Rats with adenine-induced chronic renal failure develop low-renin,salt-sensitive hypertension and increased aortic stiffness

Lisa Nguy,1,2 Maria E. Johansson,2 Elisabeth Grimberg,1 Jaana Lundgren,1 Tom Teerlink,3

Mattias Carlström,4 Jon O. Lundberg,4 Holger Nilsson,2 and Gregor Guron1

1Department of Molecular and Clinical Medicine/Nephrology, Institute of Medicine, Sahlgrenska Academy at the Universityof Gothenburg, Sweden; 2Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy at theUniversity of Gothenburg, Sweden; 3Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, TheNetherlands; and 4Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

Submitted 5 December 2012; accepted in final form 12 March 2013

Nguy L, Johansson ME, Grimberg E, Lundgren J, Teerlink T,Carlström M, Lundberg JO, Nilsson H, Guron G. Rats withadenine-induced chronic renal failure develop low-renin, salt-sensi-tive hypertension and increased aortic stiffness. Am J Physiol RegulIntegr Comp Physiol 304: R744–R752, 2013. First published March20, 2013; doi:10.1152/ajpregu.00562.2012.—Rats with adenine-in-duced chronic renal failure (A-CRF) develop metabolic and cardio-vascular abnormalities resembling those in patients with chronickidney disease. The aim of this study was to investigate the mecha-nisms of hypertension in this model and to assess aortic stiffness invivo. Male Sprague-Dawley rats were equipped with radiotelemetryprobes for arterial pressure recordings and received either chowcontaining adenine or normal control diet. At 7 to 11 wk after studystart, blood pressure responses to high NaCl (4%) diet and differentpharmacological interventions were analyzed. Aortic pulse wave ve-locity was measured under isoflurane anesthesia. Baseline 24-h meanarterial pressure (MAP) was 101 � 10 and 119 � 9 mmHg in controlsand A-CRF animals, respectively (P � 0.01). After 5 days of ahigh-NaCl diet, MAP had increased by 24 � 6 mmHg in A-CRFanimals vs. 2 � 1 mmHg in controls (P � 0.001). Candesartan (10mg/kg by gavage) produced a more pronounced reduction of MAP incontrols vs. A-CRF animals (�12 � 3 vs. �5 � 5 mmHg, P � 0.05).Aortic pulse wave velocity was elevated in A-CRF rats (5.10 � 0.51vs. 4.58 � 0.17 m/s, P � 0.05). Plasma levels of creatinine weremarkedly elevated in A-CRF animals (259 � 46 vs. 31 � 2 �M, P �0.001), whereas plasma renin activity was suppressed (0.6 � 0.5 vs.12.3 � 7.3 �g·l�1·h�1, P � 0.001). In conclusion, hypertension inA-CRF animals is characterized by low plasma renin activity and isaggravated by high-NaCl diet, suggesting a pathogenic role for so-dium retention and hypervolemia probably secondary to renal insuf-ficiency. Additionally, aortic stiffness was elevated in A-CRF animalsas indicated by increased aortic pulse wave velocity.

chronic kidney disease; adenine; hypertension; pulse wave velocity;aortic stiffness

CARDIOVASCULAR (CV) DISEASE is the major cause of morbidityand mortality in patients with chronic kidney disease (CKD)(26). There is a graded and inverse relationship betweenestimated glomerular filtration rate (GFR) and CV risk that isapparent already when GFR falls below 60 ml·min�1·1.73 m2

(8), and the majority of CKD patients die from CV eventsbefore developing end-stage renal disease (25). Although thepathophysiological mechanisms causing increased CV risk inCKD are complex, elevated aortic pulse wave velocity (PWV),

a measure of aortic stiffness, is per se an independent powerfulpredictor of CV death in patients with end-stage renal disease(1). In CKD patients, aortic PWV has been shown to becorrelated to vascular calcification indices (14, 23, 29), which,in turn, are associated with disorders in bone and mineralmetabolism that develop with declining GFR (15). However,increased aortic PWV is associated with mortality also inpatient groups with normal kidney function and even in healthysubjects (34).

We have recently analyzed vascular function in a model ofsevere chronic renal failure produced in Sprague-Dawley ratsby feeding animals with chow supplemented with adenine (22),i.e., adenine-induced chronic renal failure (A-CRF). Theseanimals develop tubulointerstitial kidney injury (33) and amore pronounced reduction in GFR compared with the com-monly used model of 5/6 nephrectomy (20–22). In addition,animals with A-CRF develop hypertension, disordered mineralmetabolism and secondary hyperparathyroidism, oxidativestress, and a marked reduction in the rate of aortic relaxationwhen assessed ex vivo (22). In view of the resemblance withthe uremic syndrome in patients, we consider this model wellsuited for examining pathophysiological mechanisms thatcause CV disease in CKD. The overall objective of this studywas to characterize the hemodynamic changes in this modelfurther. The specific aims were first to determine by whichmechanisms A-CRF animals develop hypertension, concentrat-ing on the role of the renin-angiotensin system and nitric oxide.Secondly, we hypothesized that the marked decrease in aorticrelaxation rate that we previously have observed ex vivo wouldbe associated with increased aortic stiffness in vivo. Reducedaortic compliance may have deleterious effects on the heart byincreasing ascending aortic pressure and left ventricular after-load. To examine aortic stiffness we assessed aortic pulse wavevelocity in anesthetized animals.

