Monophosphate and Interstitial Hydrostatic Pressure

Reinforcing Feedback Loop of Renal Cyclic Guanosine 3ⴕ5ⴕ-Monophosphate and Interstitial Hydrostatic Pressure in Pressure-Natriuresis David C. Lieb, Brandon A. Kemp, Nancy L. Howell, John J. Gildea, Robert M. Carey

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Abstract—This study addresses the hypothesis that renal interstitial (RI) cGMP, a modulator of pressure-natriuresis, exerts its effect through a relationship with renal interstitial hydrostatic pressure (RIHP). Increasing renal perfusion pressure in Sprague-Dawley rats led to increases in RIHP (5.2⫾0.6 to 10.9⫾1.6 mm Hg; P⬍0.01), urine sodium excretion (0.062⫾0.009 to 0.420⫾0.068 ␮mol/min per gram; P⬍0.01), and RI cGMP (3.5⫾0.8 to 9.5⫾1.7 fmol/min; P⬍0.01), and these effects were blocked by partial renal decapsulation. Infusion of cGMP into the RI compartment of decapsulated animals restored natriuresis (0.067⫾0.010 to 0.310⫾0.061 ␮mol/min per gram; P⬍0.01). These changes were independent of changes in glomerular filtration rate . Artificially increasing RIHP in normotensive animals increased RI cGMP (4.1⫾0.6 to 6.9⫾0.7 fmol/min; P⬍0.01) and urine sodium excretion (0.071⫾0.013 to 0.179⫾0.039 ␮mol/min per gram; P⬍0.05). Coinfusion of organic anion transport-inhibitor probenecid, or soluble guanylyl cyclase inhibitor 1-H(1,2,4) oxadiazolo-(4,2)quinoxalin-1-one, abolished these effects. Infusion of cGMP into the RI compartment of normotensive animals increased RIHP (6.7⫾0.4 to 10.3⫾0.9 mm Hg; P⬍0.001). Exogenous RI cGMP delivery did not affect total, cortical, or medullary renal blood flow. These studies suggest that extracellular RI cGMP is required for the natriuresis observed after increases in renal perfusion pressure and RIHP and that cGMP acts via a tubule mechanism. The results support an intrarenal positive-feedback loop wherein RI cGMP increases RIHP, which, in turn, increases RI cGMP, contributing to the reinforcement of pressure-natriuresis. (Hypertension. 2009;54: 1278-1283.) Key Words: sodium 䡲 kidney 䡲 cyclic GMP 䡲 natriuresis 䡲 hydrostatic pressure

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increase in RPP.2,7 This may occur through the transmission of high pressure from the renal medullary vasculature to the RI compartment.8 The increased RIHP may inhibit passive renal tubular Na⫹ reabsorption and stimulate the production of natriuretic substances.2,9 The following studies explored the relationship between RI cGMP and RIHP, 2 modulators of P-N. They tested the hypothesis that RI cGMP stimulates P-N by modulating RIHP and introduced the concept of a positive feedback loop that reinforces P-N.

ressure-natriuresis (P-N), wherein a rise in renal perfusion pressure (RPP) leads to natriuresis and a diuresis, is a primary mechanism by which the kidney regulates blood pressure.1 Multiple factors are involved in this response, but the mechanisms linking them are unknown.2 cGMP is an important modulator of P-N. Previous in vivo studies demonstrated that renal interstitial (RI) cGMP levels and urine sodium (Na⫹) excretion (UNaV) increased in parallel in response to an increase in RPP. These responses were abolished when 1-H(1,2,4) oxadiazolo-(4,2)quinoxalin1-one (ODQ), an inhibitor of the cGMP-producing enzyme soluble guanylyl cyclase (sGC), was infused into the RI compartment.3 Blocking intracellular cGMP export into the extracellular compartment with the organic anion transporterinhibitor probenecid (PB) inhibited Na⫹ transport in vitro in human renal proximal tubule (RPT) cells and abolished P-N in vivo in the rat.4,5 These studies suggested that the production of cGMP and its transport into the RI compartment are critical steps in P-N. RI hydrostatic pressure (RIHP) is another modulator of P-N.6 Studies in rats demonstrate that RIHP increases after an

Methods General Methods Please see the online Data Supplement at http://hyper.ahajournals. org for details.

