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Background: Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR), that has been implicated in the pathogenesis of hypertension. We recently developed a novel mouse model with mutation in the cleavage site of the PRR using CRISPR/Cas9 such that sPRR is not generated. Since the gene encoding PRR is on the X-chromosome, male mutant mice have markedly lower plasma sPRR levels (control: 21.5 ± 2.5 vs mutant: 0.2 ± 0.03 ng/ml, p+ or K+ excretion with Ang-II infusion while female mutant sPRR mice had enhanced urinary Na and K excretion compared to controls on day 2 and day 7 of Ang-II infusion. Both male and female mutant sPRR mice had attenuated vasoconstrictor response to Ang-II in isolated mesenteric arteries despite similar Ang-II receptor expression. We are currently examining differences in renin angiotensin aldosterone system markers between male and female control and mutant sPRR mice following Ang-II/DOCA-salt hypertension. Conclusion: These results suggest that sPRR plays a role in Ang-II induced hypertension in male and female mice through modulation of urinary Na+/K+ excretion and vascular reactivity. sPRR is also involved in regulating BP with DOCA-salt treatment in male but not female mice. NHLBI This is the full abstract presented at the American Physiology Summit 2023 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.

Hypertension (HTN) is associated with an increase in pro-inflammatory cytokines. Sentinel renal dendritic cells (rDCs) form an intricate network within the kidney cortex. DCs can activate immune cells and secrete pro-inflammatory cytokines. We hypothesize that loss of renal-specific DCs (rDCs) prevents salt-sensitive hypertension (SS HTN) and reduces intra-renal chemokine and cytokine production. To test this hypothesis, male mice lacking a functional CX 3 CR 1 chemokine receptor (CX 3 CR 1 - EGFP+/+ ) or wild-type (Wt) were used. CX 3 CR 1 is required for DC localization to the kidney; thus these mice have a renal-specific DC-depletion. Mice were subjected to L-NAME (0.5mg/mL, 2 wks) and then a wash-out (1-2wks), followed by HS (4%) or normal chow (NC) for the remainder of the study. Mice were implanted with telemetry devices and mean arterial pressure (MAP) assessed. Total kidney mRNA was isolated and pro-inflammatory chemokine/cytokine levels were determined using RT-PCR (n=4-6). Wt mice exhibited a progressive increase in MAP, with rDC-depleted mice having significantly lower MAP by week 3 (121±4 vs . 114±3mmHg, *p

Two recent clinical trials, using sodium glucose cotransporter (SGLT2) or endothelin-A receptor (ET-A) blocker, reported the first efficacious treatments in 18 years to slow progression of diabetic kidney disease (DKD). We hypothesized that combined inhibition of SGLT2 and ET-A receptor may confer greater protection against renal injury than either agent alone. Uninephrectomized male db/db mice were randomized to four groups: vehicle, SGLT2 inhibitor (dapagliflozin (dapa), 1 mg/kg/day), ET-A blocker (atrasentan (atra), 5 mg/kg/day), or dual treatment from 10 weeks until 22 weeks of age. At 10 weeks of age, no differences were observed in body weight, blood glucose or urinary albumin excretion among the four groups. At 16 and 22 weeks of age, body weight was lower and blood glucose levels higher in the vehicle and atra groups compared with dapa- and dual-treated groups. No notable differences were observed among the four groups in urinary albumin excretion at weeks 16 and 22. Histological analysis showed mild glomerulosclerosis and tubular injury (

Rationale: Cleavage of the extracellular domain of the PRR ([pro]renin receptor) yields a soluble fragment (sPRR). Although changes in plasma sPRR levels have been reported in hypertension, the causal role of sPRR in blood pressure regulation is unknown. Objective: Determine the role of sPRR in blood pressure regulation at baseline and following Ang II (angiotensin II)–induced hypertension. Methods and Results: CRISPR-Cas9 was used to mutate the cleavage site of the PRR such that sPRR is not generated. Because the gene encoding PRR is on the X-chromosome and male mutant sPRR mice are infertile, only male mice were studied. Mutant sPRR mice had virtually undetectable plasma sPRR levels compared with littermate controls. Mutant sPRR mice had normal survival and development and no apparent histological abnormalities in the kidney, heart, or aorta despite lower body weight. During normal Na + intake, no differences in food or water intake, urinary water or Na + excretion, or acid-base status were observed between control and mutant sPRR mice. Compared with controls, mutant sPRR mice had lower blood pressure at baseline and an attenuated hypertensive response to 2 weeks of Ang II infusion (400 ng/kg per minute) which was partially reversed by infusion of mouse recombinant sPRR. Mutant sPRR mice also had lower albuminuria, renal tubular injury, and oxidative stress relative to control mice post-Ang II infusion. Furthermore, mesenteric arteries from mutant sPRR mice displayed reduced Ang II–induced vasocontraction and greater acetylcholine, but not sodium nitroprusside, evoked vasorelaxation under baseline conditions. Conclusions: Loss of sPRR reduces blood pressure at baseline and decreases Ang II–induced hypertension and renal injury. These effects of sPRR loss are associated with greater endothelium-dependent but not independent vasorelaxation of resistance-sized arteries.

