J Am Soc Nephrol 10: 2631–2636, 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 (83 Ϯ 2 mmHg), but led to significantly greater values in both means for assessing renal function in anesthetized mice and to GFR and RPF (1.35 Ϯ 0.14 versus 1.01 Ϯ 0.1 ml/min ⅐ g and characterize the renal hemodynamic responses to acute volume 11.26 Ϯ 1.39 versus 6.29 Ϯ 0.5 ml/min ⅐ g, respectively). expansion and how these responses are altered by concurrent Infusion of AngII during volume expansion led to significant angiotensin II (AngII) infusions. Inulin and para-aminohippu- elevations of MAP (100 Ϯ 3 mmHg, P Ͻ 0.05) and prevented rate clearances were used to assess GFR and renal plasma flow the increases in GFR and RPF elicited by volume expansion (RPF) in three groups of male C57Bl/6 mice anesthetized with (0.77 Ϯ 0.08 and 5.35 Ϯ 0.48 ml/min ⅐ g, respectively). inactin (100 mg/kg, intraperitoneally) and ketamine (10 mg/ Volume expansion also elicited marked increases in absolute kg). To avoid the hypotension associated with repeated blood and fractional sodium excretion (6.1 Ϯ 1.0 versus 0.62 Ϯ 0.2 sampling, a single blood sample was taken after three timed ␮Eq/min ⅐ g and 3.1 Ϯ 0.7 versus 0.4 Ϯ 0.1%, respectively). urine collections. Renal function and mean arterial pressure AngII infusion attenuated the absolute and fractional sodium (MAP) were measured under euvolemic conditions (2.5 ␮l/ excretion responses to volume expansion (3.4 Ϯ 1.2 ␮Eq/min min, intravenously, n ϭ 7) during isotonic saline volume ⅐ g and 2.5 Ϯ 0.5%, respectively). The present findings dem- expansion (12.5 ␮l/min, intravenously, n ϭ 5) and during onstrate that anesthetized mice exhibit marked renal hemody- volume expansion with concurrent AngII infusion (5 ng/min ⅐ namic and excretory responses to isotonic saline volume ex- g, n ϭ 5). MAP in the control group was 77 Ϯ 2 mmHg; pansion. Concomitant AngII infusion attenuates these volume expansion alone did not change MAP significantly responses in spite of greater increases in arterial pressure.

Recent advances in transgenic and gene-targeted manipulations It has been shown that inhibition of endogenous AngII leads of the murine genome have provided new and potentially to natriuresis after extracellular volume expansion (ECVE) powerful approaches for studying the structural, biochemical, (13) and that maintaining intrarenal AngII levels by infusion of functional, and pathophysiologic roles of different genes and exogenous AngII minimizes natriuretic responses to ECVE their products. Indeed, a vast array of gene knockout and (14,15). However, little information is available regarding re- transgenic mouse models have been developed, and some of nal functional responses to ECVE in mice, although Field et al. these have demonstrated altered renal structure and function (16) evaluated the GFR, but not RPF, responses to isotonic (1–10). Nevertheless, a systematic description of the changes volume expansion. In the present study, we determined the in renal hemodynamics has been infrequent because of the renal responses to ECVE with isotonic saline and the responses problems associated with assessing renal plasma flow (RPF) during concurrent administration of AngII. The small size of and GFR in mice. Recently, Gross et al. (11,12) defined the mice and their exquisite sensitivity to loss of even minor pressure-natriuresis-diuresis relationships in normotensive and quantities of blood pose a challenge to clearance studies, which deoxycorticosterone acetate-salt hypertensive mice. However, normally require sequential assessment of plasma concentra- the absence of data for RPF and GFR prevented a more tions of inulin and para-aminohippurate (PAH). In some stud- detailed assessment of the mechanisms responsible. Thus, there ies, GFR has been assessed by using radioisotope-labeled are still many uncertainties regarding normal baseline values inulin (16,17). To use standard colorimetric procedures, how- for renal function in mice and the renal functional responses to ever, larger samples are needed, and we evaluated an approach experimental manipulations such as volume expansion and that does not involve blood sampling until the end of the urine angiotensin II (AngII) infusions. collections. Using this approach, we assessed RPF, GFR, and sodium excretory function in three groups of anesthetized mice: euvolemic mice, volume-expanded mice, and volume- Received December 31, 1998. Accepted June 8, 1999. Correspondence to Dr. L. Gabriel Navar, Department of Physiology SL39, expanded mice receiving AngII infusions. Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. Phone: 504-588-5251; Fax: 504-584-2675; E-mail: navar@ mailhost.tcs.tulane.edu Materials and Methods 1046-6673/1012-2631 The studies described were performed in accordance with the Journal of the American Society of Nephrology guidelines and practices established by the Tulane University Animal Copyright © 1999 by the American Society of Nephrology Care and Use Committee. Because the C57Bl/6 mice are commonly 2632 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2631–2636, 1999

