ACUTE ANGIOTENSIN II INFUSIONS ELICIT PRESSURE NATRIURESIS IN MICE AND REDUCE DISTAL FRACTIONAL SODIUM REABSORPTION

Abstract

Acute angiotensin II (Ang II) infusions into mice increase arterial pressure (AP) and elicit pressure natriuresis. We used this model of pressure natriuresis to delineate the distal nephron responses to AP mediated increases in distal sodium delivery (DSD). In the first group, we measured changes in urinary sodium excretion (U_NaV) in male C57/BL6 anesthetized mice (n=9) before and during acute Ang II infusions (5 ng/g Body Weight (BW)/min). Acute Ang II infusions increased AP (98±3 mm Hg to 126±5 mm Hg, P<0.001), urine flow (2.7±0.5 μl/min to 6.0±0.8 μl/min, P<0.01) and U_NaV (0.6±0.2 μEq/min to 1.3±0.2 μEq/min, P<0.05). There were significant relationships between U_NaV and urine flow (y = 0.207x + 0.3, P<0.0001) and between U_NaV and AP (y = 0.027x - 2.1). In a separate series, DSD and fractional reabsorption of DSD (FRDSD) were determined in control (n=12) and Ang II infused mice (n=8) by comparing U_NaV before and following blockade of the two major distal nephron sodium transporters with amiloride (5 mg/kg BW) plus bendroflumethiazide (12 mg/kg BW). A positive relationship was found between U_NaV (y = 0.015x - 1.1, P<0.0001) or DSD (y = 0.027x - 0.9, P<0.0001) and AP. An inverse relationship was found between FRDSD and AP (y = -0.511x + 128.3, P<0.01). These data indicate that Ang II mediated pressure natriuresis involves an increase in distal sodium delivery combined with a reduced distal nephron fractional sodium reabsorption suggesting that increased AP prevents the distal nephron transport mechanisms from accommodating to the increased distal delivery.

Introduction

It is well known that increases in arterial pressure (AP) lead to the increases in renal sodium excretion, a phenomenon commonly referred to as pressure natriuresis (1-8). The changes in renal sodium excretion in response to changes in AP provide a link between the mechanisms regulating sodium excretion and those regulating AP (1). Because glomerular filtration rate (GFR) and filtered sodium load are autoregulated over the same AP range, the mechanism of pressure natriuresis involves decreased tubular sodium reabsorption in response to increased AP (6); however, the relative contributions of the various nephron segments responsible have not been fully defined (5, 9-12). Acute angiotensin II (Ang II) infusions in mice and rats increase AP and elicit pressure natriuresis suggesting that it is a useful model to delineate the relative contributions of the various nephron segments to increases in AP.

The development of a mouse model with minimal surgical interventions to study pressure natriuresis would allow evaluation of the roles of specific tubular transport systems by utilizing various gene targeted models. While several studies have focused on the AP effects on proximal nephron segments (13-14), the distal nephron segments consisting of distal convoluted tubule, connecting tubule, cortical and medullary collecting ducts are ultimately responsible for the fine regulation of sodium excretion, even though the proximal tubule is responsible for reabsorbing the bulk of the glomerular filtrate (15). Sodium reabsorption at distal nephron segments is mainly mediated by amiloride (AM) sensitive-epithelial sodium channels and bendroflumethiazide (BFTZ) sensitive-Na⁺-Cl^- cotransporters (NCC). In the presence of AM plus BFTZ to block most of sodium transport at distal nephron segments, sodium excretion provides a collective measure of sodium delivery to distal nephron segments (9, 16). Sodium reabsorption by distal nephron segments can thus be determined from the difference between Urinary sodium excretion (U_NaV) during distal blockade and U_NaV in the absence of blockade of epithelial sodium channels and NCC. Although it has been clearly shown that increases in AP elicit increases in distal sodium delivery, the final effect of such changes on sodium excretion would be minimized by the predictable ability of the distal nephron transport mechanisms to accommodate the increased sodium delivery by increasing reabsorption rate (17). However, a failure to accommodate the increased distal sodium delivery would augment the natriuretic responses (9, 12).

