Renal Interstitial Fluid Angiotensin
Modulation by Anesthesia, Epinephrine, Sodium Depletion, and Renin Inhibition
Abstract Using a microdialysis technique, we monitored changes in right and left renal interstitial fluid angiotensins in anesthetized and conscious dogs (both n=5) in response to right renal interstitial epinephrine (0.2 mg/kg per minute) administration. Renal interstitial and plasma angiotensin levels also were monitored in conscious dogs (n=4) in response to dietary sodium deprivation (10 mmol/d) for 5 consecutive days. Changes in renal interstitial and plasma angiotensins in response to interstitial administration of a specific renin inhibitor, ACRIP (0.5 μg/kg per minute for 20 minutes), were monitored on day 5 of sodium depletion. At basal levels, there were no significant differences between the right and left renal interstitial immunoreactive angiotensin levels in anesthetized dogs. Renal interstitial epinephrine administration caused a significant increase in renal interstitial immunoreactive angiotensin concentrations in both anesthetized and conscious dogs (P<.01). However, anesthetized dogs had significantly higher renal interstitial immunoreactive angiotensin levels basally and in response to epinephrine than conscious dogs (P<.05). Renal interstitial immunoreactive angiotensin concentrations increased significantly and progressively during exposure to a low sodium diet from 3.9±1 nmol on day 1 to 740±332 nmol on day 5 (P<.01). Renal interstitial immunoreactive angiotensin decreased significantly to 124±37 nmol (P<.01) in response to intrarenal renin inhibition at the end of day 5 of sodium depletion. Plasma immunoreactive angiotensin increased significantly (P<.01) in response to sodium depletion, and no change occurred during intrarenal renin inhibition. We conclude that anesthesia, epinephrine, sodium depletion, and renin inhibition modulate renal interstitial angiotensin, which may serve as an important physiological regulator.
The renin-angiotensin system plays an important role in body fluid volume, electrolyte balance, and arterial pressure.1 The mechanisms whereby these actions occur remain incompletely understood. All of the elements needed for the formation of angiotensin II (Ang II) are present within the kidney. Renin and angiotensinogen are synthesized within the kidney as demonstrated by direct measurement and by the presence of specific mRNA.2 Converting enzyme is present in renal vascular endothelium, and intrarenal formation of Ang I and Ang II has been demonstrated.1 Ang II receptors have been identified in the renal afferent and efferent arterioles, glomerular mesangium, vasa recta, and proximal tubule.3 Angiotensin generated intrarenally appears to function as a paracrine substance, locally modulating renal hemodynamic and excretory function.3
Recently, it has been shown that Ang I and Ang II are contained within renal juxtaglomerular cells and that Ang I is released from these cells.4 Angiotensins may be released from juxtaglomerular cells directly into the renal interstitium. Alternatively, Ang II may be formed within the renal interstitium after renin or Ang I release from juxtaglomerular cells. Thus, it is likely that the renal interstitium could serve as an important renal compartment for local angiotensin generation, storage, and trafficking to its cell-specific receptors.
We conducted the present study to investigate changes in renal interstitial fluid (RIF) immunoreactive angiotensin in response to halothane anesthesia, renal interstitial administration of epinephrine, sodium depletion, and renal interstitial administration of the renin inhibitor ACRIP in conscious dogs by means of renal interstitial microdialysis.
Renal Microdialysis Technique
Our laboratory developed a renal interstitial microdialysis technique.5 The microdialysis probe was made of a dialysis membrane (Clirans TH; 0.3-mm ID; molecular mass cutoff, 5000 D) connected to inflow and outflow polyethylene tubes (0.12-mm ID, 0.65-mm OD; Bioanalytical Systems, Inc). The dead space volume of the dialysis membrane and the outflow tube was 2.6 μL. A series of studies was conducted in vitro to validate the accuracy and reproducibility of the results obtained with the microdialysis probe. To calculate the relative (the percentage ratio of Ang II concentration recovered by the dialysis probe to Ang II concentration in surrounding medium) and absolute (concentration of Ang II recovered) recoveries, the inflow tube was connected to a gas-tight syringe (Hamilton Co) filled with lactated Ringer’s solution. The dialysis membrane was placed in a beaker containing 123I–Ang II. The dialysis probe was perfused with lactated Ringer’s solution (Baxter) at 1, 2, 3, 4, and 5 μL/min (20 minutes each). The dialysate was collected for each perfusion rate and counted for 123I–Ang II. This study was repeated except that the perfusion flow rate was constant at 1 μL/min, while 123I–Ang II concentration varied between 2000 and 40 000 cpm/30 μL. To test the sensitivity of the dialysis membrane to rapid changes in Ang II concentration of the surrounding media, the membrane was rapidly switched back and forth between media containing low and high 123I–Ang II levels.