METHODS

General Procedures

Seventy-three male Sprague-Dawley rats (Harlan, Horst, The Neth-erlands) weighing �300 g were used and housed in rooms with acontrolled temperature of 24–26°C and a 12:12-h dark-light cycle.Chronic renal failure was produced by feeding animals with chowcontaining adenine, as previously described (22), using a modificationof protocols employed by other investigators (16, 20–21). Longitu-dinal data on body weight, water intake, plasma creatinine concen-trations, and blood pressure in this model have been publishedpreviously (22). At study start (i.e., day 1 of the study), all animalswere randomized to, and provided with, standard pelleted rat chow

Address for reprint requests and other correspondence: L. Nguy, Dept. ofMolecular and Clinical Medicine/Nephrology, Institute of Medicine, TheSahlgrenska Academy at the Univ. of Gothenburg, Medicinaregatan 11, Box432, SE-405 30 Gothenburg, Sweden (e-mail: lisa.nguy@gu.se).

Am J Physiol Regul Integr Comp Physiol 304: R744–R752, 2013.First published March 20, 2013; doi:10.1152/ajpregu.00562.2012.

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containing adenine [adenine-induced CRF (A-CRF), n � 37] oridentical chow without adenine (pair-fed controls, n � 36). The chow(R34, Lantmännen, Kimstad, Sweden) contained 0.63% phosphorous,0.74% calcium, 0.53% potassium, and 0.22% sodium. The concen-tration of adenine in the chow was 0.5% for the first 3 wk followed by0.3% for 2 wk and 0.15%, thereafter, until animals were killed.

As pilot studies revealed a reduced food intake in animals consum-ing adenine-containing chow, controls were pair-fed and receivedtheir daily ration of chow once daily in the morning. Rats had freeaccess to tap water throughout the study. Chemicals were from Sigma(St. Louis, MO), if not stated otherwise. In protocols A and D (seeProtocols), animals were decapitated without anesthesia to eliminatethe influence of anesthesia on plasma biomarkers. All experimentswere performed in accordance with the American Physiological So-ciety’s guiding principles in the care and use of vertebrate animals inresearch and training, and they were approved by the regional ethicscommittee in Gothenburg, Sweden.

Protocols

Protocol A: analyses of arterial blood pressure by radiotelemetry.Approximately 2 wk before the start of adenine or the control diet, 18rats were anesthetized with isoflurane and equipped with radiotelem-etry transmitters (Data Sciences International, St. Paul, MN) forconscious arterial blood pressure (BP) and heart rate monitoring, asdescribed previously in detail (11, 22). Data were collected andanalyzed using Dataquest ART version 3.1 (Data Sciences Interna-tional), and the BP signal was corrected for electronic offset, asdescribed previously (22). During 24-h recordings, data were sampledat 500 Hz for 8 s every 5 min. Four out of eighteen animals wereexcluded from analyses due to weak transmitter signal, unacceptableoffset, or surgical complications leaving eight A-CRF animals and sixcontrols in the study. Data from the first 7 wk of adenine and controldiet have previously been reported (22). Results presented here arefrom experiments examining aterial blood pressure responses todifferent interventions 7–11 wk after study start. At least a 1 wkwash-out period was allowed between the interventions.

High NaCl diet. At week 7 after study start, animals received a 4%NaCl diet for 5 days. Telemetry recordings were carried out for 24 hat baseline (i.e., during 24 h immediately preceding start of high-NaCldiet) and on day 5 of high-NaCl diet. Controls were pair-fed, andhence dietary NaCl intake was matched.

Treatment with N�-nitro-L arginine-methyl ester hydrochloride. Atweek 9 after study start, animals received the nitric oxide synthaseinhibitor N�-nitro-L arginine-methyl ester hydrochloride (L-NAME)added to the drinking water (controls 200 mg/l, A-CRF 78 mg/l). Alower concentration of L-NAME to A-CRF animals was used tocorrect for increased water intake in this group. These concentrationsof L-NAME had in pilot experiments been shown to produce a dailyintake of �15 mg·kg�1·day�1 in both groups. Telemetry recordingswere performed for 24 h at baseline (i.e., during 24 h immediatelypreceding start of L-NAME) and on day 3 of L-NAME treatment.

Response to the ANG II type-1 receptor antagonist candesartan.Experiments were carried out 11 wk after study start. Measurementswere performed during two consecutive 24-h periods, before (base-line) and after administration of one dose of candesartan cilexetil(AstraZeneca, Mölndal, Sweden; 10 mg/kg, 5 ml/kg, orally by ga-vage).

One week after completion of these studies, animals were killed bydecapitation, and free-flowing trunk blood was collected. The heart,kidneys, and left tibia were excised, cleaned, and weighed, and tibialength was measured. Heparin- and EDTA-plasma were obtainedfollowing centrifugation (5,000 rpm for 10 min) and were stored at�20° or �80°C until analyzed.

Protocol B: aortic PWV, aortic pulse wave analysis, and leftventricular end-diastolic pressure. Experiments were performed on10 controls and 10 A-CRF animals 12–13 wk after study start.

