Akio Nakamura*,
Akira Imaizumi*,
Yukishige Yanagawa*,
Takao Kohsaka and
Edward J. Johns
*Department of Pediatrics, Teikyo University School of Medicine, Tokyo, Japan; Department of Immunology, National Children’s Medical Centre, Tokyo, Japan; and Department of Physiology, University College Cork, Cork, Ireland.
Correspondence to: Dr. Akio Nakamura, Department of Paediatrics, Teikyo University School of Medicine, 2–11–1 Kaga, Itabashi-ku, Tokyo 173, Japan. Phone: 03-3964-1211 ext. 1480; Fax: 03-3579-8212; E-mail: akio{at}med.teikyo-u.ac.jp
ABSTRACT. Abnormalities in the 2-adrenergic control of organfunction have been implicated in the pathogenesis of severaldisease states, such as septic shock. The objectives of thepresent study were to define the contribution of 2-adrenoceptors(2-AR) to normal renal physiology and to investigate whetheroverexpression of renal 2-AR might be potentially beneficialin preventing progressive renal damage associated with endotoxemia.Adenoviral transgenes containing the human 2-AR (Adeno-2-AR)were constructed and delivered into the rat kidney by meansof intraparenchymal injections. Administration of 109 totalviral particles of Adeno-2-AR induced an approximately threefoldincrease in 2-AR density in the renal tissue, which 2 wk afterdelivery, enhanced GFR and sodium reabsorption compared withcontrol rats. The enhanced GFR was abolished by the additionof the 2-AR antagonist, ICI 118,551. Administration of lipopolysaccharide(LPS) caused a reduction in GFR, 2-AR density, and cAMP togetherwith enhanced TNF- mRNA in the kidney. In rats overexpressing2-AR, the reduction in baseline GFR and elevation of TNF- mRNAand leukocyte infiltration into the kidney associated with theendotoxin were blocked. These findings suggested the possibilitythat a renal-specific overexpression of 2-AR preserves basalrenal function in response to a ligand-independent 2-AR activationand that the delivery of Adeno-2-AR gene is a potential noveltherapeutic strategy for treatment of acute renal failure associatedwith sepsis.
Fundamental alterations in the tissue content of 2-adrenoceptors(2-AR) and their activity may contribute to the deteriorationof the immune system and accompany the development of failurein organ function. An altered expression and/or function of2-AR has been considered to be a pathogenetic factor in somedisease states; for example, allergy (1,2), heart failure (3,4),and sepsis (5,6). It has been demonstrated that patients withseptic shock suffer from hypotension and cardiac dysfunctionthat is refractory to high doses of intravenous catecholamines(5). Bernardin et al. (6) showed that the tissue density of-AR was significantly reduced and adenylate cyclase stimulationwas heterogeneously desensitized in peripheral blood mononuclearcells freshly isolated from septic patients. There is a growingbody of evidence that the 2-AR system has an antiinflammatoryinfluence on the cytokine network during the course of immunologicresponses (7–9). Importantly, the administration of 2-ARagonists was found to attenuate the stimulation of TNF- (10,11)associated with lipopolysaccharide (LPS) (12) and Shiga toxin-2of hemolytic uremic syndrome (13,14), which is considered tobe a central mediator of the pathophysiologic changes. Theseobservations would suggest that disturbances of the 2-AR systemin an organ may be involved in the uncontrolled inflammatoryresponses that occurs during sepsis (15–17).
Endotoxemia caused by Gram-negative bacteria can result in sepsisand organ dysfunction, which includes kidney damage and renalfailure (18,19). Septic shock after surgery, trauma, burns,or severe infection is a common cause of acute renal failure,resulting in a high mortality rate (20). Pathologic examinationof the failing kidneys has revealed that there is an occurrenceof focal necrosis of the proximal tubular epithelium, eosinophiliccasts within proximal and distal tubules, and microthrombi inthe glomerular capillaries (21). Recently presented data fromthe Madrid Acute Renal Failure Study Group (22) reported thatsepsis caused acute tubular necrosis in 35% of patients in intensivecare units (ICU) and 27% of non-ICU patients. If there is animpaired regulation of 2-AR function during sepsis in the failingkidney, it may be that restoration of 2-AR function might beable to prevent renal inflammation and injury associated withsepsis.
For the purpose of activation and restoration of 2-AR function,drugs targeting 2-AR signaling, including 2-AR agonists, maybe used as a first-line approach for therapy. However, administrationof 2-AR agonists to regulate 2-AR function has an inherentlylimited efficacy, partly because of the downregulation and desensitizationof 2-AR (23). On the other hand, in vivo gene therapy usingadenoviral constructs containing the 2-AR gene has been demonstratedto be an efficient and reproducible global transgene deliverysystem that results in long-term expression in the organ, ashas been reported in the myocardium (24). Therefore, the applicationof adenoviral mediated 2-AR gene delivery to elevate 2-AR densityand prevent desensitization would be an attractive option whereby2-AR could be active over a prolonged period. Importantly, adenoviralvector (25) as well as adeno-associated virus (26) deliveredin vivo by intraparenchymal injection has been found to resultin viral transduction within renal tubular epithelial cells(25, 26), which is one of the major targets of endotoxin withinthe kidney (21, 22). With this in mind, we used adenoviral mediated2-AR gene delivery to investigate whether overexpression of2-AR could alter both biochemical and in vivo renal functionand to test the hypothesis that the 2-AR gene delivery affordsthe kidney protection against endotoxin-induced acute renalfailure.