Animal Preparation and In Vivo RIF Angiotensin Microdialysis
Experiments were conducted in 10 mongrel dogs weighing 15 to 20 kg. Surgery was performed with dogs under general halothane anesthesia. The right renal capsule was penetrated with an 18-gauge needle that was tunneled under the capsule for 2 cm and then exited. One end of the dialysis probe then was pulled through the needle until the dialysis fiber was situated in the outer renal cortex approximately 2 mm from the kidney surface. The needle then was withdrawn. The two ends of the dialysis probe (inflow and outflow tubes) were tunneled under the skin and exited near the interscapular region. In a previous study,6 a histological examination of the renal tissue 6 weeks after insertion of the dialysis probe did not show any significant inflammation, fibrosis, or scarring, which could impair free movement of substances between the probe and renal interstitium. For collection of RIF angiotensin samples, the inflow tube was connected to a gas-tight syringe filled with lactated Ringer’s solution and perfused at 1 μL/min (pump 22, Harvard Apparatus). The effluent was collected from the outflow tube for 30-minute sample periods in nonheparinized plastic tubes containing 8-hydroxyquinoline and EDTA to prevent angiotensin degradation. RIF samples were stored at −80°C until assayed.
In Vivo RIF Immunoreactive Angiotensin Response to Right Interstitial Epinephrine Administration
At least 2 weeks were allowed for the dogs to recover after surgery. The experiments were conducted in anesthetized (n=5) and conscious (n=5) dogs. In the anesthetized dogs, microdialysis probes were inserted into the cortex of the right and left kidneys, and RIF immunoreactive angiotensin levels were monitored simultaneously in both kidneys before and after right renal interstitial epinephrine administration at 0.2 mg/kg per minute for 20 minutes. Epinephrine was administered into the renal interstitium via the microdialysis probe. This study was repeated in conscious dogs except that only right RIF angiotensin levels were monitored.
Effects of Sodium Depletion and Renin Inhibition on RIF Immunoreactive Angiotensin
In this study, dogs (n=5) were maintained on a daily sodium intake of 50 mmol with a low sodium diet of 10 mmol/d (Hill’s H/D) and administration of 40 mmol sodium/d IV as normal saline (Baxter). The daily potassium intake was 40 mmol, and dogs had free access to water during the study. Dogs were kept in metabolic cages, and 24-hour urine collections were obtained daily. After 5 control days for the establishment of normal sodium balance at 50 mmol/d, dogs were placed on a low sodium intake at 10 mmol/d by discontinuation of the intravenous saline infusions for 5 consecutive days. RIF and plasma immunoreactive angiotensin levels were monitored at the end of the 5 control days of a normal sodium diet and at the end of each day of the low sodium diet. At the end of the fifth day of the low sodium diet, changes in RIF and plasma immunoreactive angiotensin levels were monitored in response to right renal interstitial administration of a specific renin inhibitor,7 ACRIP, at 0.5 μg/kg per minute for 20 minutes, a dose that inhibits intrarenal renin activity.7 ACRIP was administered into the renal interstitium with the use of the microdialysis probe.
