Renal Function in Mice: Effects of Volume Expansion and Angiotensin II

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1 J Am Soc Nephrol 10: , 1999 Renal Function in Mice: Effects of Volume Expansion and Angiotensin II LUDEK CERVENKA,* KENNETH D. MITCHELL, and L. GABRIEL NAVAR *Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic; and Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana. Abstract. The present study was performed to validate a simple means for assessing renal function in anesthetized mice and to characterize the renal hemodynamic responses to acute volume expansion and how these responses are altered by concurrent angiotensin II (AngII) infusions. Inulin and para-aminohippurate clearances were used to assess GFR and renal plasma flow (RPF) in three groups of male C57Bl/6 mice anesthetized with inactin (100 mg/kg, intraperitoneally) and ketamine (10 mg/ kg). To avoid the hypotension associated with repeated blood sampling, a single blood sample was taken after three timed urine collections. Renal function and mean arterial pressure (MAP) were measured under euvolemic conditions (2.5 l/ min, intravenously, n 7) during isotonic saline volume expansion (12.5 l/min, intravenously, n 5) and during volume expansion with concurrent AngII infusion (5 ng/min g, n 5). MAP in the control group was 77 2 mmhg; volume expansion alone did not change MAP significantly (83 2 mmhg), but led to significantly greater values in both GFR and RPF ( versus ml/min g and versus ml/min g, respectively). Infusion of AngII during volume expansion led to significant elevations of MAP (100 3 mmhg, P 0.05) and prevented the increases in GFR and RPF elicited by volume expansion ( and ml/min g, respectively). Volume expansion also elicited marked increases in absolute and fractional sodium excretion ( versus Eq/min g and versus %, respectively). AngII infusion attenuated the absolute and fractional sodium excretion responses to volume expansion ( Eq/min g and %, respectively). The present findings demonstrate that anesthetized mice exhibit marked renal hemodynamic and excretory responses to isotonic saline volume expansion. Concomitant AngII infusion attenuates these responses in spite of greater increases in arterial pressure. Recent advances in transgenic and gene-targeted manipulations of the murine genome have provided new and potentially powerful approaches for studying the structural, biochemical, functional, and pathophysiologic roles of different genes and their products. Indeed, a vast array of gene knockout and transgenic mouse models have been developed, and some of these have demonstrated altered renal structure and function (1 10). Nevertheless, a systematic description of the changes in renal hemodynamics has been infrequent because of the problems associated with assessing renal plasma flow (RPF) and GFR in mice. Recently, Gross et al. (11,12) defined the pressure-natriuresis-diuresis relationships in normotensive and deoxycorticosterone acetate-salt hypertensive mice. However, the absence of data for RPF and GFR prevented a more detailed assessment of the mechanisms responsible. Thus, there are still many uncertainties regarding normal baseline values for renal function in mice and the renal functional responses to experimental manipulations such as volume expansion and angiotensin II (AngII) infusions. Received December 31, Accepted June 8, Correspondence to Dr. L. Gabriel Navar, Department of Physiology SL39, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA Phone: ; Fax: ; navar@ mailhost.tcs.tulane.edu / Journal of the American Society of Nephrology Copyright 1999 by the American Society of Nephrology It has been shown that inhibition of endogenous AngII leads to natriuresis after extracellular volume expansion (ECVE) (13) and that maintaining intrarenal AngII levels by infusion of exogenous AngII minimizes natriuretic responses to ECVE (14,15). However, little information is available regarding renal functional responses to ECVE in mice, although Field et al. (16) evaluated the GFR, but not RPF, responses to isotonic volume expansion. In the present study, we determined the renal responses to ECVE with isotonic saline and the responses during concurrent administration of AngII. The small size of mice and their exquisite sensitivity to loss of even minor quantities of blood pose a challenge to clearance studies, which normally require sequential assessment of plasma concentrations of inulin and para-aminohippurate (PAH). In some studies, GFR has been assessed by using radioisotope-labeled inulin (16,17). To use standard colorimetric procedures, however, larger samples are needed, and we evaluated an approach that does not involve blood sampling until the end of the urine collections. Using this approach, we assessed RPF, GFR, and sodium excretory function in three groups of anesthetized mice: euvolemic mice, volume-expanded mice, and volumeexpanded mice receiving AngII infusions. Materials and Methods The studies described were performed in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee. Because the C57Bl/6 mice are commonly

