in renal injury induced by unilateral ureteral obstruction (UUO). Male 3-4-month-old CD PRR KO and CD renin KO, and their respective floxed controls were subjected to UUO surgery or sham operation for 3 days, followed by analysis of renal fibrosis, inflammation, and indices of the intrarenal RAS. Male 3-4-month-old C57BL/6 mice were subjected to the same surgical procedures and infused with vehicle or PF at 20 mg/kg/day via minipump for 3 days, followed by analysis of the same parameters. 3-day UUO in floxed mice induced renal abundance of fibronectin and α-SMA by 8.6 folds and 5.8 folds, respectively, as assessed by immunoblotting analysis. qRT-PCR detected similar magnitude of increases in mRNA expression of fibronectin, α-SMA, as well as collagen III, collagen I, and TGF-β in the obstructed kidneys. Similar results were obtained by Trichrome Masson staining. In parallel, the obstructed kidney exhibited 4-5-fold increases in renal medullary renin mRNA, renin activity, and active renin content contrasting to unaltered renin levels in the renal cortex. CD-specific deletion of PRR and renin induced comparable attenuation of various indices of renal fibrosis, inflammation, renal medullary renin levels so did PF treatment. UUO induced a 2-fold increase in plasma sPRR, which was completely blocked by PF treatment. Taken together, our results demonstrated that CD PRR/sPRR-dependent activation of renin represents a key determinant of the intrarenal RAS and thus obstruction-induced renal inflammation and fibrosis.

Abstract 098: Collecting Duct-derived (pro)renin Receptor Contributes To 2-kidney, 1-clip-induced Renovascular Hypertension And Ischemic Nephropathy In Mice

The 2-kidney, 1-clip (2K1C) Goldblatt model features overactivation of the systemic renin-angiotensin-aldosterone system (RAAS) due to increased renin release from juxtaglomerular cells. However, no prior study functionally evaluates possible involvement of the intrarenal RAAS in this model. Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) implicated in regulation of the intrarenal RAAS under physio-pathological conditions. In the present study, we employed a mouse model of CD-specific deletion of PRR (CD PRR KO) to examine the role of CD PRR in pathogenesis of 2K1C-induced renovascular hypertension and ischemic nephropathy and to further explore the underlying mechanism. Floxed and CD PRR KO mice were subjected to sham-operation or 2K1C procedure, followed by analysis of blood pressure, renal injury, and indices of the RAAS, as well as the expression of subunits of ENaC. Clipping-induced hypertension and renal injury were both attenuated in CD PRR KO mice as compared with floxed controls (MAP on day24: Floxed/2K1C 148.2±3.9 mm Hg vs. CD PRR KO/2K1C 136.9±2.5 mm Hg, n=6, p &lt; 0.05) (urinary albumin/creatinine: Floxed/2K1C 67.5±6.4 mg/g vs. CD PRR KO/2K1C 30.9±3.5 mg/g, n=6, p &lt; 0.01). Of note, the reductions of clipping-induced protein abundance of fibronectin and α-SMA in CD PRR KO mice were 70% and 55%, respectively. Similarly, clipping induced paralleled increases in renal mRNA expression of IL-1β, TNF-α, MCP-1 and TGF-β1 in floxed mice, which were all blunted in CD PRR KO mice. The protective phenotype of the null mice was paralleled with suppressed intrarenal renin and aldosterone (Aldo) levels. In contrast, clipping-induced enhancement of circulating renin and Aldo remained unchanged between the genotypes. Moreover, renal medullary α-ENaC mRNA and protein expression was elevated by clipping in floxed mice, which was respectively blunted 52% and 54% in CD PRR KO mice. Together, these results suggest that the activation of CD PRR stimulates components of the intrarenal RAAS and renal medullary α-ENaC, which result in increased tubular sodium reabsorption and thus contribute to 2K1C-induced renovascular hypertension and ischemic nephropathy.