In this study, we hypothesized that the pressure natriuresis elicited by acute Ang II infusion is dependent not only on the increased distal sodium delivery but also on a failure of the distal nephron segments to accommodate the increased delivery by augmenting reabsorption rate. We determined the relationships between AP and U_NaV, distal sodium delivery, and fractional reabsorption of the sodium delivered to distal nephron segments during acute Ang II infusions in mice. We recognize that the direct intrarenal effects of Ang II stimulate tubular sodium reabsorption and reduce sodium excretion and thus may blunt the responses to the increases in arterial pressure (15, 18-21). This is particularly true during chronic Ang II infusions which elevate intrarenal Ang II levels (20, 22). For the present study, Ang II infusions were selected so that the natriuretic effects of the elevated arterial pressure predominated over any direct antinatriuretic effects (21-23).

Methods

Animals

Studies were performed on 9- to 12-week-old male C57/BL6 mice (Jackson Laboratory, Bar Harbor, Maine) that were maintained at 12:12-hr light-dark schedule (6 AM to 6 PM) at 25°C in the vivarium at Tulane University Health Sciences Center. Normal salt diet along with tap water was provided. The protocol was approved by the Institutional Animal Care and Use Committee of Tulane University Health Sciences Center.

Experimental Protocol

In the first series of mice (n=9), we measured changes in sodium excretion in male C57/BL6 anesthetized mice before and during acute Ang II (Phoenix Pharmaceuticals, Burlingame, CA) infusions at two doses of 5 and 10 ng/g body weight (BW)/min (18). On the day of the experiment, mice were anesthetized with Inactin (thiobutabarbital sodium) injected intraperitoneally at 200 mg/kg body weight. Supplemental doses of anesthesia were administrated as required to maintain a stable plane of anesthesia. Once a stable level of anesthesia was obtained, judged by heart rate and lack of toe reflex, mice were placed on a surgical table (37°C) with servo-control of temperature to maintain body temperature at 37°C. After shaving incision site, a tracheostomy was performed with PE-90 tubing with the exterior end of the tracheal cannula placed inside a small plastic chamber into which humidified 95% O₂/5% CO₂ was continuously passed. The right carotid artery was cannulated with PE-10 tubing connected to PE-50 tubing for continuous measurement of AP and blood sampling. AP was recorded on a Grass polygraph (Grass Instrument Co., Quincy, MA) through a Statham pressure transducer (Model P23 Db, Statham-Gould, Oxnard, CA). The right jugular vein was catheterized with PE-10 tubing connected to PE-50 tubing for fluid infusion. During surgery, an isotonic saline solution containing 6% albumin (bovine; Sigma Chemical Co., St. Louis, MO) was infused at a rate of 4μl/min. The bladder was catheterized with PE-90 tubing via a suprapubic incision for urine collections. After surgery (in the left decubitus position), the intravenous infusion solution was changed to isotonic saline containing 1% albumin and was infused at 4μl/min. After a 30 minute recovery period, urine was collected for 30 minutes as control (period 1). Ang II was infused following period 1. A 5 minute period was allowed after which a urine sample was collected during acute Ang II infusion (period 2).

In the second series, renal plasma flow (RPF) and GFR were assessed by a renal clearance protocol in mice previously reported (18) in a control group (n=12) and a group (n=8) continuously infused with Ang II for the duration of the experiment. After surgery, the intravenous infusion solution was changed to isotonic saline containing 1% albumin, 4.5% polyfructosan (Inutest, Laevosan, Linz/Donau, Austria), and 1.5% para-aminohippurate (PAH, Merck Sharpe & Dohme, West Point, PA) and was infused at 4μl/min. The Ang II infused mice also received Ang II at 5 ng/g BW/min. After a 60 minute equilibration period, two 30 minute control urine samples were collected. Following the two control periods (period 1 and 2), a combination of AM (5 mg/kg BW) to block epithelial sodium channels and BFTZ (12 mg/kg BW) to block NCC was administered iv as previously suggested (9, 16) and a 15 minute period was allowed after which a 30 minute urine sample was collected (period 3). The sodium excretion during blockade was used as an estimate of sodium delivery to the distal nephron segments. When compared with sodium excretion measured during periods 1 and 2, distal nephron sodium reabsorption was determined. Terminal arterial blood samples were collected from the arterial catheter at the end of the experiment for measurements of plasma PAH, inulin, and sodium concentrations.