2 2632 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 used as the genetic background for gene-targeted mutations, this strain was used to establish our baseline values. Male C57Bl/6 mice (Charles River, Wilmington, MA) were housed in a temperature- and light-controlled room and allowed free access to standard diet (Ralston-Purina, St. Louis, MO) and tap water. On the day of the experiment, mice weighing 22 to 29 g were anesthetized with a combination of Inactin (thiobutabarbital sodium, 100 mg/kg, intraperitoneally) and Ketalar (ketamine, 10 mg/kg). Supplemental doses of anesthesia (ketamine, 5 mg/kg, intramuscularly) were administrated as required. The mice were placed on a servo-controlled surgical table that maintained body temperature at 37 C, and a tracheostomy was performed. The animals were allowed to breath air enriched with O 2 by placing the exterior end of the tracheal cannula inside a small plastic chamber into which humidified 95% O 2 /5% CO 2 was continuously passed. We have found that this procedure, which has been shown to improve the stability of arterial BP of anesthetized rats (18), also improves the stability of arterial pressure of anesthetized mice. The right carotid artery was cannulated with PE-10 tubing connected to PE-50 tubing for continuous measurement of arterial BP and blood sampling. Arterial pressure was monitored with a Statham pressure transducer (model P23 Db) and recorded on a Grass polygraph (Grass Instrument, West Warwick, RI). The right jugular vein was catheterized with PE-10 tubing for fluid infusion. The bladder was catheterized with PE-50 tubing via a suprapubic incision for urine collections. During surgery, an isotonic saline solution containing 6% albumin (bovine; Sigma Chemical Co., St. Louis, MO) was infused at a rate of 2.5 l/min. After surgery, the intravenous infusion was changed to isotonic saline containing 1% albumin, 7.5% polyfructosan (Inutest, Laevosan, Linz/Donau, Austria), and 1.5% PAH (Merck Sharpe & Dohme, West Point, PA) and infused at 2.5 l/min. This infusion rate was selected because urine flow and sodium excretion remained stable during the course of the urine collection period. After a 60-min equilibration period, three consecutive 30-min urine collections were obtained. Blood samples were not taken during the urine collections. After the third collection period, an arterial blood sample (400 l) was taken for measurements of hematocrit and plasma PAH, inulin, sodium, and potassium concentrations. The kidneys were then removed, drained, and weighed. A separate group of mice (n 5) was prepared as described above, but the intravenous infusion rate was increased to 12.5 l/min after surgery to evaluate the effects of isotonic ECVE. The clearance studies were performed in the same way. In a third group of mice (n 5) prepared in the same way as described above, the effects of isotonic ECVE (12.5 l/min) plus concurrent AngII (Sigma) infusion (5 ng/g body wt min) on MAP and renal function were examined. Analytical Procedures Blood and urine samples were analyzed for inulin, PAH, sodium, and potassium concentrations as reported previously (19). Inulin and PAH concentrations were measured using standard colorimetric techniques with 90 l of plasma. Sodium and potassium concentrations were determined by flame photometry. Plasma osmolality was determined using vapor pressure osmometry. GFR was calculated from urine inulin and plasma inulin concentrations and urine flow. Similarly, PAH clearance was used as an index of RPF. All values were calculated per gram of kidney weight. Statistical Analyses Results are expressed as mean SEM. Statistical comparisons among groups were performed using one-way ANOVA. Statistical significance was defined as P Results Baseline values for body weight, kidney weight, hematocrit, plasma sodium and potassium concentrations, and plasma osmolalities for all three experimental groups are summarized in Table 1. As expected, the volume expansion, which averaged approximately 7 to 8% body weight during the course of the experiment, decreased the hematocrit. The rest of the values were not significantly different among the groups. In our study, a single arterial blood sample was taken at the end of the third clearance period to avoid BP decreases during the urine collections due to sampling. Thus, it was necessary to establish that the urine samples used for our clearance measurements were collected under steady-state conditions. This was evaluated by comparison of infusion and excretion rates for inulin and PAH. For both inulin and PAH, there were no significant differences between the urine excretion rates and intravenous infusion rates during any of the periods. Under conditions in which excretion and infusion rates are in equilibrium, one can assume that steady state has been achieved and that plasma concentrations are remaining steady. As shown in Figure 1, the ECVE group had a slightly but not significantly higher MAP than the euvolemic group (83 2 versus 77 2). However, the group with ECVE and concurrent AngII infusion exhibited significantly higher MAP compared with euvolemic and ECVE groups (100 3 mmhg). Systemic arterial pressure did not change significantly in any Table 1. Basal values for three groups a Parameter Group 1 (n 7) Group 2 (n 5) Group 3 (n 5) Body weight (g) Kidney weight (g) Hematocrit (%) b 41 1 b Plasma sodium concentration (meq/l) Plasma potassium concentration (meq/l) Plasma osmolality (mosmol/kg) a Group 1 is the euvolemic group, group 2 is the volume-expanded group, and group 3 is the volume-expanded Ang II-infused group. b P 0.05 compared with group 1.

