Soluble Pro Renin Receptor Research Articles

Abstract P239: (Pro)Renin Receptor Contributes to Albumin Overload Nephropathy: Role of Site-1 Protease-Derived Soluble (Pro) Renin Receptor

(Pro) renin receptor (PRR) is a new member of the renin-angiotensin system (RAS) and its role in chronic kidney disease largely remains elusive. We tested the role of PRR in albumin overload (OA) nephropathy. Uninephretomized Sprague-Dawley rats were treated for 7 weeks with vehicle, bovine serum albumin (BSA) (5 g/kg/d via a single i.p. injection) or in combination with PRO20 (500 μg/kg/d via i.p. 3 times a day). The OA rat exhibited severe proteinuria (23-fold), interstitial fibrosis, and oxidative stress, accompanied with increases in urinary renin activity (3.6-fold), urinary angiotensinogen (AGT, 3.6-fold), urinary AngII(7.8-fold), renal ACE mRNA (2.4-fold), all of which were significantly attenuated by PRO20. Urinary soluble PRR (sPRR) as assessed by ELISA was increased 13.6-fold in the OA model. In cultured HK-2 cells, BSA treatment for 24 h decreased full-length PRR (76% reduction) and increased sPRR (8-fold). BSA also increased medium renin activity (3.0-fold), IL6 (2.9-fold for mRNA and 4.1-fold for medium protein) and IL8 (5.5-fold for mRNA and 2.6-fold for medium protein), all of which were attenuated by PRO20 or PRR siRNA. Although furin or ADAM19 was previously shown to mediate the generation of sPRR, inhibition of either one of these had no effect on BSA-induced cleavage of PRR. In contrast, a serine protease inhibitor AEBSF significantly suppressed the production of sPRR. Of serine proteases, proprotein convertases (PCs) mediates post-translational processing of proprotein to active protein and are of particular interest. Screening of 9 PCs led to the identification of site-1 protease (S1P) as the predominant cleavage enzyme. S1P inhibition with siRNA or PF-429242 reduced the sPRR production by 47% and 87%, respectively. PF-429242 also reduced renin activity by 40%, in parallel with suppressed IL6 and IL8 levels. Administration of a recombinant sPRR reversed all the effects of S1P inhibition. Mutagenesis of the S1P but not furin cleavage site in PRR blocked the cleavage. Together, these results suggest that PRR mediates OA-induced renal injury at least in part through S1P-derived sPRR.

Abstract P418: PPARγ Targets both (Pro)Renin Receptor and Site-1 Protease in the Collecting Duct to Mediate Rosiglitazone-induced Fluid Retention

Collecting duct (CD)-specific deletion of PPARγ attenuates the fluid retention side effect of thiazolidinedione including rosiglitazone (Rosi) but the underlying mechanism largely remains unknown. (Pro)renin receptor (PRR) is a newly described regulator of CD function. Recent results have demonstrated site-1 protease (S1P) but not furin or ADAM19 as the predominant PRR cleavage protease. A histidine-tagged soluble PRR, sPRR-His, stimulates AQP2 expression. Here, we examined involvement of PRR/S1P during Rosi-induced fluid retention. PRR promoter contains two putative PRREs at positions -834~-828 bp (AGGTTA) and -318~-312 bp (GGTGCA). A 2.0-kb mouse PRR promoter showed a 10-fold increase in luciferase activity in mpkCCD cells exposed to 10 μM Rosi. The Rosi-induced response was first mapped to -421~ -219 bp by the deletion analysis and then to the second PPRE by mutagenesis. Rosi treatment at 80 mg/kg diet in C57/BL mice rapidly induced a 1.9-fold increase in renal PRR protein, a 2.4-fold increase in urinary sPRR, a 2.0-fold increase in urinary renin activity, a 48% reduction of urine volume, and a 4.8-fold increase in renal AQP2 protein, without an effect on expression of ENaC subunits, at 36 h, all of which were diminished thereafter. On day 3, Rosi induced a 9% body weight gain (BWG) and a 15% reduction of Hct, a 20% increase of fluorescein isothiocyanate dextran (FITC-d)-measured plasma volume. In contrast, the indices of Rosi-induced fluid retention along with AQP2 upregulation were all nearly abolished by a PRR antagonist PRO20 and CD PRR deletion. The sensitivity to Rosi-induced volume expansion as assessed by Hct and FITC-d in the null mice was fully restored by a 3-d i.v. infusion of sPRR-His at 30 μg/kg/d. A 2.1-kb mouse S1P promoter also contained two PPRE sites and showed a 3-fold increase in luciferase activity in response to Rosi. Administration of a S1P inhibitor PF-429242 via minipump in C57/BL6 mice attenuated Rosi-induced FITC-d-measured plasma volume (Control 1.28 + 0.1 vs. Rosi 1.61 + 0.05 vs. Rosi + PF429242 1.37 + 0.08 ml/kg), BWG, and Hct drop. Overall, PPARγ simultaneously targets PRR and S1P to generate sPRR to increase AQP2 expression to expand plasma volume during Rosi treatment.

