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doi:10.1111/jgh.12670
H E PAT O L O G Y
Lipopolysaccharide enhanced renal vascular response to endothelin-1 through ETA overexpression in portal hypertensive rats Chiao-Lin Chuang,*,†,‡ Hui-Chun Huang,†,§ Ching-Chih Chang,*,† Fa-Yauh Lee,†,§,1 Jaw-Ching Wu,‡,¶,1 Jing-Yi Lee,§ Hsian-Guey Hsieh¶ and Shou-Dong Lee†,** *Division of General Medicine, Department of Medicine, §Division of Gastroenterology, Department of Medicine, ¶Department of Medical Research and Education, Taipei Veterans General Hospital, †Department of Medicine, National Yang-Ming University School of Medicine, ‡Institute of Clinical Medicine, National Yang-Ming University School of Medicine, and **Cheng-Hsin General Hospital, Taipei, Taiwan
Key words endothelin receptor type A, lipopolysaccharide, partial portal vein ligation, portal hypertension, renal vascular reactivity. Accepted for publication 26 May 2014. Correspondence Professor Fa-Yauh Lee, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Email: [email protected] 1
These authors contributed equally to this paper. Conflicts of interest: The authors disclose no conflicts
Abstract Background and Aim: Hypo-perfusion resulting from intense renal vasoconstriction is traditionally contributed to renal dysfunction in advanced liver disease, although cumulative studies demonstrated renal vasodilatation with impaired vascular contractility to endogenous vasoconstrictors in portal hypertension and compensated liver cirrhosis. The pathophysiology of altered renal hemodynamics remains unclear. This study, using a rat model of portal hypertension with superimposed endotoxemia, was designed to delineate the evolution of renal vascular reactivity and vaso-regulatory gene expression during liver disease progression. Methods: Rats were randomized into sham surgery (SHAM) or partial portal vein ligation (PVL). Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS) on the seventh day following surgery. Isolated kidney perfusion was performed at 0.5 h or 5 h after LPS to evaluate renal vascular response to endothelin-1. Results: In contrast to impaired vascular contractility of SHAM rats, PVL rats displayed enhanced renal vascular reactivity to endothelin-1 at 5 h following endotoxemia. There were extensive upregulations of inducible nitric oxide synthase in kidney tissues of endotoxemic rats. The changes of renal endothelin receptor type A (ETA) level paralleled with the changes of renal vascular reactivity in LPS-treated rats. Compared with SHAM rats, PVL rats showed increased renal ETA and phosphorylated extracellular-signalregulated kinases 1/2 (p-ERK1/2) at 5 h after LPS. Conclusion: LPS-induced systemic hypotension induces a paradoxical change of renal vascular response to endothelin-1 between SHAM and PVL rats. LPS-induced renal vascular hyperreactivity in PVL rats was associated with upregulation of renal ETA and subsequent activation of ERK1/2 signaling.
Introduction In patients with liver parenchymal disease and portal hypertension, renal function deteriorates as liver function worsens. Although a great advance in the researches of portal hypertension and liver cirrhosis has been achieved with highly reproducible experimental models, the mechanisms leading to altered renal blood flow and renal dysfunction in advanced liver cirrhosis are not entirely clear. The critical impediment lies mainly in the paucity of an ideal animal model to correctly mimic human disease and fulfill this complex syndrome. Usually, acute kidney injury secondary to liver dysfunction is functional in nature and occurs in the absence of significant alterations in renal histology.1,2 Traditional consensus defines the renal dysfunction in advanced cirrhotic patients as
kidneys hypo-perfusion resulting from combined intense renal vasoconstriction and decreased renal blood flow in response to generalized systemic arterial vasodilatation.3,4 However, cumulative studies demonstrated that renal vasculature of portal hypertensive and compensated cirrhotic rats had lower perfusion pressure and hypo-responsiveness to endogenous vasoconstrictors, implying renal vasodilatation.5–7 In order to illustrate the evolution of renal vascular reactivity during disease progression of liver cirrhosis, some hypotheses aimed at elucidating the potential mechanisms were proposed.8,9 Usually, the liver dysfunction appears to be an important background factor when the vasodilatation related hypovolemia can be compensated by the development of hyperdynamic circulation, such as increased heart rate, avid renal sodium and water retention, and higher cardiac output.
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Once the auto-regulatory mechanisms are overwhelmed by the precipitating events, extreme effective arterial underfilling will initiate extreme intra-renal vasoconstriction and subsequently renal impairment. Such events may include overzealous use of diuretics, large volume paracentesis, gastrointestinal bleeding, cardiomyopathy, or development of spontaneous bacterial peritonitis in ascitic patients. From our previous study, the evolution of renal vasodilatation in portal hypertensive rats has been well delineated using isolated kidney perfusion experiment.10 This study, furthermore, used a rat model of portal hypertension with superimposed endotoxemia, a common complication of cirrhosis, to mimic the pictures of clinical condition. Renal vascular response to endotoxemia was surveyed by isolated kidney perfusion and two key vaso-regulatory systems, namely nitric oxide (NO) and endothelin-1 (ET-1), were analyzed to delineate the pathogenesis of renal dysfunction in advanced liver disease.
incubation of ETA antagonist at day 7 following PVL. Selective ETA antagonist BQ-123 (10−6 mol/L) was added into the Krebs solution 30 min before the beginning throughout the whole course of the concentration—response relationship study.