Reagents
Adenovirus Expression Vector Kit was obtained from Takara Biomedicals(Shiga, Japan). Rabbit polyclonal anti-2-AR antibody (H-20)was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz,CA). ICI 118,551 was from Funakoshi Co. (Tokyo, Japan), andglass slides precoated with siran was from Matsunami Glass Ltd.(Tokyo Japan). [125I] cyanopindolol ([125I]CYP) was obtainedfrom Perkin Elmer Life Science (Tokyo, Japan). [-33P] dCTP andcAMP ELISA kit were supplied by Amersham International Plc.(Little Chalfont, Buckinghamshire, UK). GF/C filter was fromWhatman Japan Ltd. (Tokyo, Japan). Unless stated, reagents werefrom Sigma Chemical Co. (St. Louis, MO).
Construction of Recombinant Adenovirus
The human 2-adrenoceptor–expressing adenovirus (Adeno-2-AR)and the cytoplasmic -galactosidase expressing adenovirus (Adeno-LacZ)were a kind gift from Drs. Walter J. Koch and Robert J. Lefkowitz(Departments of Surgery and of Medicine and Biochemistry inDuke University Medical Center, Durham, NC). These adenoviruseswere a replication-deficient first-generation type V adenoviruswith deletions of the E1 and E3 genes as described previously(27). Virus isolates of Adeno-2-AR, Adeno-LacZ, and no transgene(empty adenovirus: EV) were plaque-purified and propagated in293 cells given by Dr. I. Wada (Department of Biochemistry,Sapporo Medical University, Japan), isolated, concentrated,and titered using Adenovirus Expression Vector Kit.
Rat Preparation and Protocols
All procedures and protocols were approved by the Teikyo UniversityGuide for the Care and Use of Laboratory Animals. Four-week-oldWistar rats were fed a standard laboratory diet (126 mEq ofNa+/kg and 118 mEq of K+/kg food) and had free access to water.After a 7-d acclimatization period, rats were anesthetized withpentobarbitone (50 mg/kg, intraperitoneally), and the rightkidney was exposed via a retroperitoneal incision. A 50-µlsample of the virus was injected into the right kidney usinga 25-gauge needle attached to a 1-ml syringe. Briefly, the needlewas inserted at the lower pole of the right kidney just underthe capsule and parallel to the renal surface and was carefullypushed toward the upper pole. As the needle was removed slowly,injections were made along the straight line within the capsule.The flank incision was then closed by suturing the muscles andskin layers in stages. The Adeno-2-AR–treated, Adeno-LacZ–treated,and EV-treated rats rangeed in weight from 130 to 150 g, andthere was no significant difference between groups. One to fiveweeks later, animals were housed in metabolic cages for 24 hto collect urine samples. After animals were given an overdoseof pentobarbitone, blood samples were collected and the kidneyswere removed and weighed. Systolic blood pressure (BP) and heartrate (HR) were monitored weekly by means of a tail cuff sphygmomanometer,using an automated system with a photoelectric sensor (KN-201-1;Natsume Seisakusho Co., Tokyo, Japan).
To induce acute renal failure in the rats, LPS (Escherichiacoli O127:B8, 5 mg/kg) was injected intraperitoneally into rats25 d after the administration of the adenoviral vectors. Controlrats were injected intraperitoneally with an equal volume ofphysiologic saline (PBS). The 2-AR antagonist ICI 118,551 wasgiven intraperitoneally 2 h before an injection of the LPS orPBS. The rats were housed in metabolic cages for urine collectionand 24 h later were killed after an overdose of sodium pentobarbitone.Blood, urine, and kidneys were collected for assay.
Biochemical Measurements
Serum and urine creatinine levels were determined using a creatinineassay kit, according to the protocols specified by the manufacture.GFR (ml/min) was expressed as a creatinine clearance rate. Serumand urinary sodium (Na+) or potassium (K+) concentrations weremeasured using spectrophotometer (7170; Hitachi, Japan).
Renal Morphologic Analyses
Tissue for light microscopy was fixed using 3% formalin-PBSand embedded in paraffin. After sectioning, the tissues werestained with hematoxylin and eosin (H&E) for assessmentof cellular filtration and inflammation. For the -galactosidase(-gal) assay, frozen kidneys were mounted on a freezing microtome,and 10-µm sections were transferred to glass slides precoatedwith siran. Sections were fixed in 10% formalin for 2 min atroom temperature and washed twice in PBS. -gal staining wascarried out in 2 mmol/L K4Fe(CN)6, 2 mmol/L K3Fe(CN)6, 2 mmol/LMgCl2, 0.5 mg/ml X-gal (5-bromo-4-chloro-3-indoyl--D-galactopyranoside)in PBS, pH 7.4. After being stained for 2 h at 37°C, thesections were rinsed in PBS solution and taken for histologicevaluation.