Background Primary cilia regulation of renal function and blood pressure (BP) in health and disease is incompletely understood. The current study investigated the effect of nephron ciliary loss on renal physiology, blood pressure and ensuing cystogenesis. Methods Mice underwent doxycycline (DOX)-inducible nephron-specific knockout (KO) of the Ift88 gene at 2 months of age using a Cre-LoxP strategy. Blood pressure, kidney function, and renal pathology were studied 2 and 9 months after DOX (Ift88 KO) or vehicle (control). Results At 2 months post DOX, male, but not female, Ift88 KO, compared to sex-matched control, mice had reduced BP, enhanced salt-induced natriuresis, increased urinary nitrite/nitrate (NOx) excretion, and increased kidney NOS3 levels which localized to the outer medulla; the reductions in BP in male mice were prevented by L-NAME. At 9 months post DOX, male, but not female, Ift88 KO mice had polycystic kidneys, elevated BP and reduced urinary NOx excretion. No differences were observed in plasma renin concentration, plasma aldosterone, urine vasopressin or urine PGE2 between Ift88 KO and control mice at 2 or 9 months post DOX. Conclusions Nephron cilia disruption in male, but not female, mice: 1) reduces BP prior to cyst formation; 2) increases NOx production that may account for the lower BP prior to cyst formation; and 3) induces polycystic kidneys that are associated with hypertension and reduced renal NO production.

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR), which maybe involved in mediating hypertension. We recently developed a novel mouse model with mutation in the cleavage site of the PRR using CRISPR/Cas9 such that sPRR is not generated and showed that absence of sPRR attenuated angiotensin-II induced hypertension and kidney damage. In this study, we examined if sPRR alters blood pressure (BP) in angiotensin-II independent hypertension using deoxycorticosterone acetate (DOCA)-salt treatment. Mutant sPRR mice and littermate controls were treated with DOCA (50 mg/kg) and high Na + diet for 3 weeks. BP was monitored by radio-telemetry and metabolic balance studies performed at the end of the study (Day 17-18). Compared to controls, male mutant sPRR mice had markedly lower plasma sPRR levels (control: 21.5 ± 2.5 vs mutant 0.2 ± 0.03 ng/ml) and baseline BP (systolic control: 122 ± 3 vs mutant 114 ± 3; diastolic control: 94 ±5 vs mutant 82 ± 3 mm Hg). BP remained low in mutant sPRR mice relative to controls following 12 days of DOCA-salt treatment (systolic control: 141 ± 2 vs mutant 132 ± 5; diastolic control: 110 ± 4 vs mutant 95 ± 5 mm Hg). Mutant sPRR mice had lower body weight but similar food intake and urinary albumin excretion compared to controls (Table 1). Mutant sPRR mice had lower urine volume, water intake and urinary K + but not Na + excretion. No differences in renal histology were noted between control and mutant sPRR mice. Loss of sPRR attenuates DOCA-salt mediated hypertension. The mechanisms by which sPRR might regulate BP and water/Na + homeostasis in DOCA-salt hypertension are currently being investigated.

BACKGROUND: Hypertension often occurs before renal function deteriorates in autosomal dominant polycystic kidney disease (ADPKD). It is unknown whether the Pkd1 gene product polycystin-1—the predominant causal factor in ADPKD—itself contributes to ADPKD hypertension independent of cystogenesis. METHODS: We induced nephron-specific disruption of the Pkd1 gene in 3-month-old mice and examined them at 4–5 months of age. RESULTS: Kidneys from the Pkd1 knockout mice showed no apparent renal cysts, tubule dilation, or increased cell proliferation. Compared with control mice, Pkd1 knockout mice exhibited reduced arterial pressure during high salt intake; this associated with an increased natriuretic, diuretic, and kaliuretic response during the first 2–3 days of salt loading. The lower arterial pressure and enhanced natriuresis during high salt loading in Pkd1 knockout mice were associated with lower urinary nitrite/nitrate excretion and markedly increased urinary PGE(2) excretion, whereas GFR, plasma renin concentration, and urinary endothelin-1 excretion were similar between knockout and control mice. Kidney cyclooxygenase-2 protein levels were increased in Pkd1 knockout mice during high salt intake; administration of NS-398, a selective cyclooxygenase-2 inhibitor, abolished the arterial pressure difference between the knockout and control mice during high salt intake. Total kidney Na(+)/K(+)/2Cl(−) cotransporter isoform 2 (NKCC2) levels were greatly reduced in Pkd1 knockout mice fed a high salt diet compared with controls. CONCLUSIONS: These studies suggest that nephron polycystin-1 deficiency does not itself contribute to ADPKD hypertension and that it may, in fact, exert a relative salt-wasting effect. The work seems to comprise the first in vivo studies to describe a potential physiologic role for nephron polycystin-1 in the absence of cysts, tubule dilation, or enhanced cell proliferation.