Animals were anesthetized with isoflurane (Pharmacia & Upjohn,Stockholm, Sweden), mixed with air during spontaneous breathing byusing a vaporizer (Univentor-1200, Agnthos, Lidingö, Sweden). Forinduction and maintenance of anesthesia, isoflurane concentrations of�5% and 1.5% (vol/vol), respectively, were used. Rats were placedon a heating table, and rectal temperature was kept at 37°C through-out. Two venous catheters (PE-50) were placed in the femoral veinsfor fluid and drug administration, and the urinary bladder was cathe-terized. Isotonic saline was infused throughout in a volume of 6ml·kg�1·h�1 to replace fluid losses. Through the right femoral arteryand left carotid artery, two ultra-miniature fiber optic pressure sensors(Samba preclin 420, sensor diameter 0.42 mm; Harvard Apparatus,Edenbridge, Kent, UK) were placed in the distal abdominal aorta atthe level of the aortic bifurcation, and in the ascending aorta imme-diately above the aortic valve or in the left ventricle. These pressuresensors were used for aortic pulse wave analysis and measurements ofaortic PWV and left ventricular end-diastolic pressure (LVEDP) usinga sampling frequency of 1,000 Hz. In addition, a third arterial catheter[polyethylene (PE)-50] was placed in the left femoral artery forcontinuous BP monitoring throughout. After a 15-min equilibrationperiod, baseline recordings of aortic BPs were performed during 5min. Subsequently, the proximal aortic pressure sensor was gentlyinserted into the left ventricle for measurements of LVEDP before itwas withdrawn and placed in its original position in the ascendingaorta. Thereafter, aortic pressures were recorded while BP was firstlowered by sodium nitroprusside (SNP; 15 �g·kg�1·min�1 iv) andthen raised by phenylephrine (25 �g·kg�1·min�1 iv). Subsequently,rats were killed by an overdose of pentobarbital sodium, and thedistance between the tips of the pressure sensors were carefullymeasured by a fine thread and a ruler. The average distance betweenthe sensor tips was similar in the two groups (97 mm in A-CRFanimals and 96 mm in controls) The heart, kidneys, and thoracic aortawere immediately excised, weighed, and immersion-fixed in 4%neutrally buffered formaldehyde (Histolab Products AB, Gothenburg,Sweden).

Aortic and left ventricular pressure data were collected and ana-lyzed by the acquisition program Biopac MP 150 (Biopac Systems,Santa Barbara, CA). From the data recorded, heart rate, systolic BP,mean arterial pressure (MAP), diastolic BP, and pulse pressure (PP)were extracted in real-time using the built-in routines. Aortic PWV(m/s) was calculated using the foot-to-foot method, the foot beingobjectively defined by the peak of the second derivative of thepressure curve during each pressure waveform (13, 19). Augmentedaortic pressure was calculated as the difference between systolic BPand the pressure at the inflection point in the pressure waveformrepresenting the arrival of the reflected pulse wave (13). Aorticaugmentation index (AI, %) was calculated as augmented pressuredivided by PP � 100. The inflection point in the pressure waveformwas identified by the zero crossing-point of the second derivative asdescribed previously (13). Left ventricular end-diastolic pressure wasdetermined by identifying the peak of the second derivative of the leftventricular pressure curve during each pressure waveform. AorticPWV and AI, and LVEDP, were determined by postprocessing of datausing the Biopac MP 150 features. These variables were determinedfor all pressure waveform(s) during 4–6 consecutive respiratorycycles (corresponding to �25–40 pressure waveforms) for eachanimal and during each intervention (i.e., baseline, SNP, and phenyl-ephrine), and average values are presented.

Protocol C: kidney function and perfusion fixation of the aorta.Experiments were performed on eight controls and eight A-CRFanimals 12–13 wk after study start. Rats were placed individually inmetabolic cages and after 24 h of equilibration measurements wereperformed during two consecutive 24-h periods. At the end of exper-iments, blood was sampled from the tail vein for plasma analyses ofcreatinine and electrolytes, and animals were transferred to normalcages.

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Subsequently, these animals were anesthetized with pentobarbitalsodium (60 mg/kg ip) and underwent perfusion fixation via the leftventricle using a continuous perfusion pressure of 110 mmHg. After2 min of perfusion with Dulbecco’s PBS, the perfusate was switchedto 4% neutrally buffered formaldehyde (Histolab Products AB,Gothenburg, Sweden) for 10 min. The thoracic aorta was excised andfixed for 24 h in 4% neutrally buffered formaldehyde after which itwas stored in 70% ethanol at 4°C.

Protocol D: analysis of plasma nitrite. Experiments were per-formed on a separate group of 10 controls and 9 A-CRF animals 10wk after study start. Animals were killed by decapitation and free-flowing trunk blood was collected into vials containing EDTA andimmediately centrifuged at 5,000 rpm for 10 min before plasma wasstored at �80°C until analyzed. Plasma nitrite was determined afterreductive cleavage and subsequent determination of the NO releasedinto the gas phase by chemiluminescence, as previously described (2).A rapid-response chemiluminescence NO system (ECO Physics, Du-ernten, Switzerland) was used to detect the NO signals.

Histological Analyses

Morphometry. Perfusion-fixed thoracic aortas were divided intoeight consecutive segments comprising the full length of the vesseland from each segment, 4-�m-thick transverse sections were preparedand stained with hematoxylin and eosin. Images (�10 magnification)of whole aortic rings were derived from all eight levels using micro-scope Olympus BX60 (camera Olympus DP72) and the imagingsoftware cellSens Dimensions (Olympus). BioPix iQ 2.0 imagingsoftware (Gothenburg, Sweden) was used for objectively measuringlumen diameter, cross-sectional area of the intima-media, and intima-media thickness. Intima-media thickness was calculated assuming acircular vessel shape. The intima-media were defined as the distancebetween the endothelial cell layer and the most peripheral elasticlamellae of the media. All measurements and scorings were made byan investigator blinded to the treatment group.