-AR Binding Assay
Membrane fractions were extracted following the method describedby Lefkowitz et al. (27,28) with minor modifications. Membranepreparations (25 µg) were incubated with [125I] CYP (15to 315 pmol/L) in binding buffer either alone or 20 µmol/Lalprenolol, which was used for determination of nonspecificbinding. The incubation was carried out at 37°C for 1 hin a total volume of 500 µl followed by rapid filtrationon GF/C filters and three washings with 750 µl of ice-coldbinding buffer. -AR density (Bmax) was determined using linearregression analysis of saturation isotherm data linearly transformedto give a Scatchard plot. Receptor density (measured in femtomoles)was normalized to milligrams of membrane protein. The proteinconcentration was assayed using a micro protein determinationkit.
2-AR Immunohistochemistry
Frozen kidney sections were cut 10-µm-thick for the immunofluorescencestudies. Sections were rinsed in PBS, then with PBS containing0.05% Triton X-100 (triton-PBS), blocked with serum diluent(10% goat serum in PBS with BSA and 0.1% sodium azide), andthen rinsed for 15 min in Triton-PBS before overnight incubationat 4°C with a primary rabbit antihuman 2-AR antiserum (1:500dilution in serum diluent). The sections were then washed fourtimes for 10 min in Triton-PBS at room temperature and incubatedfor 1 h in FITC-conjugated goat anti-rabbit IgG (1:50 dilutionin serum diluent). After five 3-min rinses in PBS, the sectionswere mounted with sodium iodide (25 g/L) in 1:1 PBS/glycerolsolution and photographed using a confocal laser-scanning microscope(CLSM; BIO-RAD, Hemel Hempstead, UK).
Analysis of TNF- mRNA and cAMP Activity
We estimated mRNA levels using Northern blot hybridization analysisas described in our previous study (29). For Northern blot hybridization,the 546-bp cDNA for TNF- (30) and the 420-bp Hinf I fragmentof human -actin (National Children’s Research Centre,Japan, Tokyo) were labeled using the oligo-labeling method inthe presence of [-33P] dCTP and used as a hybridization probe.All mRNA samples (10 µg) were applied to a Biodyne A membrane,hybridized simultaneously, and exposed for the same time. The-actin cDNA probe was used as a loading control after the TNF-probe was stripped from the membrane. Urinary and renal cAMPlevels were estimated using a commercially available ELISA kitin which the assay was based on the competition between unlabeledcAMP and a fixed quantity of peroxidase-labeled cAMP for a limitednumber of binding sites on a cAMP-specific antibody. For measurementof renal cAMP levels, the frozen kidney samples were lysed usinga liquid phase extraction method (31), and the supernatantswere taken for the analysis of renal cAMP levels according tothe manufacturer’s manual. Renal cAMP levels were expressedas pmol/kidneys weight (g).
Statistical Analyses
Statistical analyses were undertaken using ANOVA followed bya Bonferroni and Dunnett test for multiple comparisons. Theunpaired t test was used for comparisons of GFR, -AR density,and urinary or renal cAMP between LPS-treated and untreatedrats. Results were expressed as mean ± SE.
Adenoviral-Mediated 2-AR and -gal Expression
The Adeno-LacZ was delivered to assess the efficacy of adenovirus-mediatedtransfer of the marker gene LacZ. Figure 1 shows the -gal stainingof a cross-section of a right kidney taken 1 to 5 wk after intraparenchymaldelivery of 109 total viral particles (t.v.p.) of Adeno-LacZ.-galactosidase expression was measurable in the renal proximaland distal tubules at 1–3 wks but could not be detectedat 5 wk. The gene was also expressed in the liver but not inother organs, such as left kidney (data not shown), and wasprobably transferred via the blood stream. Once the in vivoglobal renal transgene delivery had been demonstrated with Adeno-LacZ,the delivery of the therapeutic transgene Adeno-2-AR was assessed.Figure 2A shows the immunofluorescence staining of 2-AR expressedin the right kidney 4 wk after intraparenchymal injection (109t.v.p) of Adeno-2-AR. Diffuse 2-AR overexpression was evidentthroughout the kidney, in the proximal and distal tubules andglomeruli. By contrast, control sections of the right kidneytreated with equivalent doses of EV revealed a low level of2-AR expression in the renal tubules (Figure 2B).
Figure 1. -gal staining in a cross-section of the right kidney injected with 109 total viral particles (t.v.p.) of Adeno-LacZ. Expression of -galactosidase in the right kidney taken at 1 wk (A), 3 wk (B), 5 wk (C) was measured after in vivo intraparenchymal delivery of adenovirus. A part of the section (A) was magnified and is shown in the panel.
Figure 2. Immunohistochemical detection of 2-AR expressed in the right kidney 4 wk after intraparenchymal injection (109 t.v.p.) of Adeno-2-AR–treated rats (A) and empty adenovirus (EV)–treated rats (B). Adeno-2-AR was not only expressed in tubules but also in glomeruli as indicated by the circle (A). Scale bars: 100 µm in A and B.