Abstract P236: Site-1 Protease-derived Soluble (Pro) Renin Receptor Mediates Angiotensin II-induced Hypertension via Activation of Intrarenal Renin System

We have previously shown that collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) attenuates angiotensin II (Ang II)-induced hypertension, accompanied with reduced soluble PRR (sPRR) that exerts antidiuretic action. Recent preliminary and published results demonstrated site-1portease (S1P) but not furin or ADMA19 as the predominant PPR cleavage enzyme. In the present study, we evaluated involvement of S1P-derived sPRR in Ang II-induced hypertension. By radiotelemetry, CD PRR KO mice exhibited reduced MAP on day 7 of Ang II infusion at 300 ng/kg/min as compared with floxed mice (MAP: 118±5 vs. 137±3 mmHg, N=5, p<0.05). Administration of sPRR-His, a histidine-tagged sPRR, at 120 μg/kg/d via i.v. infusion to CD PRR KO mice for additional 7 days largely restored the sensitivity to Ang II (MAP: 139±6 mmHg in sPRR-His +Ang II group vs. 116±5 mmHg in Ang II group, N = 4, p<0.05). The i.v. infusion was achieved via placement of a catheter in jugular vein with the other end connected to mimipump. In C57/BL6 mice, administration of a S1P inhibitor PF429242 (PF) via mini pump infusion at 30 mg/kg/d for 7 days attenuated Ang II-induced increases in MAP (day 7: 125±5 in Ang II+ PF group vs. 142±3 in Ang II group; N=6, p<0.05), urinary sPRR excretion (27±4 vs. 63±9 pg/24h; N=6, p<0.05). In parallel, urinary renin levels were elevated by Ang II, which was blunted by PF (renin activity: 0.17±0.03 in Ang II+PF vs. 0.80±0.081 in Ang II vs. 0.12±0.016 ng/24h in Control, N=6, p<0.01; active renin content: 15.2±2.7 vs. 236.0±23.2 vs. 5.6±1.3 ng/24h, N=6, p<0.01; prorenin content: 9.6±3.1 vs. 27.8±6.1 vs. 6.2±1.8 ng/24h, N=6, p<0.05; total renin content: 24.8±5.2 vs. 263.8±27.0 vs. 11.8±3.0 ng/24h, N=6, p<0.01). An intravenous infusion of sPRR-His counteracted the blood pressure-lowering effect of PF in Ang II-infused mice (MAP: 147±3 in PF+sPRR vs. 126±4 mmHg in PF; N=4, p<0.05). Together, these results suggest that S1P-derived sPRR contributes to Ang II-induced hypertension through activation of intrarenal renin-angiotensin system.

Sodium butyrate suppresses angiotensin II-induced hypertension by inhibition of renal (pro)renin receptor and intrarenal renin-angiotensin system.

Butyrate, a short-chain fatty acid, is the end product of the fermentation of complex carbohydrates by the gut microbiota. Recently, sodium butyrate (NaBu) has been found to play a protective role in a number of chronic diseases. However, it is still unclear whether NaBu has a therapeutic potential in hypertension. The present study was aimed to investigate the role of NaBu in angiotensin II (Ang II)-induced hypertension and to further explore the underlying mechanism. Ang II was infused into uninephrectomized Sprague-Dawley rats with or without intramedullary infusion of NaBu for 14 days. Mean arterial blood pressure was recorded by the telemetry system. Renal tissues, serum samples, and 24-h urine samples were collected to examine renal injury and the regulation of the (pro)renin receptor (PRR) and renin. Intramedullary infusion of NaBu in Sprague-Dawley rats lowered the Ang II-induced mean arterial pressure from 129 ± 6 mmHg to 108 ± 4 mmHg (P < 0.01). This corresponded with an improvement in Ang II-induced renal injury, including urinary albumin, glomerulosclerosis, and renal fibrosis, as well as the expression of inflammatory mediators tumor necrosis factor α, interleukin 6. The renal expression of PRR, angiotensinogen, angiotensin I-converting enzyme and the urinary excretion of soluble PRR, renin, and angiotensinogen were all increased by Ang II infusion but decreased by NaBu treatment. In cultured innermedullary collecting duct cells, NaBu treatment attenuated Ang II-induced expression of PRR and renin. These results demonstrate that NaBu exerts an antihypertensive action, likely by suppressing the PRR-mediated intrarenal renin-angiotensin system.

Activation of Renal (Pro)Renin Receptor Contributes to High Fructose-Induced Salt Sensitivity.