196-OR: (Pro) renin Receptor Signaling in Tyrosine Hydroxylase Neurons in the Hypothalamic Paraventricular Nucleus Is Required for Obesity-Associated Glucose Metabolic Impairment

We recently reported that neuron-specific (pro) renin receptor (PRR) deletion reduces fasting blood glucose (BG) levels and improves glucose tolerance in high fat diet (HFD) -treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (THPVN neurons) , a largely GABAergic population that expresses PRR and projects to autonomic centers important for BG regulation. To delete PRR from THPVN neurons, we injected into the PVN of PRR-loxp mice either an adeno-associated virus expressing cre recombinase under the control of the rat TH promoter (AAV2-TH-Cre) or AAV2-TH-eGFP as a control. After placing animals on a HFD for 6 wk, we found that the obesity-induced increase of fasting BG levels was blocked by PRR deletion from THPVN neurons (118.0 ± 3.7 vs. 139.3 ± 6.6 mg/dL, n=6/group, p= 0.04) , accompanied by a trend towards improved i.p. glucose tolerance (AUC: 12990 ± 1136 vs. 9467 ± 1298, n=6, p=0.07) . To further investigate the role of THPVN neurons in glucose homeostasis, we employed a Designer Receptors Exclusively Activated by Designer Drugs (DREADD) approach whereby AAV2-TH-Cre was injected bilaterally into the PVN of either hM3Dq-loxp (excitatory) or hM4Di-loxp (inhibitory) mice, and continuous glucose monitoring was performed in conscious, freely moving mice. Following i.p. injection of clozapine-N-oxide (CNO, 1 mg/Kg) or vehicle, we found that whereas basal BG levels were not altered by THPVN neuron activation, THPVN neuron inhibition significantly increased basal BG levels compared to vehicle in male (ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03) but not female mice. These findings establish a role for THPVN neuron PRR signaling in the pathogenesis of obesity-associated glucose metabolic impairment, and show that in male mice, basal THPVN neuron activity is required for normal glucose homeostasis. Disclosure L.Ac. souza: None. C.J.Worker: None. A.Gayban: None. M.W.Schwartz: None. Y.Feng earley: None. Funding National Institutes of Health (R01HL122770, R35HL155008, RDK83042, RDK124238)

Deletion of Prorenin Receptor in the Rostral Ventrolateral Medulla Results in Biphasic and Sex‐Dependent Pressor Responses in Deoxycorticosterone Acetate‐salt Hypertension

In models of low renin hypertension such as the deoxycorticosterone acetate (DOCA)‐salt model, the local generation of angiotensin II in the brain mediates the increase in blood pressure. The prorenin receptor (PRR) might play a potential role in the local activation of prorenin in the brain. We have new evidence that prorenin and PRR are co‐expressed in the proximity to the rostral ventrolateral region of the medulla oblongata (RVL). Therefore, we hypothesized that selective ablation of PRR targeting the RVL attenuates responses to DOCA‐salt in mice. PRR ablation was targeted to the RVL by stereotactic microinjections of adeno‐associated virus (AAV) expressing Cre recombinase in PRR‐flox mice (PRRRVL‐KO). AAV mCherry was used as a control virus (WT). Pressor response to L‐glutamate in the injection site served as confirmatory stereotactic target hit. During the first week of DOCA‐salt treatment, RVL‐targeted ablation of PRR resulted in lower pressor responses to DOCA‐salt in females (WT=115±3 vs KO=104±4 mmHg; p&lt;0.05; n=8), but not males. However, after week 2 on DOCA‐salt treatment, female PRRRVL‐KO unexpectedly exhibited an exaggerated increase in systolic blood pressure (WT=149±3 vs KO=163±3 mmHg; p=0.004; n=8) and this was accompanied by a trending increase in urinary norepinephrine (WT=177±28 vs KO=270±38 ng/ml; p=0.07; n=7‐8). Next, female mice were challenged with an intraperitoneal hypertonic saline injection equivalent to 10% of their body weight followed by 4 hours of urine collection at baseline and on DOCA‐salt treatment. Urinary sodium excretion in female PRRRVL‐KO was significantly lower when compared to WT in both conditions (p&lt;0.05). Finally, using nuclear magnetic resonance/bioimpedance spectroscopy we assessed body fluid compartmentalization. We observed no difference in total body fluid among groups at any time point, but there was a significant mobilization of extracellular fluid to the intracellular compartment in PRRRVL‐KO. Our data indicate that the role of PRR in the RVL is sex‐dependent and biphasic. That is, PRR contributes to the pressor response during the initial stage of DOCA‐salt hypertension in females, presumably by facilitating the generation of angiotensin peptides in the RVL, while it plays a protective role by promoting renal sodium excretion and preventing blood pressure elevation during the maintenance stage of DOCA‐salt hypertension. This study also suggests that distinct PRR expressing cell populations within the RVL might elicit diverging physiological functions.