-AR Density in the Kidneys
To test how long 2-AR transgene overexpression was supportedin the renal tissue, -AR density levels were measured in theright and left kidneys during a 5-wk period after intraparenchymalgene delivery (Figure 3A). There was a sharp increase in -ARdensity level 2 wk after intraparenchymal gene delivery (109t.v.p of Adeno-2-AR) in the right kidney, which was sustaineduntil the 4-wk time point. Furthermore, measurable -AR overexpressionwas also observed in the contralateral left kidney, which waselevated at 2 wk after the gene delivery. Normal endogenous-AR density in the right and left kidneys (control group) wasunaltered over this timeframe. It was evident from Figure 3Bthat the degree of 2-AR overexpression depended on the adenoviraldose injected into the right kidney. Endogenous -AR densityin the right kidney (control group) was unaltered by deliveryof EV without the 2-AR gene. The administration of virus tothe right kidney resulted in overexpression of 2-AR in nonrenaltissue, for example liver and lung. Although the time courseof change in lung -AR density was similar to that in the leftkidney, liver -AR density reached a peak level at 1 to 2 wkand had returned to basal levels by 3 wk. On the other hand,-AR density in the heart was not significantly increased atany time point (data not shown).
Figure 3. 2-AR expression in rat kidneys after intraparenchymal delivery of Adeno-2-AR. (A) Time course of change in -AR density in right and left kidney after delivery of 109 t.v.p. of Adeno-2-AR or saline (control) via intraparenchymal injection. *P < 0.05 versus control level at the corresponding period. n = 6 to 8. (B) Dose-dependent 2-AR expression in the right kidney 4 wk after delivery of increasing doses (t.v.p.) of Adeno-2-AR. P < 0.05 versus EV (109 t.v.p.), n = 5 to 8. Data are mean ± SE.
Effects of 2-AR Overexpression on Renal Function Figure 4A shows the time course of GFR (ml/min per 100 g bodywt) after delivery of various doses of Adeno-2-AR. Althoughthere was a significant increase (P < 0.05) in GFR 2 wk afterintraparenchymal delivery of Adeno-2-AR (109 t.v.p), GFR levelsat 1, 3, and 4 wk after delivery of Adeno-2-AR (108–9t.v.p) were not changed compared with those in control rats.By contrast, the higher dose of 1010 t.v.p Adeno-2-AR produceda diminished GFR with advancing age. The changes in time courseof FENa and FEK (%) after various doses of Adeno-2-AR are shownin Figures 4B and 4C. There was a significant decrease in FENa(P < 0.05) 1 to 2 wk after intraparenchymal delivery of Adeno-2-AR(108–9 t.v.p); whereas, at 3 and 4wks after delivery ofAdeno-2-AR (108–9 t.v.p), it was not different from thatin control rats (Figure 4B). FEK levels (Figure 4C) after deliveryof Adeno-2-AR (108–9 t.v.p) were unchanged compared withthose in control rats, while there was a significant increasein FEK 3 to 4 wk after intraparenchymal delivery of the higherdose of Adeno-2-AR (1010 t.v.p). These results indicate thatGFR, FENa, and FEK became stable approximately 3 wk after thedelivery of Adeno-2-AR (108–9 t.v.p). Furthermore, Adeno-2-AR(109 t.v.p) did not change weight, BP, or HR compared with thoseof control rats or EV-treated rats (Table 1). Thus, because109 t.v.p of Adeno-2-AR was the highest dose able to providestability to renal function and physiologic function, this dosewas chosen as the "therapeutic dose" to estimate an effect of2-AR on renal dysfunction induced by endotoxin.
Figure 4. In vivo assessment of renal function in rats treated with Adeno-2-AR. (A) Time course of GFR (ml/min per 100 g body wt) after delivery of various doses of Adeno-2-AR. (B) Time course of FENa (%) after delivery of various doses of Adeno-2-AR. (C) Time course of FEK (%) after delivery of various doses of Adeno-2-AR. Data are mean ± SE. *P < 0.02 versus control level at the corresponding period; n = 6 to 8.
Table 1. In vivo measurements in rats with 109 t.v.p. of Adeno-2-AR 25 d after 2-AR gene deliverya
Role of 2-AR in the Regulation of GFR
The mechanisms underlying the enhanced GFR 2 wk after deliveryof 109 t.v.p of Adeno-2-AR were investigated. Figure 5 showsthat the 2-AR antagonist (ICI 118,551) was able to block theincreased GFR in the Adeno-2-AR–treated rats in a dose-dependentmanner. This would be compatible with Adeno-2-AR–treatedrats producing an increase in GFR via a 2-AR ligand–independentmechanism and that 2-AR constitutively active receptors in thekidney were able to elevate GFR. Importantly, there was no differencein GFR levels between control rats and EV-treated rats, suggestingthat 109 t.v.p adenovirus did not of itself affect glomerularfiltration.
Figure 5. Changes in GFR (ml/min per 100 g body wt) of control (cont), EV, and Adeno-2-AR–treated rats 2 wk after delivery of 109 t.v.p. of Adeno-2-AR. Intraperitoneal injection of increasing doses of the 2-AR antagonist (ICI 118,551) given 2 h before measurement of GFR. Doses of ICI 118,551 from 31.4 µg/kg to 31.4 ng/kg were given. Data are the mean ± SE. *P < 0.05 versus control. P < 0.05 versus Adeno-2-AR rats without ICI 118,551 injection; n = 5 to 8.