A high-fructose diet is shown to induce salt-sensitive hypertension, but the underlying mechanism largely remains unknown. The major goal of the present study was to test the role of renal (pro)renin receptor (PRR) in this model. In Sprague-Dawley rats, high-fructose intake increased renal expression of full-length PRR, which were attenuated by allopurinol. High-fructose intake also upregulated renal mRNA and protein expression of sodium/hydrogen exchanger 3 and Na/K/2Cl cotransporter, as well as in vivo Na/K/2Cl cotransporter activity, all of which were nearly completely blocked by a PRR decoy inhibitor PRO20 or allopurinol treatment. Parallel changes were observed for indices of intrarenal renin-angiotensin-system including renal and urinary renin and angiotensin II levels. Radiotelemetry demonstrated that high-fructose or a high-salt diet alone did not affect mean arterial pressure, but the combination of the 2 maneuvers induced a ≈10-mm Hg increase of mean arterial pressure, which was blunted by PRO20 or allopurinol treatment. In cultured human kidney 2 cells, both fructose and uric acid increased protein expression of soluble PRR in a time- and dose-dependent manner; fructose-induced PRR upregulation was inhibited by allopurinol. Taken together, our data suggest that fructose via uric acid stimulates renal expression of PRR/soluble PRR that stimulate sodium/hydrogen exchanger 3 and Na/K/2Cl cotransporter expression and intrarenal renin-angiotensin system to induce salt-sensitive hypertension.

Abstract P209: Activation of Renal (Pro)Renin Receptor Contributes to High Fuctose-induced Salt Sensitivity

A high-fructose (HF) diet is shown to induce salt-sensitive hypertension but the underlying mechanism remains unclear. The major goal of the present study was to test the role of renal (pro)renin receptor (PRR) in this model. Male Sprague-Dawley rats were randomly divided into the following 4 groups: 1) Control, 2) Fructose, 3) Fructose + PRO20, and 4) Fructose + allopurinol, and the treatments lasted for 3 months. Fructose was added to drinking water (as 20% solution) so was allopurinol (at 30 mg/kg/d). PRO20, an antagonist of (pro)renin receptor, was administered at 700 μg/kg/day via i.p. injections. High fructose (HF) intake induced a 150% increase in renal protein expression of full-length PRR (fPRR), which were attenuated by allopurinol. HF intake also upregulated renal mRNA and protein expression of NHE3 (206% for protein) and NKCC2 (169% for protein) as well as in vivo NKCC2 activity (2-fold increases in 1-h urine volume and UNaV), all of which were nearly completely blocked by PRO20 or allopurinol treatment. HF intake induced &gt;5-fold increases in urinary renin activity, renin content, and total renin content, and a 2-fold increase in urinary AngII, which were suppressed by 60-70% with PRO20 or allopurinol, contrasting to relatively consistent values of these parameters in the plasma, evidence of involvement of intrarenal RAS . At the last week of the experimental period, radiotelemetry was performed to monitor blood pressure during one-week high salt (HS) diet (8% NaCl). The 3-mo HF intake or a 1-wk HS diet alone did not affect mean arterial pressure (MAP), but the combination of the two maneuvers induced a ~10 mm Hg increase of MAP, which was abolished by PRO20 or allopurinol treatment. In cultured human kidney 2 cells, both fructose and uric acid (UA) increased protein expression of soluble PRR (sPRR) in a time- and dose-dependent manner; fructose-induced PRR upregulation was inhibited by allopurinol. Taken together, our data suggest that fructose via UA stimulates renal expression of PRR/sPRR that stimulate NHE3 and NKCC2 expression and intrarenal RAS to induce salt-sensitive hypertension.

Abstract P210: (Pro)Renin Receptor Regulates Potassium Homeostasis via Intrarenal Aldosterone