Urine, Plasma PAH and Inulin Measurements

Urine and plasma PAH and inulin concentrations were measured using standard colorimetric techniques as reported previously adapted for a plate reader (24). RPF was estimated from PAH clearance calculated as the ratio of urine and plasma PAH concentrations times urine flow. GFR was calculated as the ratio of urine and plasma inulin concentrations times urine flow.

Urine and Plasma Sodium Measurements

Urine output was determined gravimetrically assuming a density of 1g/ml. Urine and plasma sodium concentrations were measured using flame photometry (Flame Photometer IL 973, Instrumentation Laboratory, Lexington, MA). U_NaV was normalized by 30 minutes (urine collection period).

Calculations

U_NaVc = absolute U_NaV during control periods (the average of periods 1 and 2).

U_NaV_AM + BFTZ (distal sodium delivery, DSD) = absolute U_NaV during AM + BFTZ (period 3).

Filtered sodium = GFR × plasma sodium concentration.

Fractional distal sodium delivery (FDSD) = DSD/Filtered sodium (control period). Absolute sodium reabsorption at distal nephron segments (distal sodium reabsorption, DSR) = U_NaV_AM+BFTZ - U_NaVc.

Fractional reabsorption of distal sodium delivery (FRDSD) = (U_NaVAM+BFTZ - U_NaVc)/U_NaV_AM+BFTZ.

Statistical Analysis

The statistical analysis was performed by paired t test using the GraphPad PRISM program (GraphPad, San Diego, CA) in the same individual mouse data (the first series) and unpaired t test between control group and acute Ang II infusion group. The relationships between AP and U_NaV, distal sodium delivery and fractional reabsorption of distal sodium delivery were analyzed by linear regression, respectively. The results are presented as Mean ± SEs. Significance was set at P<0.05.

Results

The Relationship between Urinary Sodium Excretion and Urine Flow or AP

Acute Ang II infusions (5 ng/g BW per minute) increased AP from 98±3 mm Hg to 126±5 mm Hg (P<0.001), and increased urine flow from 2.7±0.5 μl/min to 6.0±0.8 μl/min (P<0.01) and U_NaV from 0.6±0.2 μEq/min to 1.3±0.2 μEq/min (P<0.01). Higher doses did not significantly increase urine flow or U_NaV suggesting direct antinatriuretic effects that opposed the natriuretic effects of increased AP. As shown in Figure 1A-B, there were significant relationships between U_NaV and urine flow (y = 0.207x + 0.3, P<0.0001) and between U_NaV and AP (y = 0.027x - 2.1).

Figure 1.

The relationship between urinary sodium excretion and urine flow or AP in individual mouse. AP, arterial pressure; U_NaV, urinary sodium excretion.

Effects of Acute Ang II Infusions on AP, RPF, GFR, Urine Flow and Urinary Sodium Excretion

As shown in Table, AP was higher in the Ang II-infused group as compared with the control group (126 ± 5 mm Hg versus 90 ± 3mm Hg, P<0.001). The Ang II-infused group had reduced RPF (0.8±0.1 ml/min versus 1.3±0.2 ml/min, P<0.05), but GFR values were similar to the control group (0.21±0.01 ml/min versus 0.20±0.04 ml/min, P>0.05) reflecting the direct renal vascular effects of Ang II. The Ang II-infused group had higher urine flow (5.0±0.4 μl/min versus 2.4±0.3 μl/min, P<0.001) and U_NaV (0.88±0.13 μEq/min versus 0.25±0.05 μEq/min, P<0.001) as compared with the control group.

Table.