Semiquantitative aortic histology. Semiquantitative analyses ofthoracic aortas were performed on animals that had undergone anal-ysis of aortic PWV. All vessels were embedded in paraffin usingroutine techniques and 4-�m-thick transverse sections were preparedand stained with hematoxylin and eosin for general assessment, or vonKossa staining for the visualization of calcifications. Aortic calcifica-tions, and medial abnormalities consisting of fragmentation of elasticlamellae and disorganized vascular smooth muscle cells (VSMCs),were scored as either present or absent. Scorings were made by aninvestigator blinded to the treatment group.

Biochemical Analyses

Concentrations of creatinine, potassium, calcium, and phosphatewere determined by a Modular P800 Cobas C 701/502 analyzer(Roche/Hitachi, Roche Diagnostics, Mannheim, Germany). Creati-nine was measured by a photometric-enzymatic assay. Analysis ofplasma renin activity (DiaSorin, Stillwater, MN) was performed byradioimmunoassay. Concentrations of L-arginine, and asymmetric(ADMA), and symmetric (SDMA) dimethylarginine in plasma weremeasured with high-performance liquid chromatography, as describedpreviously (28), using modified chromatographic separation condi-tions (5). For all three analytes, the intra-assay and interassay coeffi-cients of variation were �2% and �4%, respectively.

Data Analyses and Statistics

All values are means � SD. Differences between means wereanalyzed using paired or unpaired Student’s t-test where appropriate,and a significance level of 5% was used. Bonferroni correction wasused to correct for multiple comparisons where appropriate. A 2-testwas used for categorical data. Statistical analyses were performedwith GraphPad Prism v. 5.03 (San Diego, CA) and SPSS 17.0 (SPSS,Chicago, IL).

RESULTS

Organ Weights and Plasma Analyses

There were no statistically significant differences betweengroups in body weight or tibia length (Table 1). Left ventric-ular weight was markedly elevated in A-CRF animals, whereasthere was no significant difference between groups in rightventricular weight (Table 1). Plasma concentrations of creati-nine, potassium, and phosphate were clearly elevated in A-CRF animals, while plasma calcium levels were significantlyreduced (Table 1). Plasma renin activity (PRA) was markedlysuppressed in A-CRF animals vs. controls (Table 1). BothADMA and SDMA plasma levels were significantly elevatedin A-CRF rats vs. controls, although there was no statisticallysignificant difference between groups in L-arginine concentra-tions (Table 1).

Plasma nitrite levels tended to be reduced in A-CRF animalsvs. controls, although not reaching statistical significance (Ta-ble 1; P � 0.06).

Kidney Function and Fluid-Handling

As previously reported (22), A-CRF animals showed amarked increase in urine output (4-fold vs. controls) and acorresponding elevation in water intake (Table 2). Renal cre-atinine clearance was reduced in A-CRF animals to about 10%of control values (Table 2). There were no statistically signif-icant differences between groups in the absolute rate of urinarysodium and potassium excretion (Table 2). However, fractionalurinary sodium and potassium excretion were markedly ele-vated in A-CRF animals (Table 2).

Blood Pressure and Heart Rate in Freely Moving AnimalsAssessed by Radiotelemetry

The 24-h profile of MAP and heart rate measured at 7 wkafter study start is shown in Fig. 1. Average MAP for the 24-hperiod was significantly elevated in the A-CRF group (119 �

Table 1. Organ weights and blood analyses at time ofeuthanasia

Controls (n) A-CRF (n)

BW, g 387 � 46 (16) 368 � 36 (17)Tibia length, mm 40.1 � 1.1 (15) 40.6 � 1.4 (18)LVW/tibia, mg/mm 21.9 � 2.1 (15) 26.8 � 3.8 (18)***RVW/tibia, mg/mm 4.9 � 1.1 (15) 5.1 � 1.0 (18)P-creatinine, �mol/l 31 � 4 (23) 285 � 80 (26)***P-sodium, mmol/l 143 � 3 (18) 142 � 3 (17)P-potassium, mmol/l 4.1 � 0.3 (18) 6.0 � 0.7 (17)***P-calcium, mmol/l 2.5 � 0.1 (18) 2.3 � 0.3 (17)*P-phosphate, mmol/l 1.7 � 0.2 (18) 2.5 � 1.1 (17)**PRA, �g � l�1 �h�1 12.3 � 7.3 (9) 0.6 � 0.5 (11)***ADMA, �mol/l 0.45 � 0.02 (3) 0.65 � 0.05 (9)***SDMA, �mol/l 0.32 � 0.03 (3) 1.25 � 0.12 (9)***L-Arg, �mol/l 152 � 11 (3) 137 � 16 (9)P-nitrite, nmol/l 161 � 80 (10) 104 � 32 (9)

Values are expressed as means � SD. Data from pair-fed control rats(controls) and animals with adenine-induced chronic renal failure (A-CRF) atthe time of acute experimentation 10–13 wk after study start (animals fromprotocols A, C, and D, see METHODS). The numbers of animals per group arepresented in parentheses. BW, body weight; LVW, left ventricular weight;RVW, right ventricular weight; P, plasma; PRA, plasma renin activity;ADMA, asymmetric dimethyl arginine; SDMA, symmetric dimethyl arginine;L-Arg, L-arginine. *P � 0.05, **P � 0.01, and ***P � 0.001.

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9 vs. 101 � 10 mmHg, P � 0.01) while heart rate did not differsignificantly between groups (311 � 19 vs. 311 � 21 bpm inA-CRF animals and controls, respectively). There were no statis-tically significant differences between day (light) and night (dark)values within groups for any of the measured variables.