Animal Preparation All of the studies were approved by the Animal Care and Use Committee at the University of Virginia and performed in accordance with the National Institutes of Health Guide on the Care and Use of Laboratory Animals. The experiments were conducted on

Received March 3, 2009; first decision March 21, 2009; revision accepted September 17, 2009. From the Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, Va. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health. Correspondence to David C. Lieb, 855 W Brambleton Ave, Eastern Virginia Medical School, Norfolk, VA 23510. E-mail [email protected] © 2009 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org

DOI: 10.1161/HYPERTENSIONAHA.109.131995

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12-week– old, 225- to 250-g female Sprague-Dawley rats (Harlan, Teklad; n⫽181) that were housed in a vivarium under controlled conditions (temperature: 21⫾1°C; humidity: 60⫾10%; light: 8:00 ⫹ AM to 8:00 PM) and received a normal Na diet (0.28% NaCl).

Specific Protocols

Renal Cortical Interstitial Infusion

Rats (n⫽42) were anesthetized with ketamine and xylazine, underwent an acute uninephrectomy, and were studied according to our P-N protocol. Three groups were evaluated. First was the intact renal capsule group wherein rats received RI vehicle infusion during both the 30-minute control and the 30-minute high RPP periods and served as time controls. RIHP, UNaV, and RI cGMP levels were recorded for each period. Second was the partial renal decapsulation group, wherein the renal capsule was partially removed before the onset of the control period. Partial decapsulation was performed to increase stability of the RIHP catheter (anchored to the renal capsule). Animals received RI vehicle infusion during both the 30-minute control and the 30-minute high RPP periods. RIHP, UNaV, and RI cGMP levels were recorded for each period. The third group received partial renal decapsulation⫹RI cGMP infusion: rats received an RI infusion of cGMP (18 ␮g/kg per minute) during the 30-minute high RPP period after a 30-minute control period with infusion of vehicle. UNaV and RI cGMP levels were recorded for each period. RBF and GFR were measured in a separate series of animals (n⫽24) also evaluated in the 3 groups described previously.

A microinfusion catheter (phycoerythrin-10) was inserted under the renal capsule into the cortex for RI infusion of pharmacological agent or vehicle at 2.5 ␮L/min with a syringe pump (Harvard; model 55-222). Vetbond tissue adhesive (3M Animal Care Products) was used to secure the tubing and prevent interstitial pressure loss.

RI Fluid Microdialysis Technique RI cGMP was collected using microdialysis probes that were constructed and used as described previously.10

Renal Blood Flow, Renal Cortical Blood Flow, and Renal Medullary Blood Flow Measurement Downloaded from http://hyper.ahajournals.org/ by guest on June 21, 2018

After acute uninephrectomy, a flow probe was secured around the left renal artery and connected to a dual-channel flowmeter (Transonic Systems, Inc) for measuring renal blood flow (RBF). RBF is reported as milliliters per minute per gram of kidney weight. Needle probes were placed into the cortex and medulla and connected to a laser Doppler flowmeter (Advance Laser Flowmeter ALF 21D) to measure renal cortical blood flow (RCBF) and renal medullary blood flow (RMBF).

Measurement of Glomerular Filtration Rate, Fractional Excretion of Sodium, and Fractional Excretion of Lithium Glomerular filtration rate (GFR) was measured by inulin clearance using a previously described method and is reported as milliliters per minute per gram of kidney weight.11 Tubular Na⫹ reabsorption was determined by calculating fractional excretion of sodium (FeNa), and RPT Na⫹ reabsorption was estimated using fractional excretion of lithium (FELi).

RIHP Measurement RIHP measurement was performed using a variant of a technique described by Nakamura et al.12 An opening was made in the renal capsule using a cautery device (Medtronic). A segment of PE-50 tubing was inserted 2 mm into the renal cortical interstitium and secured, whereas the other end was attached to a blood pressure analyzer. Studies that involved RIHP, UNaV, and RI cGMP measurements were separated into 2 groups to avoid potential renal trauma associated with the insertion of both an RIHP catheter and an RI microdialysis catheter. One group of rats had only an RIHP catheter, whereas a second group had an RI microdialysis probe to measure RI cGMP levels and a ureteral catheter to measure UNaV. Mean arterial pressure (MAP) was measured in both groups.