AP and Renal Function in Control and Ang II-Infused Mice before and during AM+BFTZ

	Before AM+BFTZ		During AM+BFTZ
Parameter	Control n=12	Ang II n=8	Control n=12	Ang II n=8
AP (mm Hg)	90±3	126±5‡	88±3	120±5‡§
RPF (ml/min)	1.3±0.2	0.8±0.1*	1.3±0.2	1.0±0.2*
GFR (ml/min)	0.20±0.04	0.21±0.01	0.19+-0.03	0.20±0.02
Urine Flow (μl/min)	2.4±0.3	5.0±0.4‡	5.7±0.7‖	9.2±0.8†§
UNaV (μEq/min)	0.25±0.05	0.88±0.13‡	1.44±0.17‖	2.37±0.15†‖

Values are given as mean ± SEs.

^*P<0.05

^†P<0.01

^‡P<0.001 versus control

^§P<0.01

^‖P<0.001 vs same group before AM+BFTZ.

AP, arterial pressure; RPF, renal plasma flow; GFR, glomerular filtration rate; U_NaV, urinary sodium excretion.

Distal Sodium Delivery and Distal Nephron Sodium Reabsorption

The Ang II-infused group showed an increased absolute distal sodium delivery (2.4±0.2 μEq/min versus 1.4±0.2 μEq/min, P<0.01) and fractional distal sodium delivery (7.6±0.6% versus 5.1±0.7%, P<0.05) as compared with the control group indicating that sodium reabsorption at earlier nephron segments decreased during acute Ang II infusion because filtered Na⁺ values were similar in both groups (31.9±2.2 μEq/min versus 32.3±5.2 μEq/min, P>0.05).

Although distal sodium delivery was increased in the Ang II-infused group, this did not translate to increased distal sodium reabsorption and there was not a significant difference in absolute distal sodium reabsorption between the Ang II-infused group and the control group (1.5±0.1 μEq/min versus 1.2±0.2 μEq/min, P>0.05). However, the Ang II-infused group showed a significantly decreased fractional reabsorption of the distal sodium delivery as compared with the control group (63.1±4.2% versus 83.2±4.3%, P<0.01).

The Relationship between Urinary Sodium Excretion, Distal Sodium Delivery or Fractional Reabsorption of Distal Sodium Delivery and AP

As shown in Figure 2A-C, when values from both control and Ang II-infused group were plotted together, there was a significant relationship between U_NaV and AP (y = 0.015x - 1.1, P<0.0001) similar to what was observed in the first protocol. These data confirm that the increases in U_NaV were associated with the increases in AP. There was a significant relationship between distal sodium delivery and AP (y = 0.027x - 0.9, P<0.0001). These data suggest that sodium reabsorption decreased at more proximal nephron segments in response to elevations of AP. The relationship between distal sodium delivery and AP were also analyzed depending on AP level. Interestingly, there was a significant relationship between distal sodium delivery and AP at AP below 105 mm Hg (y = 0.048x - 2.8, P<0.01). However, a positive relationship between distal sodium delivery and AP at AP above 105 mm Hg was not observed (y = 0.014x - 0.7, P=0.24). In addition, there was a significant relationship between fractional reabsorption of distal sodium delivery and AP (y = -0.511x + 128.3, P<0.01). These data suggest that the distal nephron failed to respond to the increased delivery by increasing reabsorption rate thus leading to a decreased fractional sodium reabsorption at distal nephron segments in response to the increases in AP caused by acute Ang II infusions.

Figure 2.

The relationship between urinary sodium excretion, distal sodium delivery or fractional reabsorption of distal sodium delivery and AP. There was a significant relationship between distal sodium delivery and AP at AP below 105 mm Hg (y = 0.048x - 2.8 P<0.01). However, a positive relationship between distal sodium delivery and AP at AP above 105 mm Hg was not observed (y = 0.014x - 0.7 P=0.24). AP, arterial pressure; DSD, distal sodium delivery; FRDSD, fractional reabsorption of distal sodium delivery.