Urinary sodium levels were measured by a NOVA analyzer (NOVA Biomedical). RIF immunoreactive angiotensins in the dialysate were measured by radioimmunoassay as follows. Angiotensins were measured by sample extraction followed by radioimmunoassay. Each sample was acidified (1:1) with 0.6% trifluoroacetic acid, extracted over octyldecylsilane cartridges (Sep-Pak, Waters Associates), and evaporated to dryness (SpeedVac, Savant Instruments). The dried extracts then were reconstituted in 0.01 mol/L phosphate buffer, pH 7.4, and incubated overnight in the presence of 125I–Ang II (New England Nuclear) and Ang II antibody (Dr Arthur E. Freedlender, 1.4% cross-reactive with Ang I). Free Ang I was separated from bound by charcoal addition, centrifugation, and decanting. The supernatants were then counted on a gamma counter and the counts reduced by a computer radioimmunoassay program. The sensitivity of this method (IC90) is 0.93 pg, with an intra-assay variation of 7.7% and an interassay variation of 9.6%.
Statistical Analysis of Data
Comparisons among treatments and controls were examined by ANOVA, including a repeated-measures term, using the General Linear Models procedure of SAS (Statistical Analysis System).8 Comparisons among values of corresponding periods were examined by paired t tests. Data are expressed as mean±SEM. Statistical significance was identified at a value of P<.05.
Validation of RIF Microdialysis for Ang II
Studies of in vitro recovery of labeled Ang II (Fig 1A⇓) demonstrated that a dialysate perfusion at a high speed resulted in a decrease in the percentage of relative recovery, whereas the absolute recovery of Ang II increased. Ang II relative recovery was approximately 80% with a perfusion flow rate at 1 μL/min. The Ang II relative recovery was constant at different Ang II concentrations (Fig 1B⇓) and did not deteriorate with time (Fig 2A⇓). The microdialysis probe was highly sensitive to rapid changes in Ang II concentration (Fig 2B⇓), as demonstrated by rapid changes in recovery when the concentrations were switched rapidly back and forth between low and high levels.
RIF Immunoreactive Angiotensin Response to Right Renal Interstitial Epinephrine Administration in Anesthetized Dogs
RIF immunoreactive angiotensin levels were stable during the initial pretreatment (basal) period. At basal levels, there were no significant differences between the right and left RIF immunoreactive angiotensins (Fig 3⇓). In the right kidney, basal levels of RIF immunoreactive angiotensin were 18.9±3.0 nmol and significantly increased to 154±24.1 nmol (P<.01) in response to interstitial epinephrine administration. Left RIF immunoreactive angiotensin concentrations did not change significantly during right renal epinephrine administration.
RIF Immunoreactive Angiotensin Response to Interstitial Epinephrine Administration in Conscious Dogs
Renal interstitial epinephrine administration (Fig 4⇓) caused a significant increase in RIF immunoreactive angiotensins from 8.5±5.0 to 87.2±12.4 nmol (P<.01). Comparison of the responses to epinephrine administration in anesthetized and conscious dogs (Fig 2⇑) showed that anesthetized dogs had significantly higher RIF immunoreactive angiotensin levels basally and in response to epinephrine than conscious dogs (P<.05).
RIF and Plasma Immunoreactive Angiotensins in Response to Sodium Deprivation and Intrarenal Renin Inhibition
A progressive reduction in 24-hour urinary sodium excretion was observed during a low sodium intake. Twenty-four-hour urinary sodium excretion decreased from 43±8 mmol during a normal sodium diet to 34±7, 31±8, 28±9, 18±4, and 9±4 mmol/d during days 1 through 5 of the low sodium intake, respectively. RIF immunoreactive angiotensin concentrations (Fig 5⇓) increased significantly and progressively during exposure to a low sodium diet (P<.01). RIF immunoreactive angiotensin concentrations increased approximately 200-fold by the fifth day of sodium depletion. At the end of the fifth day of sodium depletion, RIF immunoreactive angiotensins decreased significantly in response to interstitial administration of the renin inhibitor ACRIP for 20 minutes. Plasma angiotensin levels (Fig 3⇑) increased from 17±2 pmol on day 1 to 27±2 pmol on day 5 of sodium depletion (P<.01). The plasma angiotensin levels did not change significantly during renal interstitial administration of renin inhibitor.