P-N Model A variant of the P-N model of Roman and Cowley13 was used. RPP was increased during the experimental period by tying off the infrarenal aorta and clamping the superior mesenteric artery.

Assays Cyclic Nucleotides RI cGMP levels were measured using an enzyme immunoassay (Cayman Chemical).

Urinary and Plasma Naⴙ and Liⴙ Concentrations

Urinary and plasma Na⫹ and Li⫹ concentrations were measured using a flame photometer (Instrumentation Laboratory-943). Urine flow and UNaV are reported as milliliters per minute per gram of kidney weight and micromoles per minute per gram of kidney weight, respectively.

Effects of Renal Decapsulation With and Without Intrarenal cGMP Administration on RIHP, UNaV, RI cGMP, RBF, and GFR After Increasing RPP

Effects of RI Albumin Administration in the Presence and Absence of PB or ODQ on RIHP, UNaV, and RI cGMP Rats (n⫽51) were anesthetized with ketamine and xylazine and subjected to acute uninephrectomy. Three groups were evaluated, as described here. In the 2% albumin group, rats received no RI infusion during the 30-minute control period and received a 100-␮L bolus infusion of 2% albumin into the RI compartment over 30 seconds at the onset of the 30-minute experimental period (a variant of the method described by Granger et al).14 In the 2% albumin⫹PB group, rats received no RI infusion during the 30-minute control period and received a 2% albumin bolus followed by the RI infusion of PB (10 ␮g/kg per minute) throughout the 30-minute experimental period. In the 2% albumin⫹ODQ group, rats received no RI infusion during the 30-minute control period and received a 2% albumin bolus, followed by the RI infusion of ODQ (0.12 mg/kg per minute) throughout the 30-minute experimental period. RIHP, UNaV, and RI cGMP were recorded for each period.

Effects of Intrarenal cGMP Administration on RIHP, RBF, RCBF, and RMBF Rats (n⫽30) were anesthetized with pentobarbital and underwent an acute uninephrectomy. Two groups were evaluated. In the time control group, rats received RI vehicle infusion during both the 1-hour control and 1-hour experimental periods. In the cGMP group, rats received RI cGMP infusion (18 ␮g/kg per minute) for 1 hour during the experimental period after a 1-hour control infusion of vehicle. RIHP and RI cGMP levels were recorded for each period. In a separate group of animals (n⫽7), cGMP (18 ␮g/kg per minute) was infused into the RI compartment for 1 hour during the experimental period after a 1-hour control infusion of vehicle, and RBF, RCBF, and RMBF were measured during both periods.

Effects of Intrarenal cGMP Administration on UNaV, Urine Flow Rate, MAP, GFR, FENa, and FELi Rats (n⫽15) were anesthetized with pentobarbital and studied with both kidneys intact. Inulin and lithium chloride in vehicle were infused throughout the study via the internal jugular catheter. cGMP (18 ␮g/kg per minute) was infused into the RI compartment of the left (experimental) kidney for 1 hour after a 1-hour control infusion of vehicle. The right (control) kidney received RI vehicle infusion during both the 1-hour control and the 1-hour experimental periods and served as the time control. UNaV, urine flow, MAP, GFR, FENa, and FELi were recorded for each period.

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Figure 1. A, RIHP in uninephrectomized, anesthetized rats during normal or high RPP in rats with an intact renal capsule (䡺; n⫽7), after partial decapsulation (Decap; f; n⫽6), or after partial decapsulation with RI cortical infusion of cGMP during the experimental period (u; n⫽7). B, UNaV in response to normal or high RPP in rats under conditions in A (intact capsule, n⫽8; partial decapsulation, n⫽7; partial decapsulation with RI cortical infusion of cGMP during the experimental period, n⫽7). Results are reported per gram of kidney weight. C, RI cGMP levels in response to normal or high RPP in rats under conditions in A (intact capsule, n⫽8; partial decapsulation, n⫽7; partial decapsulation with RI cortical infusion of cGMP during the experimental period, n⫽7). Data are shown as mean⫾SE. ***P⬍0.001, **P⬍0.01 vs own control; †P⬍0.05 vs intact capsule (ANOVA with Dunnett).