Responses to 4% NaCl diet. A-CRF animals showed amarked increase in MAP in response to 5 days of 4% NaCl diet(from 119 � 9 to 143 � 9 mmHg, P � 0.001), whereas MAPdid not change significantly in controls (Fig. 2A). Notably,although there was a pronounced increase in MAP in A-CRFanimals, this was not accompanied by a compensatory reduc-tion in heart rate; instead, heart rate tended to increase (Fig.2A). During the 5-day period of 4% NaCl diet, there was nosignificant difference between groups in NaCl intake, as con-trols were pair-fed. However, A-CRF animals showed a morepronounced increase in body weight during the 5-day period(23 � 9 vs. 9 � 6 g in A-CRF animals and controls, respec-tively, P � 0.01), indicative of sodium and water retention.

Responses to L-NAME. Inhibition of nitric oxide synthase byL-NAME significantly increased MAP in both groups (from100 � 11 to 124 � 13 mmHg in controls, P � 0.001; and from112 � 10 to 149 � 15 mmHg in A-CRF animals, P � 0.001).The increase in MAP was significantly more pronounced inA-CRF animals vs. controls both in absolute values (Fig. 2B)and when expressed in percent change (33 � 8 vs. 25 � 4% inA-CRF animals and controls, respectively, P � 0.05). Heartrate decreased in both groups to a similar extent (Fig. 2B).

Responses to candesartan. Candesartan reduced MAP sig-nificantly in both controls (from 99 � 12 to 88 � 12 mmHg,P � 0.001) and A-CRF animals (128 � 20 to 123 � 18 mmHg,P � 0.05). However, the reduction in MAP was significantlymore pronounced in controls vs. A-CRF rats (Fig. 2C). Heartrate increased in controls (300 � 22 to 327 � 26 bpm, P �0.001) and in A-CRF animals (319 � 15 to 332 � 14 bpm,P � 0.001). The increase in heart rate was significantly moreprominent in controls vs. A-CRF animals (Fig. 2C).

Aortic Pulse Wave Velocity

Aortic PWV was significantly elevated in A-CRF animalsvs. controls at baseline (5.10 � 0.51 vs. 4.58 � 0.17 m/s, P �

0.05) and during maximal vasodilatation with SNP (4.04 �0.31 vs. 3.75 � 0.20 m/s, P � 0.05), while there was nostatistically significant difference between groups during phen-ylephrine infusion (Fig. 3). Notably, whereas MAP was sig-nificantly increased in group A-CRF during PWV measure-ments at baseline (124 � 12 vs. 108 � 10 mmHg, P � 0.05),there were no statistically significant differences betweengroups in MAP during infusion of SNP (46 � 5 vs. 48 � 6mmHg, in A-CRF and controls, respectively) and phenyleph-rine (173 � 19 vs. 161 � 7 mmHg, in A-CRF and controls,respectively). The above-mentioned MAP data are averagevalues of those recorded simultaneously in the ascending aortaand at the aortic bifurcation during PWV measurements. Therewas no statistically significant difference between groups inheart rate during baseline or during SNP or phenylephrineinfusion (data not shown).

Aortic Blood Pressures and Left Ventricular End-DiastolicPressure

Both in the ascending aorta and at the aortic bifurcation inthe distal abdominal aorta, systolic blood pressure (SBP), PP,MAP, and AI were significantly elevated in A-CRF animals vs.controls (Table 3). In addition, diastolic blood pressure (DBP)was significantly increased in A-CRF animals vs. controls at

Table 2. Fluid-handling and kidney function

Controls (n � 8) A-CRF (n � 8)

Food intake, g/day 16.5 � 0 16.6 � 3.0Water intake, ml/day 19 � 2 56 � 8***Urine volume, ml/day 9 � 1 40 � 10***UNaV, mmol/day 1.28 � 0.10 1.07 � 0.38UKV, mmol/day 2.21 � 0.13 2.27 � 0.58Ccreatinine, ml/min 2.91 � 0.29 0.27 � 0.12***FENa, % 0.23 � 0.02 2.20 � 0.57***FEK, % 14 � 1 123 � 20***FEH2O, % 0.23 � 0.04 11.94 � 2.89***

Values are means � SD. Data are average values of two consecutive 24-hcollection periods in metabolic cages performed 12–13 wk after study start (seeMethods). Note that Controls were pair-fed. There was no statistically signif-icant difference between groups in body weights. UNaV, urinary sodiumexcretion; UKV, urinary potassium excretion; Ccreatinine, creatinine clearance;FENa, fractional urinary sodium excretion; FEK, fractional urinary potassiumexcretion; FEH2O, fractional urinary water excretion. Fractional urinary excre-tion rates of Na, K, and water were calculated as (excreted amounts/filteredamounts) � 100, using Ccreatinine as a measure of glomerular filtration rate.***P � 0.001.

A

B

Fig. 1. Mean arterial pressure (A) and heart rate (B) measured by radiotelemetry (seeMETHODS) in rats with adenine-induced chronic renal failure (A-CRF; n � 8) and inpair-fed controls (n � 6). Measurements were performed 7 wk after study start. Dashedvertical lines at 1900 and 0700 indicate the 12-h night (dark) period. Data are expressedas means � SD. Average MAP for the 24-h period was significantly elevated in theA-CRF group (119 � 9 vs. 101 � 10 mmHg, P � 0.01), whereas heart rate did notdiffer significantly between groups.