BACKGROUND: The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro. METHODS: To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age. RESULTS: BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE(2) excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE(2) excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1–induced renomedullary interstitial cell contraction was observed. CONCLUSIONS: Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

The collecting duct is the predominant nephron site of prorenin and prorenin receptor (PRR) expression. We previously demonstrated that the collecting duct PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which cell type is involved remains unclear. Herein, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B1 subunit of the H+ ATPase promoters, respectively. PC KO mice had reduced renal medullary ENaC-α abundance and increased urinary Na+ losses on a low-Na+ diet compared with controls. Conversely, IC KO mice had no apparent differences in Na+ balance or ENaC abundance compared with controls. Acute treatment with prorenin increased ENaC channel number and open probability in acutely isolated cortical collecting ducts from control and IC PRR KO, but not PC PRR KO, mice. Furthermore, compared with controls, PC KO, but not IC KO mice, had increased urine volume, reduced urine osmolality, and reduced abundance of renal medullary AQP2. Taken together, these findings indicate that PC, but not IC, PRR modulates ENaC activity, urinary Na+ excretion, and water transport.

Defects in primary cilia in the nephron can cause cystic kidney disease, however, the physiological role of nephron primary cilia is largely unknown. To address this, nephron primary cilia were disrupted by targeting the intraflagellar transport 88 ( IFT88 ) gene. IFT88 f/f mice containing loxP-flanked exons 4-6 of the IFT88 gene were bred with doxycycline-inducible Pax8-rtTA/tetO7-CMV*-Cre knockin mice. Inactivation of IFT88 (KO) was induced with 2 mg/ml doxycycline in drinking water supplemented with 2% sucrose for 12 days at 2 months of age; controls were the same genotype but without doxycycline treatment; mice were studied 2-4 months after doxycycline treatment. Renal IFT88 gene recombination was observed in KO mice associated with an 88±5% decrease in whole kidney IFT88 mRNA. Metabolic cage studies were conducted under a normal-Na + (0.3% Na + , 5 days), a low-Na + (0.03% Na + , 7 days), and a high-Na + (3.2% Na + , 7 days) diet (N=10 males and 10 females per group). No significant differences were found in food intake, water intake, urine volume (UV), urinary Na + excretion (UNaV), or urinary K + excretion (UKV) between KO and control mice under either a normal-Na + or a low-Na + diet. In contrast, water intake, UV, UNaV, and UKV were higher on days 2-7 of high-Na + diet in male, but not female, KO compared with control mice (p+ diet-treated KO and control mice. Both urinary nitrite/nitrate excretion (UNOxV) and urinary prostaglandin E2 excretion (UPGE2V) were increased in high-Na + diet-treated male, but not female, KO mice (p

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR) which can promote angiotensin-II (Ang-II) formation. Although alterations in plasma sPRR levels have been reported in hypertension, the causal role of sPRR in hypertension is unknown. To investigate this, we mutated the furin cleavage site (R276A, R279A) of the PRR using CRISPR/Cas9. Because the gene encoding PRR is on the X-chromosome and male mutant mice are infertile, only male mice were studied. Mutant mice had markedly lower plasma sPRR levels (control: 16.2 ± 0.3 vs mutant 0.2 ± 0.03 ng/ml). Mutant mice had normal survival and development and no histological renal abnormalities up to 12 months of age. During normal salt intake, no differences in blood pressure, body weight, urinary water or Na + excretion, or acid-base status were observed between control and mutant mice. Compared to controls, mutant mice had an attenuated hypertensive response to 2 weeks of Ang-II infusion (400 ng/kg/min) (Figure 1). Mutant mice also had lower albuminuria (control: 327 ± 114 vs mutant: 58 ± 8 μg/day) at day 7 post Ang-II infusion. No differences in urinary Na + excretion were detected between control and mutant mice after 7 days of Ang-II infusion (control: 33.6 ± 3.1 vs mutant: 35 ± 2.7 μmol/day/gram body weight). These results suggest that sPRR plays a role in Ang-II induced hypertension and renal injury.

Activation of the intra-renal, rather than systemic renin angiotensin system (RAS) has been suggested to play a role in the development of diabetic kidney disease. In this study, we examined plasma and urinary prorenin/renin levels and urinary soluble prorenin receptor (sPRR) levels in a murine model of Type 1 diabetes. Male C57BL/6 mice were injected with streptozotocin (STZ) 50mg/kg at 3 months of age and metabolic balance studies conducted at 6 and 10 weeks post-injection. STZ injected mice developed hyperglycemia within 4 weeks of injection (STZ: 335 ± 35 vs control: 145 ± 22 mg/dL) and remained hyperglycemic at 10 weeks. At 10 weeks post-injection, STZ injected mice had significant polyuria (STZ: 20.9 ± 5.9 vs controls 1.8 ± 0.4 ml/day), increased water intake (STZ: 26.2 ± 5.9 vs controls: 6.2 ± 0.9 ml/day) and lower body weight (STZ: 28.3 ± 1.3 vs controls: 23.6 ± 1.4 grams) despite similar food intake. Compared to controls, urinary angiotensinogen, total prorenin/renin and sPRR levels were increased in STZ injected mice; these increases were proportionally greater than the trend in increased urinary microalbumin excretion (Table 1). In particular, urinary sPRR excretion was markedly increased in STZ injected mice compared to controls. In a separate experiment, 10 week old male Akita mice bearing a mutated Ins2 gene, demonstrated a 4.8 fold increase in renal medullary renin mRNA expression and a 1.5-fold increase in renal medullary PRR expression. Taken together, these results demonstrate early activation of the intra-renal RAS in Type 1 diabetes. These changes occur independent of the systemic RAS.