Statistical Analysis Results are expressed as mean⫾SE. Paired Student t test was used for comparing 2 periods within the same group of animals. Paired Student t test was also used for experiments utilizing 2 intact kidneys in the same group of animals (protocol 4). Two-sample Student t test was used when comparing 2 different groups of animals. When multiple comparisons were made across ⬎2 treatment groups, 1-way ANOVA with a repeat-measures term was used. The ANOVA test was followed by post hoc testing using the Dunnett test. P⬍0.05 was considered statistically significant.

Results Effects of Renal Decapsulation With and Without Intrarenal cGMP Administration on RIHP, UNaV, RI cGMP, RBF, and GFR After Increasing RPP Figure 1A demonstrates that, in rats with an intact renal capsule, RIHP increased (5.2⫾0.6 to 10.9⫾1.6 mm Hg; P⬍0.01) after an increase in RPP (96.5⫾3.6 to 154.3⫾4.8 mm Hg; P⬍0.001). In rats that underwent renal decapsulation, including those that received intrarenal cGMP administration, there was not a significant increase in RIHP despite a significant increase in RPP (99.6⫾2.5 to 165.6⫾5.2 mm Hg and 98.3⫾2.3 to 155.9⫾3.8 mm Hg, respectively; P⬍0.001 for each; see Figure S1A, available in the online Data Supplement, for RPP data). As shown in Figure 1B, rats with an intact capsule demonstrated an increase in UNaV (0.062⫾0.009 to 0.420⫾0.068 ␮mol/min per gram; P⬍0.01) after a significant increase in RPP (96.8⫾2.6 to 156.6⫾3.8 mm Hg; P⬍0.001). Decapsulated

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Figure 2. A, RBF in uninephrectomized, anesthetized rats during normal or high RPP in rats with an intact renal capsule (䡺; n⫽8), after partial decapsulation (Decap; f; n⫽8), or after partial decapsulation with RI cortical infusion of cGMP during the experimental period (u; n⫽8). Results are reported per gram of kidney weight. B, GFR in response to normal or high RPP in rats under conditions in A (intact capsule, n⫽8; partial decapsulation, n⫽8; partial decapsulation with RI cortical infusion of cGMP during the experimental period, n⫽8). Results are reported per gram of kidney weight. Data are shown as mean⫾SE **P⬍0.01, *P⬍0.05 vs own control.

rats had no change in UNaV despite a significant increase in RPP (98.1⫾1.9 to 158.1⫾2.3 mm Hg; P⬍0.001). However, RI infusion of cGMP in decapsulated animals restored the increase in UNaV (0.067⫾0.010 to 0.310⫾0.061 ␮mol/min per gram; P⬍0.01) after an increase in RPP (96.6⫾1.0 to 154.4⫾3.1 mm Hg; P⬍0.001). There was no significant difference between the increase in natriuresis seen in animals with an intact capsule and in those with a partial decapsulation with RI infusion of cGMP (see Figure S1B for RPP data). Figure 1C demonstrates that RI cGMP levels increased significantly (3.5⫾0.8 to 9.5⫾1.7 fmol/min; P⬍0.01) in rats with an intact capsule after an increase in RPP (96.8⫾2.6 to 156.6⫾3.8 mm Hg; P⬍0.001). There was no change in RI cGMP levels in decapsulated rats despite a significant increase in RPP (98.1⫾1.9 to 158.1⫾2.3 mm Hg; P⬍0.001). Rats that received RI infusion of cGMP had a significant increase in RI cGMP levels (1.9⫾0.4 to 11.3⫾1.1 fmol/min; P⬍0.001; see Figure S1B for RPP data). As shown in Figure 2A, RBF decreased (1.5⫾0.1 to 1.3⫾0.1 mL/min per gram; P⬍0.05) in rats with an intact capsule after an increase in RPP (93.6⫾2.8 to 146.3⫾2.2 mm Hg; P⬍0.001). There was no change in RBF in decapsulated animals after a significant increase in RPP (91.3⫾5.9 to 135.3⫾6.8 mm Hg; P⬍0.001). RBF decreased (1.9⫾0.1 to 1.5⫾0.1 mL/min per gram; P⬍0.01) after a significant increase in RPP (93.1⫾2.2 to 136.4⫾3.6 mm Hg; P⬍0.001) in animals that underwent a partial decapsulation followed by RI infusion of cGMP. There were no significant differences in RBF between the group of animals that had been decapsulated versus the animals with an intact capsule or between the animals that had been decapsulated and infused with cGMP versus the animals with an intact capsule.