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the aortic bifurcation (Table 3). As expected, DBP and MAPwere significantly lower, and AI significantly elevated, at thedistal recording site vs. the proximal site when analyzed withingroups (Table 3). However, in A-CRF animals, the increases inSBP (�14 � 7 vs. �2 � 7 mmHg, in A-CRF animals andcontrols, respectively, P � 0.001) and in PP (�20 � 7 vs. �7 � 7mmHg, in A-CRF animals and controls, respectively, P �0.001) along the length of the aorta were significantly morepronounced than in controls.

SBP and PP were significantly elevated in A-CRF animalsduring SNP infusion both in the ascending aorta (SBP: 84 � 10vs. 75 � 7 mmHg, P � 0.05; PP: 53 � 7 vs. 40 � 4 mmHg,

P � 0.001) and at the aortic bifurcation (SBP: 65 � 7 vs. 58 � 8mmHg, P � 0.05; PP: 36 � 5 vs. 23 � 5 mmHg, P � 0.001),although there were no statistically significant differences be-tween groups in heart rate or MAP at either site (data not shown).

During baseline conditions, LVEDP was almost doubled inA-CRF animals vs. controls (15.1 � 5.0 vs. 8.4 � 0.9 mmHg,P � 0.001, Fig. 4).

A B C

Fig. 2. Changes in mean arterial pressure (MAP) and heart rate in response to 4% NaCl diet (A), L-NAME (B), and candesartan (C) in rats with adenine-inducedchronic renal failure (A-CRF; n � 8) and in pair-fed controls (n � 6). All data are calculated from 24-h average values. Baseline values were compared withday 5 of 4% NaCl diet, day 3 of L-NAME treatment (15 mg·kg�1·day�1 via drinking water), and day 1 following a single dose of candesartan (10 mg/kg, bygavage) (see METHODS). Notably, during the 5-day period of 4% NaCl diet, A-CRF animals showed an increase in body weight gain compared with controls(23 � 9 vs. 9 � 6 g; P � 0.01). Data are expressed as means � SD. *P � 0.05, **P � 0.01, *** P � 0.001.

Fig. 3. Aortic pulse wave velocity (PWV) was measured in isoflurane-anesthetized rats with adenine-induced chronic renal failure (A-CRF; n � 10)and in pair-fed controls (n � 10) at baseline and during infusion of sodiumnitroprusside (SNP) and phenylephrine (PE) as described in METHODS. Meanarterial pressure was significantly increased in group A-CRF during PWVmeasurements at baseline (124 � 12 vs. 108 � 10 mmHg, P � 0.05), but therewere no statistically significant differences between groups in MAP duringinfusion of SNP or phenylephrine (see data in RESULTS). Data are expressed asmeans � SD. *P � 0.05 between groups.

Table 3. Aortic blood pressures and augmentation indexduring baseline conditions

Controls (n � 10) A-CRF (n � 10)

Heart rate, bpm 347 � 29 324 � 26Ascending aorta

SBP, mmHg 130 � 9 150 � 14*DBP, mmHg 93 � 7 101 � 11PP, mmHg 37 � 4 49 � 5*MAP, mmHg 112 � 8 126 � 12*AI, % 11 � 6 26 � 7*

Aortic bifurcationSBP, mmHg 128 � 12 164 � 17*†DBP, mmHg 84 � 11† 96 � 12*†PP, mmHg 43 � 5† 69 � 9*†MAP, mmHg 104 � 12† 123 � 13*†AI, % 36 � 16† 53 � 4*†

Values are expresssed as means � SD. Blood pressures were measuredsimultaneously in the ascending aorta and in the distal abdominal aorta at thelevel of the aortic bifurcation during isoflurane anesthesia (see METHODS).Experiments were performed 12–13 wk after study start. Presented data arefrom baseline measurements before administration of sodium nitroprusside andphenylephrine. Augmentation index (AI) was calculated as described in METHODS.A-CRF, adenine-induced chronic renal failure; SBP, systolic blood pressure;DBP, diastolic blood pressure; PP, pulse pressure; and MAP, mean arterialblood pressure. *P � 0.05 A-CRF vs. controls. †P � 0.05 ascending aorta vs.aortic bifurcation within group.

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Aortic Morphology

Morphometrical analyses of perfusion-fixed thoracic aortasrevealed no statistically significant differences between groupsin media thickness (123 � 18 vs. 105 � 16 �m, in A-CRF andcontrols, respectively) or lumen radius (818 � 145 vs. 827 �122 �m, in A-CRF and controls, respectively), although mediathickness tended to be increased in A-CRF animals. However,media thickness/lumen radius ratio was significantly elevatedin A-CRF animals vs. controls (0.152 � 0.022 vs. 0.127 �0.007, respectively, P � 0.05). Figure 5 illustrates a perfusion-fixed thoracic aorta from an A-CRF animal with clear mediathickening. Semiquantitative assessment of thoracic aortasshowed medial calcifications by von Kossa staining in only 1out of 10 A-CRF animals subjected to PWV analyses, whereasno vascular calcifications were detected in remaining animals(P � not significant between groups). However, the mediallayer of aortas from 7 out of 10 examined A-CRF animalsdisplayed an abnormal architecture with fragmentation of elas-tic lamellae and disorganized VSMCs, whereas these abnor-malities were virtually absent in aortas from controls (P � 0.01between groups, Fig. 5). The medial changes in A-CRF ani-

mals had a focal appearance and were not evenly distributedthroughout the aortic circumference. There were no apparentalterations in the intima or adventitia of A-CRF animals.