It has been shown that transgenic overexpression of human (pro)renin receptor (PRR) results in elevated aldosterone (Aldo) level with unclear functional implications. The present study examined a potential role of renal PRR during high K + (HK) loading. In normal SD rats, a 1-week HK intake (5% KCl in diet) induced a 3.4-fold increase in renal protein expression of full-length PRR and 4.2-fold increase in urinary excretion of soluble PRR (sPRR). Administration of PRO20, a decoy peptide antagonist of PRR, at 700 μg/kg/d via i.p. injections, to K + -loaded animals elevated plasma K + level (5.72+0.08 vs. 4.84±0.18 mM, p&lt;0.05) and decreased urinary K + excretion (2.52+0.11 vs. 3.43+0.19 mmol/24h, p&lt;0.05), accompanied with a 26.2% reduction of urinary aldosterone (Aldo) excretion. HK downregulated NCC protein expression (57.8%) and upregulated renal protein expression of aldosterone synthase CYP11B2 (229%), ROMK (156%), calcium-activated potassium channel subunit alpha-1 (α-BK) (367%), α-Na + -K + -ATPase (596%), and β-ENaC (155%), all of which were significantly blunted by PRO20 (by 50 - 70%). The same maneuvers were applied to adrenalectomized (ADX) rats. Although plasma Aldo was extremely low and also unresponsive to HK loading, urinary Aldo excretion was elevated by 274% with this treatment, which was abolished by PRO20. The HK-induced responses of the above K + and Na + transporting proteins in ADX rats all persisted and also remained sensitive to PRO20. Additionally, spironolactone treatment in ADX rats was still effective in inhibiting kaliuresis induced by HK loading, resulting in hyperkalemia (Plasma K+: 5.13±0.07 vs. 4.19±0.27 mM, p&lt;0.05). In primary rat IMCD cells, exposure to 10 mM KCl for 24 h augmented PRR protein expression and sPRR release in a time- and dose-dependent manner. HK upregulated Aldo release in parallel with increased CYP11B2 protein expression, which were both attenuated by PRO20 or PRR siRNA. A recombinant sPRR, sPRR-His, stimulated Aldo release and CYP11B2 expression. Taken together, we conclude that HK increased renal PRR expression that stimulates renal synthesis of Aldo that coordinates the response of renal membrane Na + and K + transporting proteins to facilitate K + secretion.

(Pro)Renin receptor regulates potassium homeostasis through a local mechanism.

(Pro)renin receptor (PRR) is highly expressed in the distal nephron, but it has an unclear functional implication. The present study was conducted to explore a potential role of renal PRR during high K+ (HK) loading. In normal Sprague-Dawley rats, a 1-wk HK intake increased renal expression of full-length PRR and urinary excretion of soluble PRR (sPRR). Administration of PRO20, a decoy peptide antagonist of PRR, in K+-loaded animals elevated plasma K+ level and decreased urinary K+ excretion, accompanied with suppressed urinary aldosterone excretion and intrarenal aldosterone levels. HK downregulated Na+-Cl- cotransporter (NCC) expression but upregulated CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2), renal outer medullary K+ channel (ROMK), calcium-activated potassium channel subunit α1 (α-BK), α-Na+-K+-ATPase (α-NKA), and epithelial Na+ channel subunit β (β-ENaC), all of which were blunted by PRO20. After HK loading was completed, urinary, but not plasma renin, was upregulated, which was blunted by PRO20. The same experiments that were performed using adrenalectomized (ADX) rats yielded similar results. Interestingly, spironolactone treatment in HK-loaded ADX rats attenuated kaliuresis but promoted natriuresis, which was associated with the suppressed responses of β-ENaC, α-NKA, ROMK, and α-BK protein expression. Taken together, we discovered a novel role of renal PRR in regulation of K+ homeostasis through a local mechanism involving intrarenal renin-angiotensin-aldosterone system and coordinated regulation of membrane Na+- and K+-transporting proteins.

Adipocyte (Pro)Renin-Receptor Deficiency Induces Lipodystrophy, Liver Steatosis and Increases Blood Pressure in Male Mice.

Adipose tissue dysfunction related to obesity is overwhelmingly associated with increased risk of developing cardiovascular diseases. In the setting of obesity, (pro)renin receptor (PRR) is increased in adipose tissue of mice. We sought to determine the physiological consequences of adipocyte-PRR deficiency using adiponectin-Cre mice. We report a unique model of adipocyte-PRR-deficient mice (PRR(Adi/Y)) with almost no detectable white adipose tissues. As a consequence, the livers of PRR(Adi/Y) mice were enlarged and demonstrated a marked accumulation of lipids. Adipocyte-specific deficiency of PRR increased systolic blood pressure and the concentration of soluble PRR in plasma. To determine whether adipocyte-PRR was involved in the development of obesity-induced hypertension, mice were fed a low-fat or a high-fat diet for 16 weeks. Adipocyte-PRR-deficient mice were resistant to diet-induced obesity. Both high-fat- and low-fat-fed PRR(Adi/Y) mice had elevated insulin levels. Interestingly, adipocyte-PRR deficiency improved glucose tolerance in high-fat-fed PRR(Adi/Y) mice. In response to feeding either low-fat or high-fat diets, systolic blood pressure was greater in PRR(Adi/Y) mice than in control mice. High-fat feeding elevated soluble PRR concentration in control and PRR(Adi/Y) mice. In vitro knockdown of PRR by siRNA significantly decreased mRNA abundance of PPARγ (peroxisome proliferator-activated receptor gamma), suggesting an important role for PRR in adipogenesis. Our data indicate that adipocyte-PRR is involved in lipid homeostasis and glucose and insulin homeostasis, and that soluble PRR may be a predictor of metabolic disturbances and play a role in systolic blood pressure regulation.