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Figure 4. RIHP in uninephrectomized, anesthetized rats during control and experimental periods in response to RI cortical infusion of D5W vehicle (V) during both periods (time control, 䡺; n⫽8) or cGMP during the experimental period (f; n⫽10). Data are shown as mean⫾SE. ***P⬍0.001 vs own control; †††P⬍0.001 vs time control (2-sample Student t test).

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Figure 3. A, RIHP in uninephrectomized, anesthetized rats during control and experimental periods in response to RI bolus of 2% albumin (Alb) during the experimental period (䡺; n⫽6), RI bolus of albumin followed by RI infusion of PB (f; n⫽7), and RI bolus of albumin followed by RI infusion of ODQ (u; n⫽8). B, UNaV in response to RI infusions as for A (albumin bolus alone, n⫽8; albumin bolus⫹PB, n⫽7; albumin bolus⫹ODQ, n⫽13). Results are reported per gram of kidney weight. C, RI cGMP levels in response to RI infusions as for A (albumin bolus alone, n⫽10; albumin bolus⫹PB, n⫽6; albumin bolus⫹ODQ, n⫽13). Data are shown as mean⫾SE. ***P⬍0.001, **P⬍0.01, *P⬍0.05 vs own control; †P⬍0.05 vs albumin bolus alone (ANOVA with Dunnett).

Figure 2B demonstrates that GFR did not differ significantly in the 3 groups of animals (intact capsule, decapsulated, or decapsulated with RI cGMP infusion; see Figure S2 for RPP data).

Effects of RI Albumin Administration in the Presence and Absence of PB or ODQ on RIHP, UNaV, and RI cGMP As seen in Figure 3A, RIHP increased in rats that received an RI bolus of 2% albumin alone, 2% albumin⫹PB, or 2% albumin⫹ODQ (4.8⫾0.4 to 9.5⫾0.7 mm Hg, P⬍0.001; 4.9⫾0.3 to 9.2⫾0.6 mm Hg, P⬍0.001; 5.2⫾0.2 to 9.7⫾0.5 mm Hg, P⬍0.001, respectively). MAP was not significantly different between the control and experimental periods for each group of animals. The MAP for animals receiving albumin and ODQ was significantly different from the MAP for animals receiving an albumin bolus alone (see Figure S3A for MAP data). Figure 3B demonstrates that RI infusion of 2% albumin increased UNaV (0.071⫾0.013 to 0.179⫾0.039 ␮mol/min per gram; P⬍0.05), whereas there was no change in rats that received 2% albumin⫹PB or 2% albumin⫹ODQ. UNaV was significantly different between the animals receiving albumin and ODQ and those receiving an albumin bolus alone, whereas it was not significantly different between the animals receiving albumin and PB and

those receiving only an albumin bolus. MAP was not significantly different between the control and experimental periods for each group of animals (see Figure S3B for MAP data). Figure 3C demonstrates that the RI bolus of 2% albumin increased RI cGMP levels (4.1⫾0.6 to 6.9⫾0.7 fmol/min; P⬍0.01), whereas there was no effect after RI infusion of 2% albumin⫹ODQ. The RI infusion of 2% albumin⫹PB significantly decreased RI cGMP levels (4.5⫾0.6 to 2.6⫾0.5 fmol/min; P⬍0.01). MAP was not significantly different between the control and experimental periods for each group of animals (Figure S3B).