DISCUSSION

The main findings in the present study were that rats withA-CRF develop hypertension that is not renin-dependent andexaggerated by the high-NaCl diet. In addition, A-CRF animalshad increased aortic PWV both during baseline conditions andduring SNP infusion, clearly indicative of elevated aorticstiffness in vivo. Notably, the increase in aortic PWV occurredalthough media calcifications were present in only 10% ofA-CRF animals, suggesting that other pathophysiologicalmechanisms caused the increase in aortic stiffness.

The model of A-CRF is characterized by severe tubulo-interstitial kidney injury and marked reductions in GFR, and ithas frequently been used to investigate disorders in mineral andbone metabolism, as well as mechanisms of vascular calcifi-cations in severe renal failure (10, 12, 16, 18, 20–22). How-ever, although increased BP has been reported in a few studieson animals with A-CRF (17, 22), we are unaware of previousstudies investigating the pathophysiological mechanisms ofhypertension in this model. In the present study, PRA wassuppressed in hypertensive A-CRF animals during baselineconditions when animals had free access to chow with normalNaCl content. In addition, in response to the ANG II type-1receptor antagonist candesartan A-CRF animals showed only aminor reduction in MAP that was less pronounced than thedecrease observed in normotensive controls. These resultsclearly indicate that the hypertension in A-CRF animals is notmediated by the renin-angiotensin system. During 5 days of the4% NaCl diet, A-CRF animals showed a marked increase inMAP that was accompanied by a significant weight gaincompared with controls. These findings indicate that the in-crease in MAP during a high-NaCl diet was at least partiallycaused by sodium retention and extracellular fluid volumeexpansion. It is reasonable to hypothesize that sodium retention

Fig. 4. Left ventricular end-diastolic pressure (LVEDP) was measured inisoflurane-anesthetized rats with adenine-induced chronic renal failure (A-CRF; n � 8) and in pair-fed controls (n � 10) during baseline conditions asdescribed in METHODS. Data are means � SD. ***P � 0.001 between groups.

A B

Fig. 5. Hematoxylin- and eosin-stained sec-tions of thoracic aortas from control (A) andanimal with adenine-induced chronic renalfailure (A-CRF; B). Note the thickened mediain the A-CRF aorta. The medial layer of theA-CRF aorta displays fragmentation of elas-tic lamellae and disorganized smooth musclecells. There were no detectable alterations inthe intima or adventitia of A-CRF aortas. Toppanels show �4.5 magnification, and bottompanels show �20 magnification.

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and extracellular fluid volume expansion contributed to hyper-tension also when dietary NaCl intake was normal, consideringthe marked reduction in GFR (Ccreatinine was 10% of controlvalues) and the suppressed PRA in these animals. Hypotheti-cally, another explanation for the low PRA levels in A-CRFanimals could be the damage of renin-producing juxtaglomer-ular cells caused directly by adenine or its metabolites. How-ever, by light microscopy, we have been unable to detect anysignificant abnormalities in renal arterial or arteriolar morphol-ogy in A-CRF animals (unpublished observation). The pre-served renal arterial morphology was anticipated as adenine,via its metabolite 2,8-dihydroxyadenine causes renal failure bytubular obstruction and subsequent tubulointerstitial injury.Interestingly, the increase in MAP during the high-salt diet inA-CRF animals was accompanied by an increase in heart rate,while the expected baroreflex-mediated decrease in heart ratewas observed in response to L-NAME. These observationssuggest an impaired baroreflex control of heart rate, particu-larly during high-salt intake in A-CRF animals. Similar find-ings of salt-induced impairments in baroreflex functions havebeen demonstrated in salt-sensitive hypertensive models (3, 9).

Confirming our previous results in this model (22), plasmalevels of ADMA, an analog of L-arginine that inhibits nitricoxide synthase (30), and SDMA, which may interfere withnitric oxide synthesis by competing with L-arginine for trans-membrane transport (4), were both significantly elevated inA-CRF animals. In addition, plasma nitrite levels tended to bereduced in A-CRF animals, lending further support for thenotion that this model is associated with reduced nitric oxidebioavailability. Hence, we speculated that nitric oxide defi-ciency could contribute to the hypertension observed in A-CRFanimals and that these rats would show an attenuated increasein BP in response to L-NAME treatment. However, on thecontrary, A-CRF animals showed a more pronounced increasein MAP compared with controls in response to L-NAME,suggesting that nitric oxide bioavailability in A-CRF animalswas at least similar to that in controls. A hypothetical expla-nation could be that L-NAME-induced vasoconstriction inhypertensive A-CRF animals produced a more pronouncedincrease in vascular resistance compared with that in controlsas a consequence of structural adaptation of resistance arterieswith increased wall-to-lumen ratios (7). However, additionalstudies are clearly needed to explain this finding.

Unexpectedly, there was no difference between day andnight regarding BP and heart rate data collected by radiotelem-etry, indicating a disturbed circadian rhythm. Notably, this lackof circadian rhythm was observed in both A-CRF animals andcontrols and is most likely explained by the fact that foodintake and feeding patterns were altered as a consequence ofthe experimental protocol. Food intake is moderately reducedin A-CRF animals consuming a chow containing 0.15% ade-nine (our unpublished observations) and averaged 16–17 g perday in the present study. In addition, pair-fed rats were admin-istered their daily ration of chow once daily, which obviouslyhad a major impact on the feeding pattern of these animals.Previous studies have shown that food restriction markedlyattenuates circadian variations in BP and heart rate (31).