(Pro)renin Receptor Knockdown in Tyrosine Hydroxylase neurons in the PVN Protects Against the Development of HFD‐Induced Hyperglycemia and Increases Energy Expenditure in Mice

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin‐angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy‐expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)‐induced metabolic physiology is not well understood. We recently showed that neuron‐specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)‐containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD‐induced hyperglycemia. To test this hypothesis, we injected both male and female TdTomato‐PRR‐LoxP mice with adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre) to ablate PRR in the THPVN neurons (THPVNPRRKO). The Tdtomato‐LoxP mice received the same virus were used as control. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following 6‐week HFD (60% calories from fat). Mouse metabolic parameters were measured over 48 hours in Sable Promethion metabolic cages with ad libitum access to HFD. In male mice, PRR‐LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, P= 0.0059) at the end of the six week HFD treatment. Interestingly, PRR ablation in the THPVN did not improve FBG and glucose handling in female mice. Metabolic cage measurements revealed that THPVN PRRKO mice had elevated oxygen consumption (VO2, ml/minute/g body weight) (0.047 ± 0.009 vs. 0.043 ± 0.0008 P= 0.027) and carbon dioxide emission (VCO2, ml/minute/g body weight) (0.035 ± 0.0005 vs. 0.032 ± 0.0005, P=0.0125) versus control HFD‐fed mice over 48 hours, indicating enhanced metabolic rate. The energy expenditure was significantly higher (0.0125 ± 0.00017 vs 0.0116 ± 0.00014, kCal/Hr/g body weight P=0.0026) in the THPVN PRRKO mice compared with their control counterparts. Locomotor activity over 48 hours was similar between the two groups (59.94 ± 19.6 vs. 103.5 ± 42.42 total meters traveled, P=0.204), and no significant difference in food intake (0.254 ± 0.011 vs. 0.274 ± 0.012 g, P=0.090) or body weight (42.7 ± 3.65 vs. 43.6 ± 1.41 g, P=0.842) was observed. This data suggests that PRR knockout in THPVN neurons protects against glucose dysregulation and elevates energy expenditure in mice following HFD. We conclude that PRR THPVN neurons play a key regulatory role in glucose and metabolic regulation.

Soluble (Pro)Renin Receptor as a Negative Regulator of NCC (Na+-Cl- Cotransporter) Activity.

[Figure: see text].

(Pro)Renin Receptor in the Hypothalamic Tyrosine Hydroxylase‐Containing Neurons Contributes to High Fat Diet Induced Hyperglycemia

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin-angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy-expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)-induced metabolic physiology is not well understood. We recently showed that neuron-specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)-containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD-induced hyperglycemia. To test this hypothesis we injected male PRR-LoxP mice with adeno-associated virus serotype 2 (AAV2)-mediated Cre-recombinase driven by a rat TH promoter (AAV2-TH-Cre) to ablate PRR in the THPVN neurons (THPVN PRRKO) or AAV2-eGFP (Control) prior to 6 week HFD (60% calories from fat) treatment. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following HFD. PRR-LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (139.3 ± 6.551 vs. 118.0 ±3.715 mg/dl, P= 0.0409) and trended towards improved glucose tolerance when compared with the control mice following the treatment period (Area under the curve-AUC: 12990 ± 1494 vs. 9035 ± 1354, P=0.0782), suggesting that THPVN PRRKO protects against hyperglycemia and may improve glucose tolerance after 6 weeks HFD treatment. No significant difference between the two groups was observed in the insulin tolerance test (AUC: 10833 ± 727.7 vs. 10135 ± 729.5, P=0.7142), suggesting a possible mechanism of THPVN PRR glycemia regulation independent of insulin resistance. These data indicate a critical role of THPVN PRR in the regulation of HFD-induced hyperglycemia and a potential pathway for understanding the mechanism behind the metabolic action of neuronal PRR.