Effects of Intrarenal cGMP Administration on RIHP, RBF, RCBF, and RMBF Figure 4 demonstrates that RI infusion of cGMP increased RIHP (6.7⫾0.4 to 10.3⫾0.9 mm Hg; P⬍0.001), whereas RI vehicle infusion had no effect. MAP was not significantly different between the control and experimental periods (Figure S4A). RI cGMP infusion significantly increased RI cGMP levels (4.1⫾0.7 to 8.8⫾0.5 fmol/min; P⬍0.001), whereas RI vehicle infusion had no effect (Figure S4B). MAP was not significantly different between the control and experimental periods for these animals (Figure S4C). RI infusion of cGMP had no effect on total RBF (Figure S5A), RCBF, or RMBF (Figure S5B), and MAP was not significantly different between the control and experimental periods for these animals (Figure S5C).

Effects of Intrarenal cGMP Administration on UNaV, Urine Flow Rate, MAP, GFR, FENa, and FELi RI infusion of cGMP increased UNaV (0.052⫾0.007 to 0.107⫾0.012 ␮mol/min per gram; P⬍0.001), whereas RI vehicle infusion had no effect (Figure S6A). RI infusion of cGMP increased urine flow rate (0.0017⫾0.0002 to 0.0032⫾0.0003 mL/min per gram; P⬍0.001), whereas RI vehicle infusion had no effect (Figure S6B). MAP was not influenced by cGMP infusion (Figure S6C). RI infusion of either vehicle or cGMP had no significant effect on GFR (Figure S6D). RI infusion of cGMP significantly increased FENa (0.14⫾0.04% to 0.24⫾0.03%; P⬍0.05), whereas RI

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vehicle infusion had no effect (Figure S6E). RI infusion of cGMP significantly increased FE Li (22.0⫾2.0% to 35.7⫾3.4%; P⬍0.01), whereas RI vehicle infusion had no effect (Figure S6F).

Discussion


RI cGMP is an important modulating factor in P-N. Studies from our laboratory have suggested that RI cGMP acts through a mechanism that is independent of changes in renal hemodynamics and involves protein kinase G.3,15 In vitro studies in human RPT cells and in vivo studies in rats demonstrate that the production of cGMP by sGC and its export into the extracellular compartment through a PBsensitive organic anion transporter are critical mechanisms for natriuretic responses to increased RPP.4,5,15 RIHP is another modulating factor of P-N. Increases in RPP lead to increases in RIHP, and the subsequent natriuresis is abolished by renal decapsulation.7 Increases in RIHP made by RI volume expansion lead to increases in natriuresis.6,16 Our studies address the relationship between these 2 important modulating factors of the P-N relationship. The major findings of the present studies are described here. First, decreasing RIHP by renal decapsulation prevents the rise in RI cGMP levels and UNaV in response to increased RPP, and natriuresis can be restored in decapsulated animals with exogenous RI cGMP replacement. Second, directly increasing RIHP without changing MAP increases RI cGMP and UNaV, both of which can be blocked by inhibiting transport of cGMP from the intracellular to the extracellular compartment with PB or by decreasing cGMP production with the sGC inhibitor ODQ. Third, exogenous RI cGMP administration increases both RIHP and UNaV. These findings are observed in the absence of increases in GFR, RBF, RCBF, and RMBF, suggesting a tubular rather than a hemodynamic mechanism. These findings suggest that extracellular RI cGMP modulates P-N at least in part by augmenting RIHP and that an increase in RIHP is required for the rise in RI cGMP levels observed after an increase in RPP. The mechanism by which RI cGMP increases RIHP is unclear. Some investigators have suggested that an increase in RPP leads to increased RIHP through changes in RMBF.2,8,17 Intrarenal infusions of vasodilatory substances are associated with increases in RIHP that are necessary for natriuresis.18,19 Although cGMP is known to be involved in vasodilation in other vascular beds, our experiments do not support this mechanism for the increase observed in RIHP. Here we show that intrarenal infusion of cGMP does not increase RBF, RCBF, and RMBF. Additional studies are currently underway in our laboratory evaluating relationships among RI cGMP, RCBF, and RMBF during P-N. Of note, in our studies we measure cortical RIHP. Previous studies have shown that, whereas renal decapsulation significantly reduces cortical RIHP, it does not have as great an effect on medullary RIHP.7 In fact, medullary RIHP still increases significantly when RPP is increased, even in the absence of a renal capsule. It will be very important that we differentiate between cortical and medullary interstitial pressures in future studies to help understand the roles that each