Rats with A-CRF developed increased aortic PWV, indica-tive of elevated aortic stiffness, independently of vascular wallcalcifications. In addition, pulse wave analysis revealed thataortic AI was elevated both in the ascending aorta and at the

aortic bifurcation of A-CRF animals, demonstrating that re-flected pressure waves from the periphery contributed more toaortic PP and SBP in this group of animals compared with incontrols. It is reasonable to hypothesize that elevated aorticPWV at least partly contributed to increased AI by causing amore rapid return of reflected pressure waves from the periph-ery, which, in turn, led to a considerable overlap of forward-directed waves and reflected pressure waves in the aortaalready during systole. Importantly, as MAP per se has a majorimpact on aortic PWV (32), we were able to demonstrate thataortic PWV, as well as aortic PP and SBP, were significantlyelevated in A-CRF animals during SNP infusion when MAPlevels were similar to those in controls. Thus, our observationsindicate that elevated aortic stiffness in A-CRF animals wasindependent of vascular calcifications and increased MAP.However, from the present experiments we cannot rule out thathypertension in A-CRF animals contributed to the develop-ment of increased aortic stiffness.

Evidently, the elevations in aortic AI and SBP in A-CRFanimals caused an increase in left ventricular afterload that wasassociated with left ventricular hypertrophy and increasedLVEDP. The increase in LVEDP indicates diastolic dysfunc-tion that could be a consequence of impaired myocardialrelaxation or decreased compliance. Interestingly, our resultsare in line with clinical studies demonstrating that enhancedarterial stiffness is an independent risk factor for the develop-ment of left ventricular diastolic dysfunction (24).

We have previously shown, using an identical protocol as inthe present study, that thoracic aortas from A-CRF animalsdisplay a marked reduction in the rate of relaxation ex vivo inresponse to a variety of different vasodilator stimuli (22). Thisabnormality was independent of the endothelium and was notpresent in mesenteric resistance arteries, suggesting an abnor-mality in vascular smooth muscle cells of the aorta (22).Hence, we speculate that the increase in aortic stiffness ob-served in A-CRF animals in the present study could be causedby the same functional abnormality in vascular smooth musclefunction that was responsible for reduced aortic relaxation ratein our previous report (22). We are at present elucidating thesemechanisms further. However, also morphological changes inthe aortic wall could at least partly contribute to the increase inaortic stiffness observed in the present study. In addition to anincrease in media thickness to lumen radius, A-CRF animalsshowed fragmentation of elastic lamellae and disorganizedsmooth muscle cells in the media. Elastin is abundant in theextracellular matrix of the aorta and contributes importantly tothe passive elastic properties of large “Windkessel” arteries(6). Hence, the fragmentation of elastic lamellae that wasobserved in aortas of A-CRF animals may be a factor causingincreased vascular stiffness. In our model of A-CRF the vastmajority of rats do not develop aortic media calcifications incontrast to rats in other studies (27), in which adenine often hasbeen used specifically to cause severe alterations in mineraland bone metabolism accompanied by vascular calcifications.The discrepant findings are likely explained by the higherconcentration of adenine in the chow (0.75%), which routinelyhas been used by others, leading to more severe renal failureand more pronounced hyperphosphatemia and hyperparathy-roidism (27). In general, other investigators have also usedchow with higher concentrations of calcium and phosphorous.

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In conclusion, hypertension in A-CRF animals is not renin-dependent and is exaggerated by increased dietary NaCl intake.In addition, A-CRF animals develop increased aortic PWV,clearly indicative of elevated aortic stiffness, in the absence ofaortic calcifications.

Perspectives and Significance

The major cause of death in CKD is cardiovascular disease,and hypertension and indices of elevated aortic stiffness arepowerful risk factors. The experimental model used in thepresent study bears many resemblances to the clinical syn-drome of uremia and may serve as a promising tool forinvestigating cardiovascular disease mechanisms in CKD. Ourresults provide novel insights into the mechanisms causinghypertension in this model. Future studies using this modelmay identify novel more effective antihypertensive therapies inpatients with reduced kidney function. In addition, we demon-strated that A-CRF animals developed increased aortic stiff-ness, which underlines the clinical relevance of this model.Interestingly, aortic stiffness was elevated in the absence ofvascular calcifications, indicating that other mechanisms areinvolved. On the basis of our previous results, we hypothesizethat abnormalities in aortic vascular smooth muscle function,which need to be examined further, cause this increase in aorticstiffness.

ACKNOWLEDGMENTS

The technical assistance of Lisbeth Selven and Mohamed Ibrahim isacknowledged.

GRANTS

This study was supported by grants from the Swedish Heart-Lung Foun-dation, the Swedish Government under the agreement concerning research andeducation of medical doctors (ALF), Göteborg Medical Society, SwedishMedical Society, Swedish Association for Kidney Patients, Swedish Society ofNephrology, Inger Bendix Foundation, Paul Frankenius Foundation, BrittWennerström’s Research Foundation, and IngaBritt and Arne LundbergsResearch Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: L.N., M.E.J., H.N., and G.G. conception and designof research; L.N., E.G., J.L., T.T., M.C., and J.O.L. performed experiments;L.N., J.L., T.T., M.C., J.O.L., and G.G. analyzed data; L.N., M.E.J., J.L., T.T.,J.O.L., H.N., and G.G. interpreted results of experiments; L.N. preparedfigures; L.N. drafted manuscript; L.N., M.E.J., H.N., and G.G. edited andrevised manuscript; L.N., M.E.J., E.G., J.L., T.T., M.C., H.N., and G.G.approved final version of manuscript.

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