Abstract P030: Dct (pro)renin Receptor Via Its Soluble Product Inhibits Ncc Activity During Regulation Of Blood Pressure And Potassium Homeostasis In Mice

Sodium-chloride cotransporter (NCC), uniquely located at the distal convoluted tubule (DCT), is the target of thiazide diuretics and critically involved in renal handling of both Na + and K + . However, the mechanism of how NCC activity is regulated is incompletely understood. Here we report a novel role of (pro)renin receptor (PRR) and its cleavage product soluble PRR (sPRR) via site-1 protease (S1P) as negative regulators of NCC during high-salt or high-K + loading. Mice with DCT-specific deletion of PRR (termed as DCT PRR KO) was generated by crossing PRR floxed mice with a transgenic mouse line expressing Cre under the control of the parvalbumin (PV) promoter. Radiotelemrty demonstrated that under basic condition, DCT PRR KO mice exhibited modest hypertension (~5 mmHg) associated with reduced urinary Na + (Floxed: 0.20±0.02, KO: 0.12±0.01 mmol/24h, p&lt;0.001), K + (Floxed: 0.29±0.02, KO: 0.18±0.03 mmol/24h, p&lt;0.05), and Cl - (Floxed: 0.30±0.03, KO: 0.21±0.02 mmol/24h, p&lt;0.01) excretion and increased in vivo NCC activity as assessed by hydrochlorothiazide (HCTN)-induced natriuresis ( by 61%). Renal protein abundance of pNCC-T53 (by 51%) but not total NCC was elevated in the null mice. Following a high salt diet, DCT PRR KO mice were salt sensitive as evidenced by a ~30 mm Hg increase of mean arterial pressure (MAP) which was sensitive to HCTN (ΔMAP, Floxed+HS: -2.05±1.50, KO+HS: -12.77±2.6 mmHg, p&lt;0.05), contrasting to salt resistance in the floxed control. The null mice also exhibited impaired kaliuresis (Floxed+HK: 3.24±0.13, KO+HK: 2.32±0.23 mmol/24h, p&lt;0.001) and hyperkalemia (Floxed+HK: 3.75±0.05, KO+HK: 4.40±0.08 mM, p&lt;0.001) after high K + intake. This phenotype was recapitulated by treatment of C57/BL6 mice with S1P inhibitor PF429242. In cultured Flp-In T-REx 293 NCC cells, S1P-derived sPRR directly dephosphorylated NCC (by 40%) via activation of angiotensin II receptor type 1 (AT1R) independently of aldosterone. 1 / 2 Taken together, the present study has demonstrated that S1P-derived sPRR via AT1R negatively regulates NCC activity in the DCT to render salt resistance and to promote K + excretion. 2 / 2

The neuronal (pro)renin receptor and astrocyte inflammation in the central regulation of blood pressure and blood glucose in mice fed a high-fat diet.

We report here that the neuronal (pro)renin receptor (PRR), a key component of the brain renin-angiotensin system (RAS), plays a critical role in the central regulation of high-fat-diet (HFD)-induced metabolic pathophysiology. The neuronal PRR is known to mediate formation of the majority of angiotensin (ANG) II, a key bioactive peptide of the RAS, in the central nervous system and to regulate blood pressure and cardiovascular function. However, little is known about neuronal PRR function in overnutrition-related metabolic physiology. Here, we show that PRR deletion in neurons reduces blood pressure, neurogenic pressor activity, and fasting blood glucose and improves glucose tolerance without affecting food intake or body weight following a 16-wk HFD. Mechanistically, we found that a HFD increases levels of the PRR ligand (pro)renin in the circulation and hypothalamus and of ANG II in the hypothalamus, indicating activation of the brain RAS. Importantly, PRR deletion in neurons reduced astrogliosis and activation of the astrocytic NF-κB p65 (RelA) in the arcuate nucleus and the ventromedial nucleus of the hypothalamus. Collectively, our findings indicate that the neuronal PRR plays essential roles in overnutrition-related metabolic pathophysiology.