Do Sweeteners Affect the Expression of (Pro)renin Receptors in the Rat Kidney?

Excess amounts of sugars can cause diabetes and chronic kidney diseases. Recently stevia and saccharin have been used to lower calorie intake and reduce blood sugar. Aqueous extracts of stevia may reduce blood pressure and could be a useful hypotensive agent. The mechanism has not yet been determined but may relate to the renin angiotensin system (RAS). A key protein in the system is the (Pro)renin receptor (PRR) that binds both renin and prorenin. PRR may lead to upregulation of genes associated with hypertension and chronic kidney disease. Recent studies have shown that sweeteners affect RAS; however their effect on PRR is unknown. PRR is a transmembrane protein with a large extracellular domain. The extracellular domain can be cleaved in the Golgi by furin to make a soluble form of PRR. Additionally the mRNA of PRR has a long 3′UTR and may be targeted by microRNAs. We performed a preliminary study to test the effect of sweeteners on PRR expression in the rat kidney. Rats were fed standard Osmolite diets with supplements of sucrose, stevia or saccharin. Kidneys were obtained and small RNAs and mRNAs were purified from the tissues using the mirVana kit. Then the expression of PRR mRNA was measured. At the mRNA level, PRR mRNA expressions were not statistically different in the experimental samples versus the control sample. Then we tested the expression of miRNAs that can potentially target PRR protein expression. miRNA‐132 showed a decrease in expression in the stevia and saccharin samples; however, these samples were not statistically different from the control samples. miRNA‐152 was found to be expressed in the rat kidney and showed the same trend as miRNA‐132. Saccharin and stevia samples did not show any statistical differences from the control. This suggests that sweeteners do not affect PRR mRNA expression or the expression of miRNAs that might target PRR protein expression. In the third study, PRR protein expression was measured in the samples using Western blotting. The blot showed the presence of both intact and soluble PRR proteins. The blot suggested a significant decrease in the protein expression of the soluble PRR in the kidneys of rats fed stevia and sucrose. Increased levels of sPRR have been associated with increased hypotensive affects. However, more samples will be tested to further determine if sPRR is further downregulated or unregulated in the saccharin, stevia and sucrose diets. Our preliminary study indicates that sweeteners may play a role in the regulation of soluble PRR that may further affect kidney diseases.Support or Funding Information2013 and 2014 Minnesota State University, Mankato Foundation Grants

(Pro)Renin Receptor Mediates Antidiuretic Action of Vasopressin/Prostaglandin EP4 Subtype

Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD), particularly in intercalated cells and is regulated by the EP4 subtype. Here we investigated the function of CD PRR during antidiuresis and defined its relationship with the EP4 subtype as well as vasopressin (VP). Water deprivation (WD) elevated renal PRR expression and urinary soluble PRR (sPRR) excretion in rats. Intrarenal infusion of a PRR decoy peptide PRO20 to rats partially attenuated the decrease in urine volume and the increase in urine osmolality in response to 48‐h WD, in parallel with suppressed aquaporin‐2 expression; a similar effect was seen with EP4 antagonism. The mice lacking PRR in the CD exhibited increased urine volume and decreased urine osmolality at basal condition and exhibited impaired urine concentrating capability after WD, accompanied with severe volume loss and a dangerous level of plasma hyperosmolality. In primary culture of rat inner medullary collecting duct (IMCD) cells, exposure to 10 nM prorenin for 24 h remarkably induced AQP2 protein expression, which was blunted by a PRR decoy inhibitor PRO20 and PRR siRNA. VP treatment for 24 h similarly induced aquaporin‐2 (AQP2) expression, accompanied with increased release of PGE2 and sPRR; the AQP2 upregulation was blunted by antagonism of EP4 or PRR. Overall, we have defined a VP/PGE2/EP4/PRR/AQP2 pathway that appears to play an important role in regulation of urine concentrating capability.Support or Funding InformationThis work was supported by National Institutes of Health Grants DK104072 and DK094956, VA Merit Review from the Department of Veterans Affairs, and National Natural Science Foundation of China Grant No. 91439205 and No. 31330037. T. Yang is a Research Career Scientist in the Department of Veterans Affairs.

Antidiuretic Action of Collecting Duct (Pro)Renin Receptor Downstream of Vasopressin and PGE2 Receptor EP4.