Renal Function in Adeno-2-AR Treated Rats after Sepsis
A study was undertaken to examine whether 2-AR overexpressionin the kidney prevented the renal failure induced by endotoxin.This was done using intraperitoneal injection of LPS (5 mg/kg)to induce renal failure in control and Adeno-2-AR treated rats.The experiment was performed on the 25th day after the deliveryof 109 t.v.p of Adeno-2-AR, which was chosen as the therapeuticdose. All rats in this study survived, but the LPS (5 mg/kg)decreased weight gain in both control and the Adeno-2-AR–treatedrats (a weight loss of 20 to 30 g). It can be seen in Figure 6that GFR in the Adeno-2-AR treated rats was not changed bythe injection of LPS while in the control rats it was significantly(P < 0.05) depressed by the LPS challenge. The addition ofthe antagonist, ICI 118,551 blocked the ability of the Adeno-2-AR–treatedrats to maintain GFR, suggesting that constitutive 2-AR activityplays an important role in preserving renal function againstthe endotoxin. The changes in the BP and HR after LPS injectionin Adeno-2-AR–treated rat were not different from thosein control rats (Table 2), suggesting that these parameterswere not involved in the regulation of GFR in Adeno-2-AR–treatedrats. In addition, GFR, FENa, and FEK were monitored over aperiod of 1 mo after the LPS challenge, showing that the Adeno-2-AR–treatedrats did not have a significant change in renal function (Table 3).
Figure 6. Rescue of endotoxin-induced renal dysfunction in the Adeno-2-AR rats. LPS (5 mg/kg intraperitoneally) was injected into control and Adeno-2-AR–treated rats on the 25th day after delivery of the Adeno-2-AR (109 t.v.p.). Intraperitoneal injection of various doses of the Adeno-2-AR antagonist (ICI 188,551) was given 2 h before the LPS challenge. Dose of 10-1 or 10-2 or ICI 118,551 represents administration of 3.14 µg/kg or 0.314 µg/kg, respectively. Data are mean ± SE. *P < 0.05 versus control without any treatment; P < 0.05 versus Adeno-2-AR rats without any treatment. n = 5 to 8.
Table 3. Long-term effects of 2-AR overexpression on kidney functiona
cAMP level and -AR Density in Failing Kidneys Figure 7A indicates that -AR density measured in a combinedsample of both right and left kidneys from control rats wassignificantly (P < 0.05) depressed 24 h after the LPS challenge.The renal -AR density in Adeno-2-AR–treated rats, althoughhigher than the control rats, was also decreased by the injectionof LPS (P < 0.05). Furthermore, the LPS challenge suppressed-AR densities in the liver and lung of both groups, but -ARdensity in the heart was unchanged by the LPS challenge. UrinarycAMP levels were not altered by any of the treatments (Figure 7B).On the other hand, renal cAMP content (of both right andleft kidneys) was depressed (P < 0.05) by the LPS challengein the control rats but not in the Adeno-2-AR–treatedrats (Figure 7C). The responses in renal cAMP level inducedby the LPS in both groups correlated with the changes in renal-AR density.
Figure 7. Changes in -AR density (A), urinary cAMP (B), and renal cAMP (C) after injection of LPS (5 mg/kg intraperitoneally). -AR density and renal cAMP levels were recorded from samples of combined right and left kidneys. LPS (5 mg/kg intraperitoneally) was injected into control and Adeno-2-AR rats on the 25th day after delivery of Adeno-2-AR (109 t.v.p.), and samples were collected after 24 h. Data are the mean ± SE. *P < 0.05 versus control without any treatment. P < 0.05 versus Adeno-2-AR rats without any treatment. n = 6 to 8.
Histologic Findings in Kidneys Exposed to LPS Figure 8 shows a cross-section (H&E staining) through theright kidney 24 h after PBS injection in a control rat (A),after LPS (5 mg/kg) injection into an Adeno-2-AR–treatedrat (B) and into an untreated rat (C). Although the controlrats exposed to LPS demonstrated a minor inflammatory responsein the renal interstitium, signified by cellular infiltration(C), the Adeno-2-AR–treated rat had little, if any, cellularinfiltration in the kidney (B).
Figure 8. Cross-section (H&E staining) of the right kidney 24 h after saline injection to control rates (A) and LPS (5 mg/kg) injection to Adeno-2-AR–treated rats (B) or untreated rats (C). Adeno-2-AR–treated rats were used on the 25th day after delivery of 109 t.v.p. of Adeno-2-AR. Cellular infiltration (arrow) was found in the renal interstitium in the LPS-injected control rat. Original magnifications: x200 in A and B, x100 in C.
Renal TNF- mRNA Levels in Adeno-2-AR–Treated Rats Challenged with LPS Figure 9 presents the levels of TNF- mRNA in both left and rightkidneys 24 h after PBS or LPS (5 mg/kg) injection into untreatedcontrol rats or Adeno-2-AR–treated rats. The LPS increasedthe level of TNF- mRNA in control rats, but this was significantlydepressed (by some 39%, P < 0.05) by prior treatment withAdeno-2-AR. Importantly, the suppression of TNF- mRNA in Adeno-2-AR–treatedrats could be prevented by the addition of the antagonist, ICI118,551 in a dose-dependent manner. There was no differencein TNF- mRNA levels between control rats and Adeno-2-AR–treatedrats in the absence of exposure to LPS.