Background In vitro studies suggest that nephron nitric oxide synthase 3 ( NOS 3) modulates tubule Na + transport. Methods and Results To assess nephron NOS 3 relevance in vivo, knockout (KO) mice with doxycycline‐inducible nephron‐wide deletion of NOS 3 were generated. During 1 week of salt loading, KO mice, as compared with controls, had higher arterial pressure and Na + retention, a tendency towards reduced plasma renin concentration, and unchanged glomerular filtration rate. Chronic high salt‐treated KO mice had modestly decreased total NCC and total SPAK / OSR 1 versus controls, however percent phosphorylation of NCC (at T 53 ) and of SPAK / OSR 1 was increased. In contrast, total and phosphorylated NKCC 2 (at T 96/101 ) were suppressed by 50% each in KO versus control mice after chronic salt intake. In response to an acute salt load, KO mice had delayed urinary Na + excretion versus controls; this delay was completely abolished by furosemide, partially reduced by hydrochlorothiazide, but not affected by amiloride. After 4 hours of an acute salt load, phosphorylated and total NCC were elevated in KO versus control mice. Acute salt loading did not alter total NKCC 2 or SPAK / OSR 1 in KO versus control mice but increased the percent phosphorylation of NKCC 2 (at T 96/101 and S 126 ) and SPAK / OSR 1 in KO versus control mice. Conclusions These findings indicate that nephron NOS 3 is involved in blood pressure regulation and urinary Na + excretion during high salt intake. Nephron NOS 3 appears to regulate NKCC 2 and NCC primarily during acute salt loading. These effects of NOS 3 may involve SPAK / OSR 1 as well as other pathways.

The endothelin (ET) and prorenin/renin/prorenin receptor (PRR) systems have opposing physiological effects on collecting duct (CD) salt and water reabsorption. It is unknown if the CD ET and renin/PRR systems interact, hence we examined the effects of deleting CD renin or nephron PRR on CD ET system components. PRR knockout (KO) mice were polyuric and had markedly increased urinary ET-1 and inner medullary CD (IMCD) ET-1 mRNA. PRR KO mice had greatly increased IMCD ETA receptor mRNA and protein, while ETB mRNA and protein were decreased. Water loaded wild-type mice with similar polyuria as PRR KO mice had modestly increased urinary ET-1 excretion and inner medullary ET-1 mRNA, while inner medullary ETA and ETB mRNA or protein expression were unaffected. In contrast to PRR KO, CD prorenin/renin KO did not alter ET system components. Taken together, these results suggest that the nephron PRR is involved in regulating CD ET system expression, but this effect may be independent of CD-derived renin.

In vitro studies have suggested that nephron nitric oxide synthase 3 (NOS3) plays a role in the regulation of urinary Na + excretion (UNaV) and potentially contributes to blood pressure (BP) control. To assess whether NOS3 is indeed involved in the physiological regulation of UNaV and BP, mice with doxycycline-inducible nephron-wide deletion of NOS3 were generated. These mice were homozygous for loxP-flanked exons 9-12 of the NOS3 gene (contains the calmodulin binding site) and hemizygous for Pax8-rtTA and LC-1 transgenes (Pax8 promoter-rtTA confers nephron-specific targeting and LC-1 transgene contains doxycycline/rtTA-inducible Cre recombinase). Mice were treated with either vehicle (controls) or doxycycline (knockouts, KO) at 1 month of age for 12 days and studied at ~3 months of age. Nephron-specific NOS3 KO mice had renal-specific NOS3 gene recombination and reduced mRNA levels in microdissected nephron segments. Renal vascular NOS3 mRNA levels were markedly increased in KO mice, presumably as a compensatory response to nephron NOS3 KO. KO mice had modestly increased mean arterial pressure (100 ± 2 mmHg in controls and 103 ± 1 mmHg in KO, N=6, p+ ) salt diet that increased further during high (3.2% Na + ) salt intake (103 ± 2 mmHg in controls and 118 ± 3 mmHg in KO, N=6, p+ retention compared to controls over the 7 days of high salt feeding. PRC tended to be lower in KO mice on a high salt diet compared to controls (85.8 ± 13.5 ng/ml in controls and 60.1 ± 10.3 ng/ml in KO, N=12, p+ excretion and BP during high salt intake. Taken together with previous studies showing that collecting duct-specific KO of NOS3 does not alter UNaV or BP, our data suggest that thick ascending limb is at least partly involved – further mechanistic studies are ongoing.

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na+and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na+or K+excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na+channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na+and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na+and water excretion in general or vasopressin regulation of CD function in particular.