Within the kidney, the (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD), particularly in intercalated cells, and it is regulated by the PGE2 receptor EP4 Notably, EP4 also controls urinary concentration through regulation of aquaporin 2 (AQP2). Here, we tested the hypothesis that sequential activation of EP4 and PRR determines AQP2 expression in the CD, thus mediating the antidiuretic action of vasopressin (AVP). Water deprivation (WD) elevated renal PRR expression and urinary soluble PRR excretion in rats. Intrarenal infusion of a PRR decoy peptide, PRO20, or an EP4 antagonist partially prevented the decrease in urine volume and the increase in urine osmolality and AQP2 expression induced by 48-hour WD. In primary cultures of rat inner medullary CD cells, AQP2 expression induced by AVP treatment for 24 hours depended on sequential activation of the EP4 receptor and PRR. Additionally, mice lacking PRR in the CD exhibited increased urine volume and decreased urine osmolality under basal conditions and impaired urine concentrating capability accompanied by severe volume loss and a dangerous level of plasma hyperosmolality after WD. Together, these results suggest a previously undescribed linear AVP/PGE2/EP4/PRR pathway in the CD for regulation of AQP2 expression and urine concentrating capability.

Renal medullary (pro)renin receptor contributes to angiotensin II-induced hypertension in rats via activation of the local renin-angiotensin system.

Background(Pro)renin receptor (PRR) is a new component of the renin–angiotensin system and regulates renin activity in vitro. Within the kidney, PRR is highly expressed in the renal medulla where its expression is induced by angiotensin II infusion. The objective of the present study was to test a potential role of renal medullary PRR during angiotensin II-induced hypertension.MethodsA rat AngII infusion model (100 ng/kg/min) combined with renal intramedullary infusion of PRO20, a specific inhibitor of PRR, was builded. And the intravenous PRO20 infusion serve as control. Mean arterial pressure was recorded by radiotelemetry for one week. Further anaylsis of kidney injury, inflammation, biochemical indices and protein localization were perrformed in vivo or in vitro.ResultsRadiotelemetry demonstrated that AngII infusion elevated the mean arteria pressure from 108 ± 5.8 to 164.7 ± 6.2 mmHg. Mean arterial pressure decreased to 128.6 ± 5.8 mmHg (P < 0.05) after intramedullary infusion of PRO20, but was only modestly affected by intravenous PRO20 infusion. Indices of kidney injury, including proteinuria, glomerulosclerosis, and interstitial fibrosis, inflammation, and increased renal medullary and urinary renin activity following angiotensin II infusion were all remarkably attenuated by intramedullary PRO20 infusion. Following one week of angiotensin II infusion, increased PRR immunoreactivity was found in vascular smooth muscle cells. In cultured rat vascular smooth muscle cells, angiotensin II induced parallel increases in soluble PRR and renin activity, and the latter was significantly reduced by PRO20.ConclusionRenal medullary PRR mediates angiotensin II-induced hypertension, likely by amplifying the local renin response.

Abstract 023: Collecting Duct (Pro)Renin Receptor Mediates Angiotensin II-induced Hypertension

Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the intercalated cells (IC) of collecting duct (CD) where its expression is induced by angiotensin II (AngII). Here we examined the function of PRR in the CD by analyzing mice with CD-specific deletion of PRR (CD PRR KO) using AQP2-Cre which has recently been shown to target both IC and principal cells (PC). Radiotelemetry demonstrated that the null mice were largely resistant to AngII-induced hypertension (MAP on day 7: Floxed/AngII 137.4 ± 3.5 vs. KO/AngII 121.2 ± 1.1 mmHg, p&lt;0.05, n=4), accompanied with reduced urinary soluble PRR (sPRR) and aldosterone levels. Electrophysiology analysis demonstrated that within minutes activation of PRR by 10 nM prorenin induced a transient increase in amiloride-sensitive Na+ transport in cultured mpkCCD cells (Ieq: 1.85 ± 0.17 vs. 1.30 ± 0.06 μA/cm2, p&lt;0.05). Interestingly, this was followed by a second phase of ENaC activation after 6 h, which reached the plateau activation at 24 h, accompanied with increased aldosterone release as assessed by ELISA (14.41 ± 0.92 vs. 5.45 ± 0.28 pg/ ml/μg protein, p&lt;0.05). The chronic but not acute phase of ENaC activation was abolished by eplerenone. Both phases of ENaC activation depended on Nox4-derived reactive oxygen species (ROS). Immunostaining using an antibody against sPRR (the N terminus) showed exclusive labeling in the principle cells (PC) whereas the labeling with the C-terminal antibody was exclusively found in IC. A recombinant histidine-tagged sPRR, termed sPRR-His, in the nanomolar range induced a similar dual effect on ENaC activation as prorenin. Intravenous infusion of sPRR-His in CD PRR KO mice for 5 days completely restored the hypertensive response to AngII (MAP: 135.5 ± 7.5 vs. 116.7 ± 5.7 mmHg, p&lt;0.05). We conclude that: 1) CD PRR mediates AngII-induced hypertension; 2) PRR activation in the CD leads to increased ENaC activity acutely through the direct action of ROS and chronically through local generation of aldosterone; 3) sPRR derived from IC may act in a paracrine fashion to stimulate Na+ transport in PC.