Figure 9. Representative Northern blots for renal TNF- mRNA and -actin mRNA at 24 h after PBS or LPS (5 mg/kg) injection into control or Adeno-2-AR–treated rats. Adeno-2-AR–treated rats were used on the 25th day after delivery of 109 t.v.p. of Adeno-2-AR. Dose of 10-1 or 10-2 or ICI 118,551 represents administration of 3.14 µg/kg or 0.314 µg/kg, respectively. RNA was extracted from right and left kidneys in each rat. mRNA units in each group are the mean ± SE and are expressed as amount of TNF- mRNA over the amount of -actin. *P < 0.05 versus Adeno-2-AR–treated rats injected LPS along. n = 5 to 6.
The model presented herein is of in vivo gene transfer of Adeno-2-ARinto the kidney, which has been demonstrated to be an efficientand reproducible global delivery of transgene to the renal glomeruliand tubular epithelial cells of the rat. In this model, we presentnovel findings indicating that constitutive 2-AR activation,independent of the receptor ligand, was implicated in the regulationof glomerular filtration and tubular sodium reabsorption. Inaddition, we found that renal overexpression of 2-AR using genetransfer with Adeno-2-AR was effective in preventing endotoxin-inducedrenal injury. This finding was intriguing in that the sepsis-inducedrenal failure occurred with the decreases in 2-AR density andcAMP activity, which suggested an impaired renal 2-AR signalingsystem.
The method of in vivo2-AR gene transfer using intraparenchymalinjection utilized herein had an enhanced effectiveness comparedwith other delivery methods, that is intravenous or percutaneousinjections, which were utilized in preliminary studies (unpublisheddata). Intraparenchymal injection of 2-AR gene elevated -ARdensity in the kidney at a more rapid rate and achieved higherdensities compared with the other methods. In addition, thetechnique of direct intraparenchymal injection was the mosteffective way to ensure consistent uptake of the 2-AR gene intothe kidney. The elevation of -AR density in the right kidneybegan quickly after the intraparenchymal injection and reachedpeak levels 3 to 4 wk after the delivery. Interestingly, -ARdensity in the left non-injected kidney was also increased,but this took place slowly over 1 to 2 wk. This implied thatthe adenovirus from the right kidney had spread to other organs,including the left kidney. In fact, an increased 2-AR expressionwas also found in the liver and lung. Whereas 2-AR gene deliveryincreased expression in the glomeruli, -galactosidase was notexpressed in glomeruli after gene delivery. The discrepancymay be due to a difference in transfer efficiency or transductioninto the kidney via the blood stream. This would help explainto some degree the observation that 2-AR was overexpressed inthe contralateral left kidney while -gal expression was notdetected. It is likely that the adenovirus encoding 2-AR passedinto the systemic circulation after intraparenchymal injection,which would have resulted in deposition in the contralateralkidney glomeruli and caused expression in that area.
It was also evident that BP, HR, and growth rate in Adeno-2-ARrats were not influenced by the 109 t.v.p. dose of Adeno-2-AR,as GFR and FENa became stable 3 wk after the administration.Furthermore, histologic examination of the kidney indicatedthat the 109 t.v.p. dose of the Adeno-2-AR produced no evidenceof inflammation within the tissue as had been reported withhigher doses of adenovirus (32). Interestingly, although itis well known that 2-AR agonists given acutely will cause hypokalemia(33), the adenoviral treatment (108–9 t.v.p.) in the presentstudy had no influence on serum potassium levels or the levelof FEK (%). In addition, as shown in Table 3, there was no long-termeffect on renal function in the Adeno-2-AR–treated rats.Thus, although further evaluation will be required, intraparenchymalinjection of 109 t.v.p. of Adeno-2-AR into the kidney was takento be efficient, comparatively nonpathogenic. and potentiallytherapeutic.
Previous reports (34,35) demonstrated that renal 2-AR in normalrats were predominantly localized to the apical and sub-apicalcompartments of proximal and, to a lesser extent, distal tubularepithelia and the membranes of smooth muscle cells from renalarteries. From this morphologic evidence, it was proposed that2-AR may regulate glomerular function and thereby sodium andwater balance in the different nephron segments. This hypothesiswas supported by the present observations using Adeno-2-AR–treatedrats that overexpressed 2-AR in the proximal and distal tubulesand in the glomeruli as evaluated using an immunohistologicapproach. The important finding arising from the present studywas that constitutive 2-AR activation in the Adeno-2-AR ratsplayed an important physiologic role in causing an increasein GFR and sodium absorption in the kidney. Elevation in 2-ARactivity may contribute to an increase in tissue blood flow(36), which in the kidney might lead to a subsequent increasein GFR and decrease in FENa. However, the -AR density in theAdeno-2-AR rats could not be correlated with the elevation inGFR and sodium absorption, suggesting that the mechanisms wherebyrenal function was modulated through intracellular signals via2-AR were complex (37).