used as the genetic background for gene-targeted mutations, this strain Analytical Procedures was used to establish our baseline values. Male C57Bl/6 mice Blood and urine samples were analyzed for inulin, PAH, sodium, (Charles River, Wilmington, MA) were housed in a temperature- and and potassium concentrations as reported previously (19). Inulin and light-controlled room and allowed free access to standard diet (Ral- PAH concentrations were measured using standard colorimetric tech- ston-Purina, St. Louis, MO) and tap water. On the day of the exper- niques with 90 ␮l of plasma. Sodium and potassium concentrations iment, mice weighing 22 to 29 g were anesthetized with a combination were determined by flame photometry. Plasma osmolality was deter- of Inactin (thiobutabarbital sodium, 100 mg/kg, intraperitoneally) and mined using vapor pressure osmometry. GFR was calculated from Ketalar (ketamine, 10 mg/kg). Supplemental doses of anesthesia (ket- urine inulin and plasma inulin concentrations and urine flow. Simi- amine, 5 mg/kg, intramuscularly) were administrated as required. The larly, PAH clearance was used as an index of RPF. All values were mice were placed on a servo-controlled surgical table that maintained calculated per gram of kidney weight. body temperature at 37°C, and a tracheostomy was performed. The

animals were allowed to breath air enriched with O2 by placing the exterior end of the tracheal cannula inside a small plastic chamber into Statistical Analyses Results are expressed as mean Ϯ SEM. Statistical comparisons which humidified 95% O2/5% CO2 was continuously passed. We have found that this procedure, which has been shown to improve the among groups were performed using one-way ANOVA. Statistical Ͻ stability of arterial BP of anesthetized rats (18), also improves the significance was defined as P 0.05. stability of arterial pressure of anesthetized mice. The right carotid artery was cannulated with PE-10 tubing con- Results nected to PE-50 tubing for continuous measurement of arterial BP and Baseline values for body weight, kidney weight, hematocrit, blood sampling. Arterial pressure was monitored with a Statham plasma sodium and potassium concentrations, and plasma os- pressure transducer (model P23 Db) and recorded on a Grass poly- molalities for all three experimental groups are summarized in graph (Grass Instrument, West Warwick, RI). The right jugular vein Table 1. As expected, the volume expansion, which averaged was catheterized with PE-10 tubing for fluid infusion. The bladder was catheterized with PE-50 tubing via a suprapubic incision for urine approximately 7 to 8% body weight during the course of the collections. During surgery, an isotonic saline solution containing 6% experiment, decreased the hematocrit. The rest of the values albumin (bovine; Sigma Chemical Co., St. Louis, MO) was infused at were not significantly different among the groups. a rate of 2.5 ␮l/min. After surgery, the intravenous infusion was In our study, a single arterial blood sample was taken at the changed to isotonic saline containing 1% albumin, 7.5% polyfruc- end of the third clearance period to avoid BP decreases during tosan (Inutest, Laevosan, Linz/Donau, Austria), and 1.5% PAH the urine collections due to sampling. Thus, it was necessary to (Merck Sharpe & Dohme, West Point, PA) and infused at 2.5 ␮l/min. establish that the urine samples used for our clearance mea- This infusion rate was selected because urine flow and sodium excre- surements were collected under steady-state conditions. This tion remained stable during the course of the urine collection period. was evaluated by comparison of infusion and excretion rates After a 60-min equilibration period, three consecutive 30-min urine for inulin and PAH. For both inulin and PAH, there were no collections were obtained. Blood samples were not taken during the significant differences between the urine excretion rates and urine collections. After the third collection period, an arterial blood intravenous infusion rates during any of the periods. Under ␮ sample (400 l) was taken for measurements of hematocrit and conditions in which excretion and infusion rates are in equi- plasma PAH, inulin, sodium, and potassium concentrations. The kid- librium, one can assume that steady state has been achieved neys were then removed, drained, and weighed. A separate group of and that plasma concentrations are remaining steady. mice (n ϭ 5) was prepared as described above, but the intravenous infusion rate was increased to 12.5 ␮l/min after surgery to evaluate the As shown in Figure 1, the ECVE group had a slightly but not Ϯ effects of isotonic ECVE. The clearance studies were performed in the significantly higher MAP than the euvolemic group (83 2 same way. In a third group of mice (n ϭ 5) prepared in the same way versus 77 Ϯ 2). However, the group with ECVE and concur- as described above, the effects of isotonic ECVE (12.5 ␮l/min) plus rent AngII infusion exhibited significantly higher MAP com- concurrent AngII (Sigma) infusion (5 ng/g body wt ⅐ min) on MAP pared with euvolemic and ECVE groups (100 Ϯ 3 mmHg). and renal function were examined. Systemic arterial pressure did not change significantly in any

Table 1. Basal values for three groupsa

Group 1 Group 2 Group 3 Parameter (n ϭ 7) (n ϭ 5) (n ϭ 5)

Body weight (g) 24.8 Ϯ 0.47 23.5 Ϯ 0.48 26.1 Ϯ 0.8 Kidney weight (g) 0.30 Ϯ 0.01 0.27 Ϯ 0.01 0.33 Ϯ 0.01 Hematocrit (%) 47 Ϯ 139Ϯ 1b 41 Ϯ 1b Plasma sodium concentration (mEq/L) 149 Ϯ 3 151 Ϯ 2 144 Ϯ 3 Plasma potassium concentration (mEq/L) 4.6 Ϯ 0.8 3.4 Ϯ 0.6 3.8 Ϯ 0.8 Plasma osmolality (mosmol/kg) 292 Ϯ 4 293 Ϯ 3 290 Ϯ 5