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Objectives An association between the renin-angiotensin system and the pathogenesis of pregnancy-induced hypertension has been reported. The prorenin receptor was discovered in 2002, and Wanatabe et al. reported that women with plasma soluble prorenin receptor concentrations above the 75th percentile at delivery had a significantly increased risk of preeclampsia. We evaluated plasma prorenin concentrations during pregnancy, and we assessed the incidence of pregnancy-induced hypertension. Methods We measured plasma prorenin concentrations in 430 pregnant women (565 samples). Regression analysis was performed to determine the associations between the plasma prorenin level and maternal/neonatal complications. Results The plasma prorenin concentration and gestational age had a positive correlation in non-pregnancy-induced hypertension in women with singleton pregnancies (Spearman rank-correlation coefficient, −0.215; p p p = 0.0107). Conclusions The plasma prorenin levels during pregnancy may be adversely correlated with the prorenin receptor, and low prorenin levels during late pregnancy were significantly associated with pregnancy-induced hypertension. Disclosures Y. Mikami: None. T. Narita: None. Y. Takai: None. K. Baba: None. H. Seki: None. K. Matsuoka: None.

Association of Soluble (Pro) Renin Receptor with Gestational Diabetes Mellitus

There is a need to identify biomarkers for gestational diabetes mellitus (GDM). Recently the soluble pro-renin receptor (s[Pro]RR) has been shown to be associated with GDM. We investigated the association of s(Pro) RR levels in Asian Indians with GDM. We recruited 222 pregnant females, 147 without GDM (non-GDM) and 75 with GDM visiting antenatal clinics in Tamilnadu in South India. We included singleton pregnancy and excluded those with pre-existing diabetes mellitus or hypertension. Oral glucose tolerance tests (OGTTs) were performed, and GDM was diagnosed using the International Association of Diabetes and Pregnancy Study Group criteria. s(Pro)RR was measured by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves were used to identify s(Pro) RR cut-off points to identify GDM. The mean levels of the s(Pro)RR were significantly higher in subjects with GDM (34.0 ± 12 ng/mL, P<.001) compared to non-GDM (21.4 ± 6.5 ng/mL). The proportions of subjects with GDM were 11 (15%) in the first tertile of s(Pro)RR (<19.61 ng/mL), 20 (27%) in the second (19.62-26.8 ng/mL), and 44 (59%) in the third tertile (>26.8 ng/mL). In multiple logistic regression analysis, s(Pro)RR showed a significant association with GDM (odds ratio [OR]: 1.201, 95% confidence interval [CI]: 1.065-1.355, P = .003) after adjusting for potential confounders. A s(Pro)RR cut-off of 23.3 ng/mL had a C statistic of 0.828 (95% CI: 0.738-0.918, P<.001), sensitivity of 68%, and specificity of 70% to identify GDM. s(Pro)RR levels are higher in females with GDM, and this could be used as a potential biomarker.

Abstract 158: Role of Prorenin Receptor in the Enhancement of Renin Activity in the Urine of Diabetic Patients

High prorenin levels in the circulation are associated with the progression of diabetic nephropathy and microvascular changes in diabetes. In spite of high plasma prorenin, the urine of diabetes mellitus patients exhibits very low pH and contains high levels of “active” renin, but low levels of prorenin. Prorenin binding to the prorenin receptor (PRR) results in non-proteolytic activation of prorenin and stimulation of intracellular synthesis of transforming growth factor-beta (TGF-β). Furthermore, the PRR cytosolic domain is associated with the vacuolar H+-ATPase, which is involved in acid-base homeostasis by renal intercalated cells. In diabetes, the collecting duct is the major source of prorenin and PRR is upregulated in the kidneys of diabetic rats. We examined the relationships among urine levels of renin (uRen), prorenin (uPro), soluble PRR (sPRR) and TGF-β (uTGF-β) in 93 subjects, including non-diabetes mellitus (Non-DM: n=64); and type-2 diabetes mellitus (DM; n=29) patients. Urine albumin (uAlb), creatinine (uCr) and pH were also assessed. DM patients showed higher uRen levels (DM: 81±21 vs Non-DM: 29±4.5 ng AngI/mL/hr; p&lt;0.001), which were positively associated with uAlb/uCr (p&lt;0.05, r=0.21) and inversely correlated with urine pH (p&lt;0.01, r=-0.38). These values contrasted with uPro levels, which were no different between groups. DM patients showed increased uTGF-β compared to Non-DM (DM: 22±5.3 vs Non-DM: 5.6±1.5 pg/mg uCr; p&lt;0.0001). Urine sPRR levels were positively correlated with urine pH (p&lt;0.0001, r=0.56). No differences were observed in sPRR plasma and urine levels between non-DM and DM patients. However, the plasma levels of sPRR were 12.5 and 18.5-fold higher than in urine of Non-DM and DM, respectively. Treatment with RAS blockers increased sPRR in plasma (DM with RAS blockade: 24,455±2,151 vs DM without RAS blockade: 17,726±1,255 pg/mL, p&lt;0.001) but not in urine. These data suggest that the cell membrane-bound PRR, not the soluble form, might be responsible for the increases in renin activity and TGF-β, as well as in the low pH in the urine of type-2 DM patients. It is likely that prorenin-mediated activation of membrane bound PRR promotes diabetic nephropathy by increasing urinary formation of TGF-β.