Background Renin is synthesized in the collecting duct and is regulated differently than renin in the juxtaglomerular apparatus. However, the physiological relevance of collecting duct renin remains unknown, particularly with regard to its ability to regulate blood pressure. Methods We used gene targeting to generate mice with overexpression of renin in the collecting duct. A conditional mutant mouse line was created with the mouse renin transcript distal to a "transcriptional stop sequence" such that gene expression only occurred when the stop sequence was excised. These mice were bred with mice transgenic for the aquaporin-2 promoter driving Cre recombinase in order to achieve collecting duct-specific overexpression of renin. Results RNA analysis confirmed kidney-specific recombination, and medullary renin mRNA levels were increased 5-fold in collecting duct renin mice. Blood pressure was recorded by telemetry and plasma and urine was collected in 24-hour metabolic cages on normal, high-, and low-Na+ diets. Although no significant differences in 24-hour urinary Na+ excretion between targeted and control mice were detected, renin overexpresser mice had elevated blood pressure compared with controls on a high-Na+ diet. Urinary renin excretion was 2-fold higher in targeted mice as compared with controls on normal and low-Na+ diets. Plasma renin concentration was significantly suppressed in targeted mice as compared with controls on normal and high-Na+ diets. Conclusion Taken together, these results suggest that collecting duct-derived renin has the potential to modulate blood pressure independent of the systemic renin-angiotensin system.

Background Angiotensinogen (AGT) is synthesized in the liver and proximal tubule. AGT overexpression at either site might increase blood pressure (BP). We used transgenic mice with AGT overexpression in proximal tubule (K), liver (L), or both sites (KL) to determine the relative contributions of hepatic- and proximal tubule–derived AGT in modulating BP. Methods Hepatic AGT overexpression was obtained using the albumin enhancer promoter; the kidney androgen protein gene was used for proximal tubule AGT overexpression. BP and renin angiotensin system parameters were examined in male KL, K, L, and wild-type mice on normal and high-sodium diets. results Compared with wild-type mice, K and KL mice had higher BP on normal and high-sodium diets. L mice had similar BP to wild-type mice on a normal-sodium diet, but high sodium intake caused hypertension. There were no differences in plasma AGT, plasma renin concentration, urine volume, or urine sodium excretion between the groups. Urine AGT and angiotensin II (Ang II) excretion were higher in KL and K mice than in L or wild-type mice on a normal-sodium diet and increased with high sodium intake. During high sodium intake, urine AGT and Ang II were higher in all transgenic mice vs wild-type mice. conclusions Mice with liver AGT overexpression manifest salt-sensitive hypertension, whereas mice with renal AGT overexpression are hypertensive regardless of salt intake. Systemic AGT may stimulate endogenous renal AGT synthesis during high sodium intake, leading to hypertension in L mice. This suggests that systemic and renal AGT may interact to modulate BP.

Nitric oxide (NO) inhibits collecting duct (CD) Na+and water reabsorption. Mice with CD-specific knockout (KO) of NO synthase 1 (NOS1) have salt-sensitive hypertension. In contrast, the role of NOS3 in CD salt and water reabsorption is unknown. Mice with CD NOS3 KO were generated with loxP-flanked exons 9–12 (encodes the calmodulin binding site) of the NOS3 gene and the aquaporin-2 promoter-Cre transgene. There were no differences between control and CD NOS3 KO mice, irrespective of sex, in food intake, water intake, urine volume, urinary Na+or K+excretion, plasma renin concentration, blood pressure, or pulse during 7 days of normal (0.3%), high (3.17%), or low (0.03%) Na+intake. Blood pressure was similar between genotypes during DOCA-high salt. CD NOS3 KO did not alter urine volume or urine osmolality after water deprivation. In contrast, CD NOS3 KO male, but not female, mice had lower urine volume and higher urine osmolality over the course of 7 days of water loading compared with control mice. Male, but not female, CD NOS3 KO mice had reduced urinary nitrite+nitrate excretion compared with controls after 7 days of water loading. Urine AVP and AVP-stimulated cAMP accumulation in isolated inner medullary CD were similar between genotypes. Western analysis did not reveal a significant effect of CD NOS3 KO on renal aquaporin expression. In summary, these data suggest that CD NOS3 may be involved in the diuretic response to a water load in a sex-specific manner; the mechanism of this effect remains to be determined.

The renal tubular (pro)renin receptor (PRR) has been shown to modulate water balance, blood pressure and Na + homeostasis. We recently reported that inducible nephron wide deletion of the PRR results in Na + wasting, reduced epithelial Na + channel (ENaC) expression in the kidney and attenuated hypertensive response to angiotensin-II (Ang-II) infusion. In this study, we examined the effects of PRR deletion in collecting duct (CD) specific mouse models targeting either the principal cells (PC) or intercalated cells (IC). PC-specific PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring AQP-2 Cre recombinase. Compared to floxed mice, PC specific KO PRR mice had no differences in PRR immunostaining but had 50% reduction in PRR mRNA in micro-dissected cortical CDs. No differences in blood pressure were observed between the two groups at baseline or following Ang-II infusion at 600 ng/kg/min. Similarly, plasma renin concentration and renal expression of ENaC protein isoforms were comparable between the two groups. To achieve IC-specific PRR deletion, floxed PRR mice were bred with mice expressing B-1 Cre recombinase. Compared to floxed controls, IC-specific PRR KO mice were smaller (KO body weight: 5.9 ± 1.3 g vs controls: 11.1± 1.2 g) and did not survive beyond 30 days after birth. IC-specific PRR KO mice also demonstrated marked reduction in renal medullary PRR immunostaining along with decreased renal expression of ENaC-α protein (50% reduction compared to controls), similar to the findings in nephron wide deletion of PRR. Taken together, these findings suggest that IC specific deletion of PRR but not PC-specific deletion modulates renal ENaC expression. Further studies evaluating ENaC activity in isolated cortical CDs from PC and IC specific PRR KO mice will help delineate the functional role of CD PRR in Na + homeostasis.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na+ excretion and investigated the signaling mechanisms by which PRR regulates Na+ balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na+ diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na+ balance with normal- and low-Na+ intake. PRR KO mice had an attenuated hypertensive response and reduced Na+ retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na+ channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na+ wasting and reduces the hypertensive response to ANG II.