The additional important finding arising from this study wasthat transfection with Adeno-2-AR to a large degree blockedthe renal dysfunction associated with endotoxemia. Administrationof LPS on the 25th day after the Adeno-2-AR gene delivery wasfound to protect renal function from the LPS-induced insult.Because GFR and FENa were stable from 3 wk after the deliveryand the peak level of renal 2-AR expression was observed between3 and 4 wk after the delivery, we chose day 25 as the experimentalday for LPS injection. The question arose as to what mechanismswere involved in the 2-AR–mediated protective effect.2-AR activation causes the generation of the second-messengercAMP via the activation of adenylate cyclase (38). Indeed, itwas observed that there was an increase in the renal contentof cAMP in the Adeno-2-AR–treated rats. Importantly, renalcAMP content levels in the Adeno-2-AR–treated rat werenot depressed after the exposure to LPS unlike those in thecontrol rat which were significantly decreased. The fall inrenal cAMP level was correlated with the depression in GFR causedby the LPS challenge, suggesting the possibility that the protectiveeffect of 2-AR activation was exerted through the intracellularcAMP/cAMP-dependent protein kinase (PKA) pathway. This wouldbe compatible with previous reports that cAMP-PKA activationplays an important role in the protection against acute renalfailure (39,40).
Another mechanism that may be implicated after 2-AR activationis an antiinflammatory action. On the basis of the histologicfindings, it was apparent that the LPS-induced leukocyte infiltrationwas absent in the kidney sections of the Adeno-2-AR–treatedrats. Moreover, it was possible to demonstrate that the LPS-inducedTNF- mRNA in the kidney was suppressed in the Adeno-2-AR–treatedrats. Another possibility is that the kidney could be influencedindirectly by 2-AR overexpression in nonrenal tissue. Therewas no evidence that the 2-AR system was overexpressed in theheart, so it could not have mediated the reduction in GFR afterthe LPS challenge. However, the possibility remains that overexpressionin other organs could modify renal function. It is also recognizedthat activation of 2-AR can inhibit perimicrovessel edema formation(41, 42) as it has been reported that the 2-AR agonist, dopexamine,attenuated endotoxin-induced vascular permeability in rat mesentery(41). Indeed, a similar mechanism may operate as a consequenceof 2-AR activation in the present study, conferring some protectionfrom the LPS-induced reduction in renal function. Taken together,these findings suggest that constitutive 2-AR activation isable to protect renal function through several mechanisms, includingthe cAMP-PKA pathway. The replacement of lost receptors as aconsequence of sepsis may represent a novel therapeutic approach.
Acknowledgments
This study was supported by grants from the Human Science Foundationin Japan.
Ping P, Anzai T, Gao M, Hammond HK: Adenyl cyclase and G protein receptor kinase expression during development of heart failure. Am J Physiol 273: H707–H717, 1997
Silverman HJ, Penaranda R, Orens JB, Lee NH: Impaired beta-adrenergic receptor stimulation of cyclic adenosine monophosphate in human septic shock: Association with myocardial hyporesponsiveness to cathecholamines. Crit Care Med 21: 31–39, 1993[Medline]
Bernardin G, Strosberg AD, Bernard B, Mattei M, Marullo S: -Adrenergic receptor-dependent and –independent stimulation of adenylate cyclase is impaired during severe sepsis in humans. Intensive Care Med 24: 1315–1322, 1998[CrossRef][Medline]
Liao J, Keiser JA, Scales WE, Kunkel SL, Kluger MJ: Role of epinephrine in TNF and IL-6 production from isolated perfused rat liver. Am J Physiol 268: R896–R901, 1995
Hetier P, Ayala J, Bousseau A, Prochiantz A: Modulation of interleukin-1 and tumor necrosis factor expression by beta adrenergic agonists in mouse ameboid microgial cells. Exp Brain Res 86: 407–413, 1991[Medline]
Panina-Bordignon PJ, Mazzeo D, Di Lucia P, D’Ambrosio D, Lang R, Fabbri L, Self C, Sinigaglia F: 2-agonists prevent Th1 development by selective inhibition of interleukin 12. J Clin Invest 100: 1513–1519, 1997[Medline]
Severn A, Rapson NT, Hunter CA, Liew FY: Regulation of tumor necrosis factor production by adrenaline and beta-adrenergic agonists. J Immunol 148: 3441–3445, 1992[Abstract]
Nakamura A, Johns EJ, Imaizumi A, Yanagawa Y, Kohsaka T: Activation of 2-adrenoceptor prevents shiga toxin-induced tumour necrosis factor production. J Am Soc Nephrol 12: 2288–2299, 2001[Abstract/Free Full Text]
Kaplan BS, Meyers KE, Schulman SL: The pathogeneis and treatment of hemolytic uremic syndrome. J Am Soc Nephrol 8: 1126–1133, 1998
Van De Kar NC, Monnens LA, Karmali MA, Van Hinsbergh VW: Tumour necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human vascular endothelial cells: Implications for the pathogenesis of the hemolytic uremic syndrome. Blood 80: 2755–2764, 1992[Abstract/Free Full Text]
Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 101: 1644–1655, 1992[Abstract/Free Full Text]
Stone R: Search for sepsis drugs goes on despite past failures. Science 264: 365–367, 1994[Free Full Text]
Warren HS: Strategies for the treatment of sepsis. N Engl J Med 336: 952–953, 1997[Free Full Text]
Kikeri D, Pennel JP, Hwang KH, Jakob JI, Richman AV, Bourgigne JJ: Endotoxemic acute renal failure in awake rats. Am J Physiol 250: F1098–F1106, 1986
Schor N: Acute renal failure and the sepsis syndrome. Kidney Int 61: 764–776, 2002[CrossRef][Medline]
Kaplan SL: Bacteremia and septic shock. In: Textbook of Pediatric Infectious Diseases, 4th ed, edited by Feigin RD, Cherry JD, Philadelphia, WB Saunders, 1998, pp 807–820
Liano F, Junco E, Pascual J. Madero R, Verde E: The spectrum of acute renal failure in the intensive care unit compared with that seen in other settings. The Madrid Acute renal Failure Study Group. Kidney Int 66 [Suppl]: S16–S24, 1998
Emilien G, Maloteaux J-M: Current therapeutic uses and potential of beta-adrenoceptor agonists and antagonists. Eur J Clin Pharmacol 53: 389–404, 1998[CrossRef][Medline]
Maurice LP, Hata JA, Shah AS, White DC, McDonald PH, Dolber PC, Wilson KH, Lefkowitz RJ, Glower DD, Koch WJ: Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary 2-adrenergic receptor gene delivery. J Clin Invest 104: 21–29, 1999[Medline]
Ortiz PA, Hong NJ, Plato CF, Varela M, Garvin JL: An in vivo method for adenovirus-mediated transduction of thick ascending limbs. Kidney Int 63: 1141–1149, 2003[CrossRef][Medline]
Lipkowitz MS, Hanss B, Tulchin N, Wilson PD, Langer JC, Ross MD, Kurtzman GJ, Klotman PE, Klotman ME: Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors. J Am Soc Nephrol 10: 1908–1915, 1999[Abstract/Free Full Text]
Kypson AP, Peppel K, Akhter SA, Lilly RE, Glower DD, Lefkowitz RJ, Koch WJ: Ex vivo adenovirus-mediated gene transfer to the adult rat heart. J Thorac Cardiovasc Surg 115: 623–630, 1998[Abstract/Free Full Text]
Koch WJ, Rockman HA, Samama P, Hamilton R, Bond RA, Milano CA, Lefkowitz RJ: Cardiac function in mice overexpressing the –adrenergic receptor kinase or a ARK inhibitor. Science 268: 1350–1353, 1995[Abstract/Free Full Text]
Nakamura A, Johns EJ: Effect of renal nerves on expression of renin and angiotensinogen genes in rat kidneys. Am J Physiol 266: E230–E241, 1994
Nakamura A, Johns EJ, Imaizumi A, Yanagawa Y, Kohsaka T: 2-adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin. Nephrol Dial Transplant 24: 1121–1129, 2000
Mayer SE, Stull JT, Wastila WB: Rapid tissue fixation and extraction techniques. Methods Enzymol 38: 1–9, 1974[Medline]
Yang Y, Ertl HCJ, Wilson JM: MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1: 433–442, 1994[CrossRef][Medline]
Brown MJ, Brown DC, Murphy MB: Hypokalemia from beta2-receptor stimulation by circulating epinephrine. N Engl J Med 309: 1414–1419, 1983[Abstract]
Taniguchi S, Watanabe T, Nakao A, Seki G, Uwatoko S, Suzuki K, Kurokawa K: Distribution of 2-adrenergic receptor mRNA expression along the hamster nephron segments. FEBS 318: 65–70, 1993[CrossRef][Medline]
Boivin V, Jahna R, Gambaryan S, Ness W, Boege F, Lohse M: Immunofluorescent imaging of 1- and 2-adrenergic receptors in rat kidney. Kidney Int 59: 515–531, 2001[CrossRef][Medline]
Rothwell NJ, Stock MJ, Sudera DK: Changes in tissue blood flow and beta-receptor density of skeletal muscle in rats treated with the beta2-adrenoceptor agonist clenbuterol. Br J Pharmacol 90: 601–607, 1987[Medline]
Nakamura A, Johns EJ, Imaizumi A, Yanagawa Y, Kohsaka T: Modulation of interleukin-6 by 2-adrenoceptor in endotoxin-stimulated renal macrophage cells. Kidney Int 56: 839–849, 1999[CrossRef][Medline]
Verghese MW, Synyderman R: Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides. J Immunol 130: 869–873, 1983[Abstract]
Begany DP, Carcillo JA, Herzer WA, Mi Z, Jackson EK: Inhibition of type IV phosphodiesterase by Ro 20–1724 attenuates endotoxin-induced acute renal failure. J Pharmacol Exp Ther 278: 37–41, 1996[Abstract/Free Full Text]
Guan Z, Miller SB, Greenwald JE: Zaprinast accelerates recovery from established acute renal failure in the rat. Kidney Int 47: 1569–75, 1995[Medline]
Schmidt W, Hacker A, Gebhard MM, Martin E, Schmit H: Dopexamine attenuates endotoxin-induced microcirculatory changes in rat mesentery: role of beta2 adrenoceptors. Crit Care Med 26: 1639–1645, 1998[CrossRef][Medline]
Davies DC: Blood-brain barrier breakdown in septic encephalopathy and brain tumours. J Anat 200: 639–646, 2002[CrossRef][Medline]
Received for publication May 30, 2003.
Accepted for publication November 14, 2003.
2-Adrenoceptor Activation Attenuates Endotoxin-Induced Acute Renal Failure
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Abstract
Introduction
2-adrenergic control of organ function have been implicated in the pathogenesis of several disease states, such as septic shock. The objectives of the present study were to define the contribution of 
mRNA in the kidney. In rats overexpressing 
