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 6 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 6 0.14 versus 1.01 6 0.1 ml/min z g and characterize the renal hemodynamic responses to acute volume 11.26 6 1.39 versus 6.29 6 0.5 ml/min z 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 6 3 mmHg, P , 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 6 0.08 and 5.35 6 0.48 ml/min z 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 6 1.0 versus 0.62 6 0.2 sampling, a single blood sample was taken after three timed mEq/min z g and 3.1 6 0.7 versus 0.4 6 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 ml/ excretion responses to volume expansion (3.4 6 1.2 mEq/min min, intravenously, n 5 7) during isotonic saline volume z g and 2.5 6 0.5%, respectively). The present findings dem- expansion (12.5 ml/min, intravenously, n 5 5) and during onstrate that anesthetized mice exhibit marked renal hemody- volume expansion with concurrent AngII infusion (5 ng/min z namic and excretory responses to isotonic saline volume ex- g, n 5 5). MAP in the control group was 77 6 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 ml 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 6 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 , 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 ml/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 ml/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 m 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 5 5) was prepared as described above, but the intravenous infusion rate was increased to 12.5 ml/min after surgery to evaluate the As shown in Figure 1, the ECVE group had a slightly but not 6 effects of isotonic ECVE.