Collecting duct (CD)-derived renin is involved in the hypertensive response to chronic angiotensin-II (Ang-II) administration. However, whether CD renin is involved in Ang-II independent hypertension is currently unknown. To begin to examine this, 12 week old male and female CD-specific renin knock out (KO) mice and their littermate controls were subjected to uni-nephrectomy followed by 2 weeks of deoxycorticosterone acetate (DOCA) infusion combined with a high salt diet. Radiotelemetric blood pressure (BP) was similar between KO and control mice at baseline; BP increased in both groups to a similar degree throughout the 2 weeks of DOCA-salt treatment. Urinary albumin excretion and plasma blood urea nitrogen were comparable between the two groups after DOCA-salt treatment. Fibrosis as assessed by Masson’s Trichrome stain/Sirius Red stain and collagen-1 mRNA expression was similar between control and KO mice. Compared to baseline, DOCA-salt treatment decreased plasma renin concentration (PRC), urinary renin excretion and medullary renin mRNA expression in both floxed and CD renin KO mice with no detectable differences between the two groups. Further, in primary culture of rat inner medullary CD, aldosterone treatment did not change renin activity or total renin content. Taken together, these data suggest that CD derived renin does not play a role in DOCA-salt hypertension.

The role of intranephron angiotensinogen (AGT) in blood pressure (BP) regulation is not fully understood. Previous studies showed that proximal tubule‐specific overexpression of AGT increases BP, whereas proximal tubule‐specific deletion of AGT did not alter BP. The latter study may not have completely eliminated nephron AGT production; in addition, BP was only assessed on a normal salt diet. To evaluate this issue in greater detail, we developed mice with inducible nephron‐wide AGT deletion. Mice were generated which were hemizygous for the Pax8‐rtTA and LC‐1 transgenes and homozygous for loxP‐flanked AGT alleles to achieve nephron‐wide AGT disruption after doxycycline induction. Compared to controls, AGT knockout (KO) mice demonstrated markedly reduced renal AGT immunostaining, mRNA, and protein levels; unexpectedly AGT KO mice had reduced AGT mRNA levels in the liver along with 50% reduction in plasma AGT levels. BP was significantly lower in the AGT KO mice compared to controls fed a normal, low, or high Na+ intake, with the highest BP reduction on a low Na+ diet. Regardless of Na+ intake, AGT KO mice had higher plasma renin concentration (PRC) and markedly reduced urinary AGT levels compared to controls. Following angiotensin‐II (Ang‐II) infusion, AGT KO mice demonstrated an attenuated hypertensive response despite similar suppression of PRC in the two groups. Taken together, these data suggest that nephron‐derived AGT may be involved in Ang‐II‐dependent hypertension, however, a clear role for nephron‐derived AGT in physiological BP regulation remains to be determined.

The nephron prorenin receptor (PRR) may modulate blood pressure (BP) and Na+ balance. Since previous models of PRR knockout (KO) mice had early lethality and/or structural defects, we developed an inducible nephron-wide PRR KO using the Pax8/LC1 transgenes. Disruption of nephron PRR at 1 month of age caused no renal histological abnormalities. On a normal Na+ diet, wild-type (WT) and PRR KO mice had similar BP and Na+ excretion. However, PRR KO mice had elevated PRC (KO- 377 ± 77 vs WT- 127 ± 19 ng Ang-I/ml/hr) and a 50% decrease in renal ENaC-α protein. Protein levels of NHE3, NKCC2, NCC and ENaC-β/γ were similar between the two groups. Treatment with mouse prorenin (10 nM for 30 min) increased ENaC channel number by 2-fold, but not open probability, in isolated split-open cortical collecting ducts (CCD) from WT mice; this was prevented by Akt inhibition (A6730) but unaffected by blockade of AT-1 (losartan), ERK1/2 (U0126) or p38 MAPK (SB203580). Addition of prorenin (10 nM) did not change isolated CCD [Ca2+]i as assessed by Fura-2 loading (10 min exposure with readings every 3 sec). On a low Na+ diet, PRR KO mice had increased Na+ excretion (Day 2: KO - 66 ± 11 vs WT- 42 ± 6 μmol/day; Day 6: KO - 39 ± 4 vs ET- 23 ± 4 μmol/day) however, no differences in BP were observed. PRC was elevated in PRR KO mice on a low Na+ diet (KO- 384 ± 40 vs WT-174 ± 12 ng/ Ang-I/ml/hr). PRR KO mice had an attenuated hypertensive response to Angiotensin-II (Ang-II) infusion at 600 ng/Kg/min for 2 weeks (MAP: KO - 117 ± 4 vs WT - 133 ± 4 mm Hg over the course of Ang-II infusion). Urinary Na+ excretion was elevated in Ang-II treated PRR KO mice as compared to WT mice (KO-344 ± 14 vs WT-268 ±30 μmol/day). Taken together, these data indicate that nephron PRR, likely via direct prorenin/renin stimulation of an Akt-dependent pathway, stimulates CCD ENaC activity. Absence of nephron PRR promotes Na+ wasting and reduces the hypertensive response to Ang-II.