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 Ͻ 0.05 compared with group 1. J Am Soc Nephrol 10: 2631–2636, 1999 Renal Function in Mice 2633 of the groups during the experimental periods, indicating car- (Figure 3) than the euvolemic or AngII infusion groups diovascular stability of the anesthetized mice prepared for (11.26 Ϯ 1.39 versus 6.29 Ϯ 0.5 and 5.35 Ϯ 0.48 ml/min ⅐ g). clearance experiments. For both GFR and PAH clearances, there were progressive As shown in Figure 2, the ECVE group exhibited signifi- increases in these values during the three periods, indicating cantly higher GFR values compared with the euvolemic group that the vasodilation elicited by the volume expansion contin- and the AngII infusion group (1.35 Ϯ 0.14 versus 1.01 Ϯ 0.1 ued during the time course of the study. The AngII infusions and 0.77 Ϯ 0.08 ml/min ⅐ g). The ECVE group also had prevented the volume expansion-induced increases in RPF and significantly higher RPF values based on PAH clearances GFR. The ECVE group exhibited significantly higher total sodium excretion and urine flow (Figure 4) compared with the euv- olemic and AngII infusion groups (6.1 Ϯ 1.0 versus 0.61 Ϯ 0.2 and 3.41 Ϯ 1.22 ␮Eq/min ⅐ g, P Ͻ 0.05). The sodium excretion did not change significantly during the course of the experi- ment in the euvolemic group (0.42 Ϯ 0.17 to 0.73 Ϯ 0.23 ␮Eq/min ⅐ 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 (3.8 Ϯ 0.6 to 9.2 Ϯ 1.5 ␮Eq/min ⅐ g, P Ͻ 0.05). The volume-expanded mice infused with AngII also exhibited significantly higher sodium excre- tion rates than the euvolemic group (3.41 Ϯ 1.22 versus 0.61 Ϯ 0.2 ␮Eq/min ⅐ 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 (3.8 Ϯ 0.4 versus 3.1 Ϯ 0.4 ␮Eq/min ⅐ 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 Figure 1. Mean arterial pressures during experimental periods under higher fractional sodium excretion than the euvolemic group euvolemic conditions (f), volume-expanded conditions (Œ), and vol- (3.13 Ϯ 0.7 versus 0.37 Ϯ 0.09%). Also, the volume-expanded ume-expanded plus AngII infusion (F). *P Ͻ 0.05 compared with mice infused with AngII had significantly higher fractional other groups.

Figure 3. Para-aminohippurate clearances under euvolemic conditions Figure 2. GFR under euvolemic conditions (f), volume-expanded (f), volume-expanded conditions (Œ), and volume expansion with conditions (Œ), and volume expansion with concurrent infusion of concurrent infusion of AngII (F). *P Ͻ 0.05 compared with other AngII (F). *P Ͻ 0.05 compared with other groups. groups. 2634 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2631–2636, 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 Ͻ 0.05 compared with other groups; ϩP Ͻ 0.05 compared with euvolemic conditions.

excretion elicited by ECVE was prevented by AngII (2.73 Ϯ 0.53 to 2.31 Ϯ 0.47%, 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 ⅐ 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.