The Dissociation of the Soluble Prorenin Receptor in Plasma and Urine of Diabetic Patients

Diabetic patients display low‐to‐normal plasma levels of renin but increased prorenin levels. In contrast, their urine contains high levels of “active” renin but low prorenin. Our hypothesis is that the soluble form of the prorenin receptor (sPRR) contributes to the enhancement renin activity in the urine of diabetic patients. We measured plasma and urinary sPRR levels by enzyme‐linked immunosorbent assays (ELISA; IBL, Japan) in 60 patients, including: Controls (CT; n=20), Diabetics (DM; n=20) and Diabetics with hypertension (DM+HTN; n=20). Plasma sPRR levels were augmented in DM compared to CT and DM+HTN (DM: 9849±828 vs. CT: 7310±715 and DM+HTN: 7121±373 pg/mL; p&lt;0.05). In contrast, in the urine of DM and DM+HTN patients, sPRR levels were decreased (DM: 576.5±130.9 and DM+HTN: 486.1±81.64 vs. CT: 1248.0±345.6 pg/mg; p&lt;0.05) but urinary renin content was increased (DM: 130±14; DM+HTN: 162±31 vs. CT: 10±1.3 ng AngI/mL/hr; p&lt;0.0001). uCre levels were similar among groups. The dissociation between plasma and urine levels of sPRR suggests that lower pH in the urine of diabetic patients may increase the capability of sPRR to enhance renin enzymatic activity. Further studies will be performed to verify it the sPRR is bound or unbound to renin/prorenin in the urine of these patients.

The Progression of Serum Prorenin Concentration during Pregnancy

Objective: An association between the renin-angiotensin system and the pathogenesis of pregnancy-induced hypertension has been reported. The prorenin receptor was discovered in 2002, and Wanatabe et al. reported that women with plasma soluble prorenin receptor concentrations above the 75th percentile at delivery had a significantly increased risk of preeclampsia. We evaluated serum prorenin concentrations during pregnancy, and we assessed the incidence of pregnancy-induced hypertension. Methods: We measured serum prorenin concentrations in 430 pregnant women (565 samples). Regression analysis was performed to determine the associations between the serum prorenin level and maternal/neonatal complications. Results: The serum prorenin concentration and gestational age had a positive correlation in non-pregnancyinduced hypertension in women with singleton pregnancies (Spearman rank-correlation coefficient, -0.215: p<0.0001). The serum prorenin concentration in women with multiple pregnancies was significantly higher than that in women with singleton pregnancies (multiple linear regression analysis, p<0.0001). Low prorenin levels in the third trimester (≤20.1 percentile) were significantly associated with pregnancy-induced hypertension (adjusted odds ratio, 18.16: 95% confidential interval, 1.95-412.41: p=0.0107). Conclusion: The serum prorenin levels during pregnancy may be adversely correlated with the prorenin receptor, and low prorenin levels during late pregnancy were significantly associated with pregnancy-induced hypertension.

Evolving concepts on regulation and function of renin in distal nephron

Sustained stimulation of the intrarenal/intratubular renin-angiotensin system in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis, and eventual renal injury. Activation of luminal AT(1) receptors in proximal and distal nephron segments by local Ang II formation stimulates various transport systems. Augmented angiotensinogen (AGT) production by proximal tubule cells increases AGT secretion contributing to increased proximal Ang II levels and leading to spillover of AGT into the distal nephron segments, as reflected by increased urinary AGT excretion. The increased distal delivery of AGT provides substrate for renin, which is expressed in principal cells of the collecting tubule and collecting ducts, and is also stimulated by AT(1) receptor activation. Renin and prorenin are secreted into the tubular lumen and act on the AGT delivered from the proximal tubule to form more Ang I. The catalytic actions of renin and or prorenin may be enhanced by binding to prorenin receptors on the intercalated cells or soluble prorenin receptor secreted into the tubular fluid. There is also increased luminal angiotensin converting enzyme in collecting ducts facilitating Ang II formation leading to stimulation of sodium reabsorption via sodium channel and sodium/chloride co-transporter. Thus, increased collecting duct renin contributes to Ang II-dependent hypertension by augmenting distal nephron intratubular Ang II formation leading to sustained stimulation of sodium reabsorption and progression of hypertension.