In the present study, we used a microdialysis technique5 9 to determine whether renal interstitial angiotensins could be measured in conscious dogs and would respond to stimuli known to increase the activity of the renin-angiotensin system. We monitored changes in RIF immunoreactive Ang II in response to halothane anesthesia, renal interstitial administration of epinephrine, dietary sodium depletion, and renal interstitial administration of the renin inhibitor ACRIP.
We first conducted studies to validate the method of RIF microdialysis for Ang II. The Ang II recovery of 80%, which did not vary with rapid changes in Ang II concentrations, compares favorably with data for brain microdialysis procedures.10 Since no angiotensin-converting enzymes or angiotensinases (MW 40 to 150 000 D) should have passed through the microdialysis membrane at a molecular weight cutoff of less than 5000 D, the formation and/or degradation of Ang II in collected dialysate fluid will not occur.
In the present study, we were able to measure RIF immunoreactive angiotensins during normal sodium balance. This suggests that angiotensin formation/metabolism is present within the renal interstitium during normal physiological conditions and exerts a role in the tonic/basal control of renal function. This is consistent with renal functional responses to interruption of the renin-angiotensin system in normal awake animals.11 Immunoreactive angiotensin levels increased significantly during anesthesia and epinephrine administration. Although the exact mechanisms by which anesthetic agents or epinephrine activates the renin-angiotensin system are not well established, the observed increase in angiotensin levels supports previous studies12 13 showing an increase in plasma renin activity in response to anesthesia and epinephrine administration. In the present study, we cannot conclude whether the increases in RIF immunoreactive angiotensins in response to epinephrine were related to direct stimulation of renin secretion by juxtaglomerular cells or caused by decreased renal blood flow secondary to local vasoconstriction induced by epinephrine. Since RIF angiotensins were measured by radioimmunoassay, the observed changes in their levels may encompass changes in different renal interstitial angiotensin peptides (eg, Ang II, Ang III, and Ang IV). The absence of changes in left RIF immunoreactive angiotensin concentrations during right renal epinephrine administration suggests that infused epinephrine was confined to the right kidney and did not enter the systemic circulation in sufficient quantities to affect the left kidney.
RIF and plasma immunoreactive angiotensin concentrations increased significantly and progressively during exposure to a low sodium diet. However, RIF angiotensin concentrations (nanomolar) were 1000-fold higher than plasma angiotensin concentrations (picomolar), suggesting that angiotensins are generated mainly within the kidney. We were able to decrease RIF immunoreactive angiotensin levels with renal interstitial administration of a renin inhibitor. In contrast, plasma immunoreactive angiotensin levels did not change significantly during administration of a renin inhibitor, suggesting that renin inhibition was mainly confined to the kidney and did not enter the systemic circulation in sufficient quantities to affect circulating angiotensin levels. These observations suggest that the renin-angiotensin system functions predominantly as a paracrine (cell-to-cell) mediator within the kidney. Thus, a local intrarenal action through the interstitial compartment may be the predominant mechanism by which angiotensins control renal function. Studies of renal function in response to manipulation of the renal interstitial concentration of different angiotensins will be required to confirm this hypothesis.
In summary, we have provided evidence that RIF angiotensins can be measured in conscious dogs. Basal RIF angiotensin concentrations are 1000-fold greater than circulating angiotensin levels. RIF immunoreactive angiotensin concentrations increased in response to anesthesia, epinephrine, and sodium depletion. RIF immunoreactive angiotensins significantly decreased in response to administration of a renin inhibitor into the renal interstitium. These findings suggest that the renin-angiotensin system within the kidney may function by means of alterations in the RIF concentration of different angiotensins.
This work was supported by grants HL-47669 and HL-49575 from the National Heart, Lung, and Blood Institute, National Institutes of Health, to H.M.S. and R.M.C. Dr Helmy M. Siragy is the recipient of a Research Career Development Award (1 K04 HL-03006) from the National Institutes of Health, Bethesda, Md.
- Received October 19, 1994.
- Revision received November 15, 1994.
- Accepted December 28, 1994.
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