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. Figure 5. Fractional sodium excretion rates under euvolemic condi- This hypotensive response thus raised the possibility of further tions (f), volume-expanded conditions (Œ), and under conditions of activation of the sympathetic nervous system and pressor hor- volume expansion with concurrent infusion of AngII (F). *P Ͻ 0.05 compared with other groups; ϩP Ͻ 0.05 compared with euvolemic monal mechanisms that could reduce renal function. To min- conditions. imize further activation of these compensatory vasoconstrictor and pressor mechanisms, the blood sampling was deferred until after completion of the urine collections. While the plasma sodium excretion than the euvolemic mice (2.47 Ϯ 0.49 versus concentrations can most accurately be used for the clearance 0.37 Ϯ 0.09%). In the euvolemic group, the fractional sodium calculations during the final urine collection period, we feel excretion did not change during the course of the experiment that the finding that the inulin and PAH excretion rates were (0.25 Ϯ 0.09 to 0.47 Ϯ 0.11%, NS). However, the ECVE equal to the corresponding infusion rates indicates steady-state group exhibited progressive increases in fractional sodium conditions. This methodologic approach is simple, does not excretion during the three clearance periods (2.12 Ϯ 0.44 to require modification of the analytical methods, and does not 4.26 Ϯ 0.7%, P Ͻ 0.05). This increase in the fractional sodium require the use of radioisotopes, which have been used in some J Am Soc Nephrol 10: 2631–2636, 1999 Renal Function in Mice 2635 previous studies (16). This approach allows routine compari- References son of renal function among groups of mice, which is often the 1. Clark AF, Sharp MGF, Morley SD, Fleming S, Peters J, Mullins main requirement when comparing genetically manipulated JJ: Renin-1 is essential for normal renal juxtaglomerular cell animals with their corresponding wild-type controls. granulation and macula densa morphology. J Biol Chem 272: It is well known that AngII plays a key role in the regulation 18185–18190, 1997 of renal hemodynamics and tubular function (20). It has also 2. Coffman TM: Gene targeting in physiological investigations: been shown that volume expansion leads to suppression of Studies of the renin-angiotensin system. Am J Physiol 274: F999–F1005, 1998 plasma and kidney AngII (21) and that inhibition of endoge- 3. Davisson RL, Kim HS, Krege JH, Lager DJ, Smithies O, Sig- nous AngII participates in the natriuresis after acute volume mund CD: Complementation of reduced survival, hypotension, expansion (13). Furthermore, it has been shown that main- and renal abnormalities in angiotensinogen-deficient mice by the tained AngII concentrations by infusion of exogenous AngII human renin and human angiotensinogen genes. J Clin Invest 99: reduces the responses to volume expansion (14,15). Because 1258–1264, 1997 these results have been obtained in rats, it seemed important to 4. Field LJ: Transgenic mice in cardiovascular research. Annu Rev characterize the renal functional responses to ECVE in mice Physiol 55: 97–114, 1993 and to determine whether AngII also exerts a major influence 5. Kim H-S, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, on these responses. In the present study, ECVE led to increases Best CF, Jennette JC, Coffman TM, Maeda N, Smithies O: in GFR and PAH clearance and proportionally greater in- Genetic control of blood pressure and the angiotensinogen locus. creases in sodium excretion. During volume expansion, there Proc Natl Acad Sci USA 92: 2735–2739, 1995 6. Okubo S, Nimura F, Matsusaka T, Fogo A, Hogan BLM, were continued increases in these indices of renal function Ichikawa I: Angiotensinogen gene null-mutant mice lack homeo- throughout the three clearance periods. static regulation of glomerular filtration and tubular reabsorption. The infusion of AngII prevented the increases in GFR and Kidney Int 53: 617–625, 1998 RPF in response to ECFV expansion. AngII infusion partially 7. Esther CR Jr, Marino