play in the P-N mechanism and to define their relationships with RI cGMP. The mechanism whereby an increase in RIHP leads to the production of cGMP is unknown. Studies have documented the presence of mRNA in rat renal tissues for the 3 isoforms of NO synthase.20 Studies in mouse inner medullary collecting duct cells have shown that shear stress, or the pressure exerted by a fluid flowing past cells in culture, can stimulate NO synthase to produce NO.21 Immunohistochemistry in human kidney preparations demonstrates the presence of neuronal NO synthase (NO synthase 1) in the cortical and medullary segments of the nephron. sGC also appears to be present in these same portions of the nephron.22 These data suggest that the enzymes necessary for cGMP production exist in the rodent and human kidney and that an increase in interstitial pressure and the resultant shear stress might stimulate NO synthase and lead to cGMP production by sGC. NO is able to diffuse across many cellular membranes, and, therefore, NO produced in the renal interstitium could diffuse into surrounding renal tubule cells.23 The precise mechanism by which cGMP inhibits Na⫹ reabsorption is unclear. Our data suggest that cGMP exits the renal tubule cell through a PB-sensitive anion channels.4,5 Multidrug-resistance protein 5 has been shown to export cGMP and localizes to the basolateral membranes of canine kidney cells.24,25 cGMP may exit the tubule cell through this channel and inhibit Na⫹ reabsorption by binding to a plasma membrane– bound receptor or the channel involved in renal Na⫹ regulation, such as Na,K-ATPase. Both in vitro and in vivo animal studies have suggested that cGMP may act at this important Na⫹ pump to reduce Na⫹ reabsorption.26 –28 Some investigators have suggested that stimulation of the NO-cGMP pathway may internalize this pump, thereby inactivating it.26 The segment of the nephron involved in RI cGMPmediated inhibition of Na⫹ reabsorption requires further delineation. Studies from our laboratory have suggested that the proximal tubule is involved, because RI cGMP administration increases both FENa and FELi.4,29 In vitro studies in human RPT cells using an Na⫹-sensitive fluorescent indicator have demonstrated that exogenous administration of cGMP decreases cellular Na⫹ uptake.5 The RPT would be an effective site of action, because this nephron segment reabsorbs the majority of filtered Na⫹.30

Perspectives These studies suggest that an increase in RIHP is required for the rise in RI cGMP and UNaV seen in the P-N response and lend support to the hypothesis that extracellular transport of cGMP is required for natriuresis. Furthermore, the direct RI administration of cGMP leads to an increase in RIHP through an unknown mechanism. These findings suggest a positivefeedback loop wherein an increase in RPP leads to increased RIHP and RI cGMP, natriuresis, and a further increase in RIHP. Such a feedback loop might allow small changes in RPP to translate into a larger and sustained natriuresis and diuresis, enhancing normal homeostatic protection against a rise in blood pressure. Additional studies are underway to determine the mechanisms by which cGMP induces natriuresis and increases RIHP.

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Acknowledgments D.C.L. is the recipient of the Warren Trust Fellowship of the Consortium for Southeastern Hypertension Control and thanks them for their support and encouragement. We also thank Drs Susanna Keller and Shetal Padia for their insightful comments during the production of this article.

Sources of Funding This research was supported by award No. F32-DK-082110 from the National Institute of Diabetes and Digestive and Kidney Diseases (to D.C.L.) and by the National Institutes of Health, grants R01-HL081891 and 5T32-DK-007646 (to R.M.C.).

Disclosures None.

References


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Reinforcing Feedback Loop of Renal Cyclic Guanosine 3′ 5′ -Monophosphate and Interstitial Hydrostatic Pressure in Pressure-Natriuresis David C. Lieb, Brandon A. Kemp, Nancy L. Howell, John J. Gildea and Robert M. Carey


Hypertension. 2009;54:1278-1283; originally published online October 19, 2009; doi: 10.1161/HYPERTENSIONAHA.109.131995 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2009 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563

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ONLINE SUPPLEMENT

REINFORCING FEEDBACK LOOP OF RENAL CYCLIC GMP AND INTERSTITIAL HYDROSTATIC PRESSURE IN PRESSURE-NATRIURESIS

David C. Lieb Brandon A. Kemp Nancy L. Howell John J. Gildea Robert M. Carey

Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908

Corresponding author: David C. Lieb, MD; 855 W. Brambleton Avenue, Eastern Virginia Medical School, Norfolk, Virginia, 23510. Email: [email protected]. Telephone (757) 446-5910. Fax: (757) 446-5970.