ELABELA antagonizes intrarenal renin-angiotensin system to lower blood pressure and protects against renal injury.

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na+ and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA), a novel endogenous ligand of apelin receptor, in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). Apelin was also found in the medullary CDs as assessed by immunofluorescence. In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of full-length PRR (fPRR), soluble PRR (sPRR), and prorenin/renin protein expression as assessed by immunoblotting and medium sPRR and prorenin/renin levels by ELISA, all of which were reversed by 8-bromoadenosine 3',5'-cyclic monophosphate. Conversely, deletion of PRR in the CD or nephron in mice elevated Apela and Apln mRNA levels as well as urinary ELA and apelin excretion, supporting the antagonistic relationship between the two systems. Administration of exogenous ELA-32 infusion (1.5 mg·kg-1·day-1, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure, systolic blood pressure, diastolic blood pressure, and albuminuria, accompanied with a reduction of urinary sPRR, angiotensin II, and prorenin/renin excretion. HS upregulated renal medullary protein expression of fPRR, sPRR, prorenin, and renin in Dahl SS rats, all of which were significantly blunted by exogenous ELA-32 infusion. Additionally, HS-induced upregulation of inflammatory cytokines (IL-1β, IL-2, IL-6, IL-17A, IFN-γ, VCAM-1, ICAM-1, and MCP-1), fibrosis markers (TGF-β1, FN, Col1A1, PAI-1, and TIMP-1), and kidney injury markers (NGAL, Kim-1, albuminuria, and urinary NGAL excretion) were markedly blocked by exogenous ELA infusion. Together, these results support the antagonistic interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.

Abstract 140: Neuronal Prorenin Receptor Deletion Attenuates Astrogliosis and Inflammation in the Arcuate Nucleus of Mice Fed With High Fat Diet

The (pro)renin receptor (PRR) is a key component of the brain renin-angiotensin system and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)-induced metabolic physiology is not well understood. We recently showed that neuron-specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the molecular mechanisms by which PRR acts remain unclear. Previous research showed that astrogliosis, characterized by enhanced glial fibrillary acid protein (GFAP) expression, is a result of HFD treatment. NF-kB signaling pathway activation leads to hypothalamic inflammation and astrogliosis. Accordingly, we hypothesized that PRR deletion in the neurons improves type II diabetes by regulating astrogliosis and inflammation in the arcuate nucleus (ARC) during HFD. To test our hypothesis, PRRKO and wildtype (WT) littermates were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 16 weeks. Brain tissues were immunolabeled with GFAP and NF-kB p65 and presented as relative fluorescence unit, RFU. We found that in the NFD groups, the GFAP immunolabelling was similar in the ARC (3.9 ± 1.1 vs. 2.2 ± 0.7) between WT and PRRKO mice, suggesting that PRRKO has no effect on astrocyte status in a NFD condition. Following 16 weeks of HFD, the GFAP intensity (7.6 ± 2.2, p=0.04) was significantly increased in the WT mice. PRR deletion in the neurons significantly reduced the HFD-induced astrogliosis in the ARC (4.0 ± 0.6, p=0.02) compared with the WT mice fed HFD. The NF-ĸB p65 level was elevated in the ARC of WT mice following 16 weeks of HFD compared with the NFD (13.1 ± 1.6 vs. 5.4 ± 0.6, p&lt;0.0001). Interestingly, PRR deletion prevented the elevation of NF-ĸB p65 levels induced by HFD (6.7 ± 0.7, p&lt;0.0001). Furthermore, astrocyte specific NF-ĸB p65 levels were higher in the WT mice fed a HFD compared to NFD (77.6 ± 1.6 vs. 14.1 ± 2.9, p&lt;0.0001). Notably, under HFD treatment, PRRKO significantly reduced the astrocytic NF-ĸB p65 levels compared with the WT mice (33.1 ± 6.3, p&lt;0.0001). Our data indicate that PRRKO attenuates HFD-induced type II diabetes and is associated with reduction in astrogliosis and NF-kB activation in the ARC.