EM, Howard TE, Machaud A, Corvol P, attenuated the volume expansion-induced increases in total Capecchi MR, Bernstein KE: The critical role of tissue angio- sodium excretion by minimizing the progressive responses in tensin-converting enzyme as revealed by gene targeting in mice. fractional sodium excretion that occurred during the periods of J Clin Invest 10: 2375–2385, 1997 urine collection. Although the dose of AngII that we used was 8. Oliverio MI, Best CF, Kim H-S, Arendshorst WJ, Smithies O, sufficient to cause increases in systemic pressure, it is not clear Coffman TM: Angiotensin II responses in AT1A receptor-defi- whether this dose is sufficient to maintain intrarenal AngII at cient mice: A role for AT1B receptors in blood pressure regula- the same levels than before volume expansion. Because it is tion. Am J Physiol 272: F515–F520, 1997 9. Tian B, Meng QC, Chen Y-F, Krege JH, Smithies O, Oparil S: well known that AngII exerts a key role in regulating tubular Blood pressures and cardiovascular homeostasis in mice having sodium reabsorption (22,23), it is thus likely that suppression reduced or absent angiotensin-converting enzyme gene function. of intrarenal AngII levels contributed to the increases in so- Hypertension 30: 128–133, 1997 dium excretion during ECFV expansion. In our study, the 10. Tsuchida S, Matsusaka T, Chen X, Okubo S, Nijmura F, Nish- isotonic solution used for expansion contained only 1% albu- imura H, Fogo A, Utsunomiya H, Inagami T, Ichikawa I: Murine min, so it is likely that decreases in colloid osmotic pressure double nullizygotes of the angiotensin type 1A and 1B receptor also contributed importantly to the natriuretic response to vol- genes duplicate severe abnormal phenotypes of angiotensinogen ume expansion. In essence, the concomitant AngII infusion nullizygotes. J Clin Invest 101: 755–760, 1998 exerted actions to reduce GFR, RPF, and sodium excretion 11. Gross V, Lippoldt A, Bohlender J, Bader M, Hansson A, Luft from the levels that were achieved in the volume-expanded FC: Cortical and medullary hemodynamics in deoxycorticoste- mice not infused with AngII. rone acetate-salt hypertensive mice. J Am Soc Nephrol 9: 346– 354, 1998 In summary, these results help to characterize renal function 12. Gross V, Lippoldt A, Luft FC: Pressure diuresis and natriuresis in the anesthetized mouse and the renal functional responses to in DOCA-salt mice. Kidney Int 52: 1364–1368, 1997 ECVE and to AngII infusions. The baseline values for GFR 13. Chevalier RL, Thornhill BA, Belmonte DC, Baertschi AJ: En- and RPF, when expressed per gram of kidney weight, are dogenous angiotensin II inhibits natriuresis after acute volume comparable to those measured in rats. Volume expansion expansion in the neonatal rat. Am J Physiol 270: R393–R397, caused marked increases in RPF and GFR along with sodium 1996 excretion, but these increases were prevented or minimized by 14. Pinilla JM, Perez MC, Hernandez I, Quesada T, Garcia-Estan J, concomitant AngII infusions. Salazar FJ: Role of intrarenal angiotensin II in mediating renal response to volume expansion. Am J Physiol 261: R619–R625, 1991 Acknowledgments 15. Pinilla JM, Alberola A, Gonzalez JD, Quesada T, Salazar FJ: This work was supported by the American Heart Association and Role of prostaglandins on the renal effects of angiotensin and by the National Heart, Lung, and Blood Institute (Grants HL26371 interstitial pressure during volume expansion. Am J Physiol 265: and HL50438). Dr. Cervenka was a postdoctoral fellow supported by R1469–R1474, 1993 a training award from the International Society of Nephrology. Dr. 16. Field LJ, Veress AT, Steinhelper ME, Cochrane K, Sonnenberg Mitchell is an Established Investigator of the American Heart Asso- H: Kidney function in ANF-transgenic mice: Effect of blood ciation. We thank Agnes C. Buffone for excellent secretarial assis- volume expansion. Am J Physiol 260: R1–R5, 1991 tance. 17. Sonnenberg H, Honrath U, Chong CK, Field LJ, Veress AT: 2636 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2631–2636, 1999