3 J Am Soc Nephrol 10: , 1999 Renal Function in Mice 2633 of the groups during the experimental periods, indicating cardiovascular stability of the anesthetized mice prepared for clearance experiments. As shown in Figure 2, the ECVE group exhibited significantly higher GFR values compared with the euvolemic group and the AngII infusion group ( versus and ml/min g). The ECVE group also had significantly higher RPF values based on PAH clearances Figure 1. Mean arterial pressures during experimental periods under euvolemic conditions (f), volume-expanded conditions (Œ), and volume-expanded plus AngII infusion (F). *P 0.05 compared with other groups. (Figure 3) than the euvolemic or AngII infusion groups ( versus and ml/min g). For both GFR and PAH clearances, there were progressive increases in these values during the three periods, indicating that the vasodilation elicited by the volume expansion continued during the time course of the study. The AngII infusions prevented the volume expansion-induced increases in RPF and GFR. The ECVE group exhibited significantly higher total sodium excretion and urine flow (Figure 4) compared with the euvolemic and AngII infusion groups ( versus and Eq/min g, P 0.05). The sodium excretion did not change significantly during the course of the experiment in the euvolemic group ( to Eq/min g). In addition to the increased sodium excretion occurring during the equilibration period, the ECVE group exhibited additional increases in sodium excretion during the course of the three urine collections ( to Eq/min g, P 0.05). The volume-expanded mice infused with AngII also exhibited significantly higher sodium excretion rates than the euvolemic group ( versus Eq/min g); however, the concurrent AngII infusion prevented the progressive increases in sodium excretion that were observed in the volume expansion group during the three periods of urine collections ( versus Eq/min g, NS). As shown in Figure 5, the changes in fractional sodium excretion that occurred were similar to the changes in absolute sodium excretion. The ECVE group exhibited significantly higher fractional sodium excretion than the euvolemic group ( versus %). Also, the volume-expanded mice infused with AngII had significantly higher fractional Figure 2. GFR under euvolemic conditions (f), volume-expanded conditions (Œ), and volume expansion with concurrent infusion of AngII (F). *P 0.05 compared with other groups. Figure 3. Para-aminohippurate clearances under euvolemic conditions (f), volume-expanded conditions (Œ), and volume expansion with concurrent infusion of AngII (F). *P 0.05 compared with other groups.