Recent studies suggest that aldosterone‐mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB‐stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin‐6c, an ETB‐selective agonist, and extracellular signal‐related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total‐ERK ratio similarly in control and aldosterone‐treated cells (aldosterone alone increased phospho/total‐ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP‐stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na+ diet, or low Na+ diet + desoxycorticosterone acetate. S6c inhibited AVP‐stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c‐stimulated cGMP accumulation in cultured IMCD, or S6c‐stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP‐dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD. Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD.

It is unknown if intrarenal generation of angiotensinogen (AGT) is important in blood pressure (BP) regulation. Previous studies showed that proximal tubule-specific overexpression of AGT increases BP, while proximal tubule-specific deletion of AGT using the KAP promoter-Cre transgene did not alter BP. The latter study may not have completely eliminated nephron AGT production; in addition, BP was only assessed on a normal salt diet. To evaluate this issue in greater detail, we developed mice with inducible nephron-wide AGT deletion. Mice were generated which were hemizygous for the Pax8-rtTA and LC-1 transgenes and homozygous for loxP flanked AGT alleles to achieve nephron-specific AGT disruption after doxycycline induction. Adult Pax8-rtTA/LC-1/floxed AGT mice at 3 months of age were treated with doxycycline 2 mg/ml in drinking water for 11 days and studied 4 weeks after treatment. Blood pressure (recorded via telemetry) and metabolic balance studies were determined during 5 days of normal, high and low Na diets. Compared to controls, AGT knockout (KO) mice demonstrated significantly lower systolic, diastolic, and mean BPs on all three diets (N=4 each group). The BP reduction was most evident on a low Na diet (mean BP 107 ± 2 mmHg in controls and 88 ± 13 mmHg in AGT KO). Plasma renin concentration was higher in the AGT KO mice as compared to controls on all three diets. There were no detectable differences in weight, urine volume, urine osmolality or urine Na excretion between the controls and KO mice on all three diets, however due to variability, small differences in these parameters may not have been detected. Taken together, these data suggest that nephron AGT may contribute to BP regulation and this is most evident during low Na intake.

Adenylyl cyclase (AC)-stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4 and 6 are expressed in the CD; while AC6 is involved in modulating CD Na and...

Recent studies propose that collecting duct (CD) renin is an important modulator of blood pressure regulation, especially in conditions such as angiotensin-II infused hypertension. We used gene targeting to generate a CD-specific renin knockout (KO) to assess if CD derived renin can regulate BP. Utilizing the Cre lox P system, exon 1 of the renin gene was ablated specifically in the CD. BP was recorded via telemetry and plasma and urine were collected in metabolic cages on normal, high and low Na diets. DNA recombination showed kidney specific recombination in KO mice. Compared to floxed mice, CD renin KO mice had 70 % lower medullary renin mRNA levels and 90% lower renin mRNA in micro-dissected cortical and inner medullary CD tubules. Urinary renin levels were significantly lower in the KO mice on normal and low Na diets (45% of floxed levels) but not with high Na intake. Plasma renin concentration was significantly higher in the KO mice on all three diets. While BP was similar between the two groups on all three diets, infusion of Ang-II delayed the increase in BP in the CD renin KO group for at least 4 days post-infusion. These findings suggest that CD renin likely plays a role in normal BP regulation (evidenced by an increase in PRC) and in response to AngII infusion.

Endothelin-1 binding to endothelin A receptors (ETA) elicits profibrogenic, proinflammatory, and proliferative effects that can promote a wide variety of diseases. Although ETA antagonists are approved for the treatment of pulmonary hypertension, their clinical utility in several other diseases has been limited by fluid retention. ETA blocker-induced fluid retention could be due to inhibition of ETA activation in the heart, vasculature, and/or kidney; consequently, the current study was designed to define which of these sites are involved. Mice were generated with absence of ETA specifically in cardiomyocytes (heart), smooth muscle, the nephron, the collecting duct, or no deletion (control). Administration of the ETA antagonist ambrisentan or atrasentan for 2 weeks caused fluid retention in control mice on a high-salt diet as assessed by increases in body weight, total body water, and extracellular fluid volume (using impedance plethysmography), as well as decreases in hematocrit (hemodilution). Mice with heart ETA knockout retained fluid in a similar manner as controls when treated with ambrisentan or atrasentan. Mice with smooth muscle ETA knockout had substantially reduced fluid retention in response to either ETA antagonist. Mice with nephron or collecting duct ETA disruption were completely prevented from ETA blocker-induced fluid retention. Taken together, these findings suggest that ETA antagonist-induced fluid retention is due to a direct effect of this class of drug on the collecting duct, is partially related to the vascular action of the drugs, and is not due to alterations in cardiac function.