General Methods Animal Preparation Animals were anesthetized with either ketamine (60 mg/kg) and xylazine (4 mg/kg) [Protocols 1,2] or pentobarbital (5 mg/100 g body weight) [Protocols 3,4] via an intraperitoneal (IP) injection. Following anesthesia a tracheostomy was performed using polyethylene tubing (PE-240) to assist respiration. Direct cannulation of the right internal jugular vein using PE-10 provided intravenous access through which 5% dextrose in water (D5W) alone was infused at 20 µL/min. D5W with inulin and lithium chloride was infused at 20 µL/min for studies involving the measurement of GFR. Cannulation of the right carotid artery using PE-50 provided arterial access for blood pressure (BP) measurements. For one-kidney models [Protocols 1-3] the right kidney was removed following a midline laparotomy and the remaining ureter was cannulated (PE-10) to collect urine for the quantification of urine sodium (Na+) excretion (UNaV). For the twokidney model [Protocol 4], both ureters were cannulated individually to collect urine for the quantification of UNaV. The right kidney served as the control kidney and received renal interstitial (RI) infusions of vehicle (V) D5W while the left kidney served as the experimental kidney and received RI infusions of pharmacologic agents. All experiments were performed at similar times each day to prevent diurnal variation in BP measurements. Kidney weights were measured in a group of 10 animals in order to normalize all data per gram of kidney weight. The mean weight per kidney was 0.978 ± 0.031 grams. We have approximated this to be 1 gram. Blood Pressure Measurements Mean arterial pressure (MAP) was measured through the carotid artery using a digital BP analyzer (Digi-Med). Pressures were recorded every 5 minutes and averaged for all periods in the study. MAP served as a surrogate for renal perfusion pressure (RPP) in our experiments. Hematocrit Measurements Hematocrit values were measured in a group of 12 animals in order to evaluate hydration status both before and after surgery. Prior to anesthesia (pentobarbital) and again 3 hours after surgery, whole arterial blood was obtained from the animal via tail stick, and spun for 3 minutes by centrifuge (Readacrit, BD Diagnostic Systems). Hematocrit was determined using a Spirocrit microhematocrit capillary tube reader (Monoject Scientific). The mean hematocrit prior to anesthesia was 46.67 ± 0.85%, and after surgery was 46.17 ± 1.30% (P=NS). Pharmacologic Agents Cyclic guanosine 3’5’-monophosphate (cGMP; BioLog) was used in this study to induce natriuresis. Probenecid (PB; Sigma) was employed to block organic anion transport and inhibit cGMP from leaving renal proximal tubule (RPT) cells. 1-H-[1,2,4] oxadiazolo-[4,2-α] quinoxalin-1-one (ODQ; Alexis Biochemicals) was

used to inhibit the cGMP-producing enzyme soluble guanylyl cyclase (sGC). 2% albumin (Sigma) constituted in 0.9% saline was used to increase RIHP artificially. Inulin from dahlia tubers (Sigma) was used to quantify glomerular filtration rate (GFR) and fractional excretion of Na+ (FENa). Lithium chloride (Sigma) was used to measure fractional excretion of lithium (Li+) (FELi).

SUPPLEMENTAL FIGURES

Figure S1. A, Renal perfusion pressure (RPP) in uninephrectomized, anesthetized rats with an intact renal capsule (white bars, n=7), after partial decapsulation (Decap) (black bars, n=6), or after partial decapsulation with RI cortical infusion of cGMP during the experimental period (gray bars, n=7). B, RPP in uninephrectomized, anesthetized rats with an intact capsule (white bars, n=8), after partial decapsulation (black bars, n=7), or after partial decapsulation with RI cortical infusion of cGMP during the experimental period (gray bars, n=7). Data are shown as mean ± SE. ***P