Discussion

The present study provides evidence that acute Ang II infusions to mice elicit pressure natriuresis due to the increases in distal sodium delivery coupled with a reduced fractional reabsorption of distal sodium delivery. However, the sodium excretion responses to Ang II infusions are complex and depend on the dose and duration of the Ang II infusions as well as the magnitude of the blood pressure responses. In this study, Ang II infusion increased AP, urine flow and U_NaV. We found a significant relationship between U_NaV and AP, suggesting that the natriuresis was due primarily to the associated changes in AP. In Leong and McDonough's study (19), there was no increase of urine output with acute infusions of captopril and Ang II in the absence of a change in AP. However, urine output increased with a 50- to 60-mm Hg elevation of AP by arterial constriction, although the effects were reduced 50% as compared with control that was without Ang II clamp. These data indicate that the natriuresis with acute Ang II infusion is primarily due to the increases in pressure while the direct effects of Ang II partially counteract the pressure natriuresis.

The effects of Ang II on renal function result from a combination of Ang II type 1 (AT₁) and Ang II type 2 (AT₂) receptor-mediated events with the AT₁ receptor-mediated effects generally predominant (25). Thus, Ang II administration results in net renal vasoconstriction (25). In this study, RPF decreased during acute Ang II infusions, whereas GFR did not significantly alter. Cervanka et al showed that RPF and GFR increased during volume expansion (18). Interestingly, the increases in RPF and GFR were inhibited during concurrent Ang II infusion with volume expansion (18). Mattson et al showed similar results in volume-expanded rats (26). It was also reported that the Ang II type 1 receptor antagonist increased RBF by 21% whereas AP fell by only 4%, suggesting that the renal vasculature is already under substantial influence mediated by endogenous Ang II (27). Ang II blockade increased GFR indicating that the kidney is under a substantial Ang II mediated influence even in normal control conditions. Thus, acute systemic Ang II infusions that raise AP probably do not make a significant impact on the intrarenal Ang II mediated effects and the major effects of acute Ang II infusions are mainly due to the associated changes in AP thus explaining the natriuretic effects of acute Ang II infusions as contrasted with the antinatriuretic effect of chronic Ang II infusions (20). In the recent study by Sandberg et al (28), acute Ang II infusions restored the apical localization of NCC that had been reduced by ACE inhibition, but did not increase apical membrane localization above that in control non ACE treated rats.