Abstract P189: The Prorenin Receptor and its Soluble From Stimulate Hepatic Triglyceride and Cholesterol Metabolism in Male Mice

Non-alcoholic fatty liver disease is strongly associated with obesity and metabolic syndrome. Our laboratory previously demonstrated that mice lacking adipose prorenin receptor (PRR) have elevated hepatic triglycerides and cholesterol contents. In addition, lack of adipose PRR induced a counter regulatory increase of hepatic PRR and plasma soluble PRR (sPRR). Therefore, our study aimed to determine whether PRR and sPRR stimulated endogenous hepatic triglycerides and cholesterol synthesis. PRR floxed male mice fed a standard diet were injected with a single dose of an adeno-associated virus thyroxine-binding globulin Cre (KO, n=5) or saline (CTL, n=6). Body weight were measured weekly for 3 weeks. The deletion of PRR in liver did not change body weight curves. Hepatic levels of TG were significantly decreased in liver PRR KO mice compared with control mice (CTL, 615 ± 196 mg/g prot; KO, 114 ± 18 mg/g prot; P=0.047). PRR KO decreased significantly PPARγ gene expression suggesting that PRR positively regulates TG contents through PPARγ pathway. Surprisingly, similar to adipose PRR KO, liver PRR KO increased significantly plasma sPRR (CTL, 5942 ± 199 pg/ml; KO, 8903 ± 559 pg/ml; P=0.001) and hepatic total cholesterol levels (CTL, 40 ± 3 mg/g prot; KO, 102 ± 6 mg/g prot; P&lt;0.0001). This elevation was associated with an increase in SREBP-2 and HMGCoA-reductase gene expression. To determine whether sPRR stimulated cholesterol synthesis, HepG2 cells were treated with sPRR and sPRR was infused in C57BL/6J mice (n=7 to 8 mice/group). Results showed that sPRR significantly up-regulated hepatic SREPB2 mRNA expression in HepG2 cells (Veh, 1.00 ± 0.04; sPRR, 1.36 ± 0.08; P=0.009) and in the liver of mice (Veh, 1.01 ± 0.07; sPRR, 1.23 ± 0.07; P=0.04). In conclusion, our results demonstrated that hepatic PRR positively regulated TG contents through a PPARγ-dependent mechanism and that sPRR stimulated cholesterol synthesis via SREBP-2 pathway. Futures studies will investigate the mechanism by which PRR and sPRR modulate lipid metabolism.

Abstract P111: ELABELA Antagonizes Intrarenal Renin to Lower Blood Pressure and Protect Against Renal Injury

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na + and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA, also known as Toddler/Apela), a novel endogenous ligand of APJ receptor (also known as APLNR) in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of fPRR (by 31.6%), soluble PRR (sPRR) (by 46.5%), and prorenin (by 45.7%) protein expression as assessed by immunoblotting, and medium sPRR (by 66.7%) and prorenin/renin (by 60%) levels by ELISA. Conversely, deletion of PRR in the CD in mice elevated ELA mRNA levels (1.5-fold), and urinary ELA excretion (2.0-fold), supporting the antagonistic relationship between the two systems. Administration of exogenous ELA (5 mg/kg/day, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure (113.0 ± 1.7 vs . 133.8 ± 1.5 mmHg, P &lt;0.001), systolic blood pressure (BP) (122.5 ± 1.8 vs . 142.9 ± 2.6 mmHg, P &lt;0.01), diastolic BP (105.8 ± 2.3 vs . 127.4 ± 0.9 mmHg, P &lt;0.001), heart rate (347.4 ± 4.9 vs . 394.1 ± 5.3 bpm, P &lt;0.001), and albuminuria (11.4 ± 1.0 vs . 5.1 ± 1.1 mg/24h, P &lt;0.01), accompanied with a reduction of urinary sPRR (by 53.3%), Ang II (by 54.6%), prorenin/renin (by 68.7%), neutrophil gelatinase-associated lipocalin (by 51.9%), and prostaglandin E2 (by 44.9%) excretion. HS upregulated renal medullary protein expression of fPRR (218.7%) and sPRR (172.4%) in Dahl SS rats, all of which were significantly blunted by exogenous ELA infusion (by 30-55%). Together, these results support the antagonist interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.