Proximal tubular function in transgenic mice overexpressing 21. Boer WH, Braam B, Fransen R, Boer P, Koomans HA: Effects of atrial natriuretic factor. Can J Physiol Pharmacol 72: 1168– reduced renal perfusion pressure and acute volume expansion on 1170, 1994 proximal tubule and whole kidney angiotensin II content in the 18. Mitchell KD, Mullins JJ: AngII dependence of tubuloglomerular rat. Kidney Int 51: 44–49, 1997 feedback responsiveness in hypertensive ren-2 transgenic rats. 22. Cogan MG: Angiotensin II: A powerful controller of sodium Am J Physiol 268: F821–F828, 1995 transport in the early proximal tubule. Hypertension 15: 451–

19. Cervenka L, Wang C-T, Navar LG: Effects of acute AT1 receptor 458, 1990 blockade by candesartan on arterial pressure and renal function 23. Mitchell KD, Navar LG: Intrarenal actions of angiotensin II in in rats. Am J Physiol 274: F940–F945, 1998 the pathogenesis of experimental hypertension. In: Hypertension: 20. Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard Pathophysiology, Diagnosis, and Management, 2nd Ed., edited LM, Mitchell KD: Paracrine regulation of the renal microcircu- by Laragh JH, Brenner BM, New York, Raven, 1995, pp 1437– lation. Physiol Rev 76: 425–536, 1996 1450