4 2634 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Figure 4. (A and B) Sodium excretion and urine flow responses under euvolemic conditions (f), volume-expanded conditions (Œ), and under conditions of volume expansion with concurrent infusion of AngII (F). *P 0.05 compared with other groups; P 0.05 compared with euvolemic conditions. excretion elicited by ECVE was prevented by AngII ( to %, NS). Absolute and fractional potassium excretion rates were also determined in the three groups. During euvolemic conditions, absolute potassium excretion rates ranged from 1.03 to 1.72 Eq/min g, while the average fractional potassium excretion rates ranged from 22 to 24% of the filtered load. Fractional potassium excretion rates were not significantly altered by the volume expansion without or with the AngII infusions. Figure 5. Fractional sodium excretion rates under euvolemic conditions (f), volume-expanded conditions (Œ), and under conditions of volume expansion with concurrent infusion of AngII (F). *P 0.05 compared with other groups; P 0.05 compared with euvolemic conditions. sodium excretion than the euvolemic mice ( versus %). In the euvolemic group, the fractional sodium excretion did not change during the course of the experiment ( to %, NS). However, the ECVE group exhibited progressive increases in fractional sodium excretion during the three clearance periods ( to %, P 0.05). This increase in the fractional sodium Discussion The aim of present study was to evaluate a relatively simple approach to the measurement of renal hemodynamics in the anesthetized mouse and the responses to isotonic ECVE with and without concomitant infusion of AngII. For the evaluation of renal function in mice, it seemed particularly important to avoid blood sampling during the course of the urine collections needed for the clearance measurements. In initial pilot studies, we observed that there was often a hypotensive response after withdrawal of even small samples in the range of 50 to 100 l. This hypotensive response thus raised the possibility of further activation of the sympathetic nervous system and pressor hormonal mechanisms that could reduce renal function. To minimize further activation of these compensatory vasoconstrictor and pressor mechanisms, the blood sampling was deferred until after completion of the urine collections. While the plasma concentrations can most accurately be used for the clearance calculations during the final urine collection period, we feel that the finding that the inulin and PAH excretion rates were equal to the corresponding infusion rates indicates steady-state conditions. This methodologic approach is simple, does not require modification of the analytical methods, and does not require the use of radioisotopes, which have been used in some

5 J Am Soc Nephrol 10: , 1999 Renal Function in Mice 2635 previous studies (16). This approach allows routine comparison of renal function among groups of mice, which is often the main requirement when comparing genetically manipulated animals with their corresponding wild-type controls. It is well known that AngII plays a key role in the regulation of renal hemodynamics and tubular function (20). It has also been shown that volume expansion leads to suppression of plasma and kidney AngII (21) and that inhibition of endogenous AngII participates in the natriuresis after acute volume expansion (13). Furthermore, it has been shown that maintained AngII concentrations by infusion of exogenous AngII reduces the responses to volume expansion (14,15). Because these results have been obtained in rats, it seemed important to characterize the renal functional responses to ECVE in mice and to determine whether AngII also exerts a major influence on these responses. In the present study, ECVE led to increases in GFR and PAH clearance and proportionally greater increases in sodium excretion. During volume expansion, there were continued increases in these indices of renal function throughout the three clearance periods. The infusion of AngII prevented the increases in GFR and RPF in response to ECFV expansion. AngII infusion partially attenuated the volume expansion-induced increases in total sodium excretion by minimizing the progressive responses in fractional sodium excretion that occurred during the periods of urine collection. Although the dose of AngII that we used was sufficient to cause increases in systemic pressure, it is not clear whether this dose is sufficient to maintain intrarenal AngII at the same levels than before volume expansion. Because it is well known that AngII exerts a key role in regulating tubular sodium reabsorption (22,23), it is thus likely that suppression of intrarenal AngII levels contributed to the increases in sodium excretion during ECFV expansion. In our study, the isotonic solution used for expansion contained only 1% albumin, so it is likely that decreases in colloid osmotic pressure also contributed importantly to the natriuretic response to volume expansion. In essence, the concomitant AngII infusion exerted actions to reduce GFR, RPF, and sodium excretion from the levels that were achieved in the volume-expanded mice not infused with AngII. In summary, these results help to characterize renal function in the anesthetized mouse and the renal functional responses to ECVE and to AngII infusions. The baseline values for GFR and RPF, when expressed per gram of kidney weight, are comparable to those measured in rats. Volume expansion caused marked increases in RPF and GFR along with sodium excretion, but these increases were prevented or minimized by concomitant AngII infusions. Acknowledgments This work was supported by the American Heart Association and by the National Heart, Lung, and Blood Institute (Grants HL26371 and HL50438). Dr. Cervenka was a postdoctoral fellow supported by a training award from the International Society of Nephrology. Dr. Mitchell is an Established Investigator of the American Heart Association. We thank Agnes C. Buffone for excellent secretarial assistance. References 1. Clark AF, Sharp MGF, Morley SD, Fleming S, Peters J, Mullins JJ: Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology. J Biol Chem 272: , Coffman TM: Gene targeting in physiological investigations: Studies of the renin-angiotensin system. 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