Blockade of the two major sodium transporters in the distal nephron segments with AM plus BFTZ leads to a marked increase in sodium excretion which then provides a collective measure of sodium delivery to distal nephron segments. In this study, sodium delivery to distal nephron segments increased during acute Ang II infusions. The increase in distal sodium delivery indicates decreased sodium reabsorption in the earlier nephron segments since filtered sodium was not significantly altered. While the technique used in this study can not delineate differences in sodium reabsorption between proximal convoluted tubule and the Loop of Henle, various studies in the rat have shown that inhibition of proximal fluid reabsorption is a primary response to acute hypertension and contributes to pressure diuresis (5, 29). The mechanisms are related to both endocytic removal of apical Na⁺/H⁺ exchangers (NHE) and basolateral Na⁺ pumps as well as decreased total Na⁺ pump activity (30). More recently, it has been shown that NHE₃ is redistributed to a domain at the base of the microvilli rather than endocytosed (31). Chou and Marsh further demonstrated that the Loop of Henle does not contribute to pressure natriuresis but actually partially compensates since there was a 40% increased flow out of the proximal tubule and only a 13% increased flow into the distal convoluted tubule (5). These studies suggest that the Loop of Henle may play a compensatory role during the process of pressure natriuresis. In contrast, Roman (11) reported that alterations in medullary hemodynamics participate in the pressure-natriuretic response by inhibiting tubular reabsorption in the proximal tubule and the thin descending limb of the loop of Henle (or both) of juxtamedullary nephrons. Thus, changes in renal perfusion pressure within the autoregulatory range may elicit changes in tubular sodium reabsorption in multiple nephron segments (32). Renal interstitial pressure could influence tubular sodium reabsorption directly or indirectly through the release of medullary humoral factors (33). Preventing renal interstitial hydrostatic pressure (RIHP) from increasing in response to increases in renal perfusion pressure attenuates pressure natriuresis and increases in RIHP decrease proximal sodium reabsorption (7). The exact mechanism whereby RIHP influences tubular reabsorption is unknown, but may be related to alterations in tight junctional permeability to sodium in proximal tubules, redistribution of apical sodium transporters, and/or release of renal autacoids such as prostaglandin E2 (7). Increases in AP stimulate increased intrarenal nitric oxide levels which have also been implicated in mediating pressure natriuresis (34). The present data also show a positive relationship between AP and distal sodium delivery, indicating that sodium reabsorption in nephron segments proximal to the distal nephron decrease with elevations of AP. However, Majid et al observed that the slope of the relation between renal AP and distal sodium delivery was markedly attenuated at renal AP above 100 mm Hg, but not below 100 mm Hg during treatment with AM plus BFTZ in dogs (9). This study suggests that at AP above 100 mm Hg, distal sodium delivery is relatively stable and the changes in sodium excretion are mediated primarily by changes in distal sodium reabsorption. In our study, fractional U_NaV rates varied from less than 1% during control conditions to ≈ 4% during dual distal nephron blockade with AM plus BFTZ suggesting predominant blockade of distal nephron transporters, epithelial sodium channels and NCC. If our dose of AM also inhibited NHE isoform NHE3, in proximal tubules, we would have expected much greater increases in urinary sodium excretion. While proximal nephron effects can not be ruled out, AM did not affect Li⁺ clearance in rats (35) and slightly increased fractional Li⁺ excretion in dogs (36). However, the effect of AM to increase Li⁺ excretion may be due to inhibition of distal Li⁺ uptake in rats (37). BFTZ did not increase Li⁺ clearance in rats, and there was actually a decrease in Li⁺ clearance with BFTZ treatment (38).

Our key novel finding is that fractional reabsorption of distal sodium delivery decreased in response to the Ang II-mediated increases in AP and an inverse relationship was found between AP and fractional reabsorption of distal sodium delivery. These findings indicate that there is not a significant compensatory increase in distal sodium reabsorption which thus allows the effects of increased distal sodium delivery to elicit proportionately greater increases in sodium excretion. We would also emphasize that the slope of the inverse relationship between AP and fractional reabsorption of distal sodium delivery is not trivial but quite substantial going from 83% to 63% which translates to a large change in sodium excretion. These data suggest that the pressure-natriuresis during acute Ang II infusions is mediated, at least partially, by an effect of elevated AP to reduce fractional reabsorption of distal sodium delivery. Sonnenberg et al (12) showed that increases in perfusion pressure led to inhibition of sodium chloride reabsorption in medullary collecting duct. McDonough (39) reported that NCC redistributed from apical plasma membrane to subapical cytoplasmic vesicles with a 50 mm Hg elevation of AP for 15-20 minutes.

In summary, acute Ang II infusions altered AP without changes in GFR. Our findings of reduced fractional reabsorption of distal sodium delivery during acute Ang II infusions provide evidence that the distal nephron segment fails to accommodate to the increased delivery and thus allow a much greater fraction of the distal delivery to be excreted, thus, contributing to the natriuresis and diuresis. These results are consistent with the concept that increases in AP inhibit sodium reabsorption by the main distal nephron sodium transporters, which are related to increased intrarenal nitric oxide levels in response to increases in AP (6, 35, 40-41).

Perspective

Recent studies of monogenetic diseases have identified mutations of distal sodium transporters involving gain of function associated with hypertension. The hypertension could be related to impaired pressure natriuresis due to inability to inhibit distal nephron sodium transport. The high efficacy of thiazide like diuretics could be due to their ability to enhance sodium excretion via replication of pressure natriuresis mechanisms. The present study develops a mouse model to study pressure natriuresis that will allow evaluation of the roles of specific tubular transport systems in mediating AP dependent changes in net tubular sodium reabsorption.