Background Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined. Methods Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed. Results Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit. Conclusions Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.

The role of proximal tubule (PT) angiotensinogen (AGT) in modulating blood pressure has previously been examined using mice expressing PT human AGT and human renin, or rat AGT. These animals are hypertensive; however, the question remains whether alterations in mouse PT AGT alone affects arterial pressure.Mouse AGT cDNA was knocked-in to the endogenous kidney androgen protein (KAP) gene using an internal ribosomal entry site (IRES)-based strategy.The KAP-mAGT animals showed kidney-specific KAP-AGT mRNA expression; renal in situ hybridization detected KAP-AGT mRNA only in PT. Urinary AGT was markedly increased in KAP-mAGT mice. On a high Na diet, radiotelemetric arterial pressure showed a systolic pressure elevation; no significant difference in arterial pressure was observed on a normal diet. Plasma renin concentration (PRC) was reduced in KAP-mAGT animals given a high Na diet, but was not different between mouse lines during normal Na intake. Plasma AGT concentration was not altered by overexpression of PT mouse AGT.In summary, PT overexpression of mouse AGT leads to salt-sensitive hypertension without recruitment of the systemic renin-angiotensin system.

Adenylyl cyclase (AC)-stimulated cAMP plays a key role in modulating transport and channel activity along the nephron. However, the role of individual adenylyl cyclase isoforms in such regulation is largely unknown. Since adenylyl cyclase 3 (AC3) is expressed throughout nephron, we investigated its role in the physiologic regulation of renal Na(+) and water transport. Mice were generated with inducible nephron knockout of AC3 (AC3 KO) by breeding mice with loxP-flanked critical exons in the Adcy3 gene with mice expressing Pax8-rtTA and LC-1 transgenes. After doxycycline treatment at 1 month of age, nephron AC3 KO mice had 100% Adcy3 gene recombination in all renal tubule segments, but not in glomeruli. Sodium intake, urinary Na(+) excretion, glomerular filtration rate, and blood pressure were similar between nephron KO mice and the controls during normal, high, and low Na(+) diets. Plasma renin concentration was not different between the two groups during varied Na(+) intake. Moreover, there were no differences in urine volume and urine osmolality between the two genotypes during normal or restricted water intake. In conclusion, these data suggested that AC3 is not involved in the physiological regulation of nephron Na(+) and water handling.

Providing primary health care is a relatively new addition to the scope of practice for certified nurse-midwives (CNMs)/certified midwives (CMs); thus, not much is known about how this component has been integrated into midwifery practice. The purpose of this descriptive study was to examine the type and extent of primary care education among CNMs/CMs and how various common health conditions are managed. In addition, perceptions of barriers to incorporating primary care into their practice were gathered. A small national survey was conducted among randomly selected members of the American College of Nurse-Midwives. No CMs participated. Although the sample size (n = 63) precludes generalizing the findings, 65% of CNMs reported lack of formal education in primary care. However, when asked about specific health conditions, more than 50% stated that they had some form of education in treating 30 of 37 common health problems. It is noteworthy that 54% of the CNM respondents indicated that they do not provide newborn care despite the fact that more than 85% of them received newborn education. Barriers such as lack of insurance reimbursement, institutional rules, and a lack of understanding of the CNM's scope of practice were cited by respondents as contributing factors. Unfamiliarity with primary care may be the most important factor in the reluctance of CNMs to incorporate primary care into practice.

Aims The role of vascular smooth muscle endothelin A receptors (ETA) in development and normal physiology remains incompletely understood. To address this, mice were generated with smooth muscle-specific knockout (KO) of ETA. Main methods Mice were homozygous for loxP-flanked exons 6–8 of the EDNRA gene (floxed) or were also hemizygous for a transgene expressing Cre recombinase under control of the smooth muscle-specific SM22 promoter (KO mice). Key findings Genotyping at 17 days postnatal yielded a 10:1 ratio of floxed:KO mice. Smooth muscle actin staining of embryos at day E10.5 revealed increased tortuosity in dorsal aortae while E12.5 embryos had mandibular, vascular and thymic abnormalities. Mice surviving to weaning developed and bred normally. ETA KO mice aged 2–3 months manifested EDNRA gene recombination in all organs tested. Aortas from KO mice had a > 90% reduction in ETA mRNA content, but no differences in ET-1 or ETB mRNA levels. Addition of 0.01–100 nM ET-1 to isolated femoral arteries from floxed, but not KO, mice dose-dependently decreased vessel diameter (up to 80% reduction in the presence of ETB blockade). Intravenous infusion of ET-1 into floxed, but not KO, mice increased mean arterial pressure (MAP) (by ~ 10 mm Hg). Telemetric analysis revealed decreased MAP in KO mice (reduced by ~ 7–10 mm Hg) when fed a high salt diet. Significance Smooth muscle ETA is important for normal vascular, mandibular and thymic development and is involved in the maintenance of arterial pressure under physiological conditions.