Materials and methods
Isolated kidney perfusion. The kidney perfusion system was performed as previously described.10 In brief, right renal artery was cannulated with a 22-gauge needle via superior mesenteric artery. At first, the kidney was perfused in situ with warm oxygenated (95% O2-5% CO2) Krebs solution by means of a roller pump (model 505S; Watson-Marlow Ltd, Falmouth, Cornwall, UK). Right renal vein was cut and ureter was transected to allow exit of perfusate. Finally, right kidney was excised from surrounding tissues, decapsulated, and placed into a chamber containing Krebs solution at 37 ± 0.5°C. Renal vascular responses were recorded as changes in renal perfusion pressure downstream from the pump through a Spectramed DTX transducer attached to the Gould model RS 3400 recorder (Gould Inc., Cupertino, CA, USA). The flow rate of perfusion solution was kept constant (5 ml/min per gm kidney weight), so the changes in perfusion pressure reflected the changes in renal vascular resistance. In each individual preparation, the contracting capability of renal vasculature was challenged with a 125 mmol/L potassium chloride solution at the end of experiments.
Animal model. Male Sprague-Dawley rats weighing 300– 350 g were maintained at 24°C with a 12-h light-dark cycle and allowed free access to food and water. Surgery and hemodynamic studies were performed under ketamine hydrochloride anesthesia (100 mg/kg, intramuscularly). Portal hypertension was induced by partial portal vein ligation (PVL) as previously described.11 Corresponding sham groups (SHAM) received the same abdominal surgery without ligation of portal vein. This study has been approved by Taipei Veterans General Hospital Animal Committee. All the experiments followed the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA and published by the National Institutes of Health (NIH publication no. 86-23, revised 1985). Experimental protocol 1. On the seventh day following surgery, rats were randomly allocated to receive intraperitoneal injection of either normal saline or lipopolysaccharide (LPS, Escherichia coli serotype O111:B4, 3 mg/kg): (i) SHAM + saline (n = 14); (ii) SHAM + LPS (n = 17); (iii) PVL + saline (n = 15); (iv) PVL + LPS (n = 17). After the measurements of body weight, mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP), each group was further randomized for isolated kidney perfusion at 0.5 h, or 5 h following LPS injection. With isolated kidney perfusion technique, cumulative concentration-response curves of renal vascular beds were determined by graded concentrations of endothelin-1 (10−10 to 3 × 10−8 M) in escalation with a constant flow rate of perfusion solution. Each concentration of perfusion solution was allowed to stabilize for 3 min before the next higher concentration was added. Left kidneys were harvested for analysis of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), endothelin receptor type A (ETA) and type B (ETB), extracellularsignal-regulated kinases 1/2 (ERK1/2), and phosphorylated ERK 1/2 (p-ERK1/2). Experimental protocol 2. To evaluate the influence of ETA in renal vascular hyperresponsiveness of endotoxemic PVL rats, another nine rats received isolated kidney perfusion with pre200
Measurement of systemic and portal hemodynamics. Under anaesthesia, right femoral artery was cannulated with polyethylene cannula (PE-50) catheter that was connected to the Spectramed DTX transducer (Spectramed Inc., Oxnard, CA, USA) to measure MAP and HR. Another PE-50 catheter connected to a Spectramed DTX transducer was inserted into the mesenteric vein to access PP. The external zero reference was placed at the level of the mid-portion of rat. Continuous recordings of MAP, HR, and PP were performed through a multi-channel recorder (model RS 3400, Gould Inc., Cupertino, CA, USA).12
Quantitative real-time polymerase chain reaction. Total RNA was extracted from frozen kidneys with SV Total RNA Isolation System (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (RT-PCR) was performed on the LightCycler 480 instrument (Roche Applied Science; Mannheim, Germany). The primer sequences were 5′-GGAAGTAGCCAATGCAGTGAA-3′ (sense) and 5′-GCCA GTCTCAGAGCCATACA-3′ (antisense) for eNOS, 5′-AGGCCA CCTCGGATATCTCT-3′ (sense) and 5′-GCTTGTCTCTGGGT CCTCTG-3′ (antisense) for iNOS, 5′-TTCCCTCTTCACTTAAG CCGAA-3′ (sense) and 5′-GCAACAGAGGCATGACTGAA AA-3′ (antisense) for ETA, 5′-TGTAGTCCAAAACCAGCAAA AA-3′ (sense) and 5′-CTGTTGGCTTCCCCTTCAC3′ (antisense) for ETB, 5′-CGCCCTAGGCACCAGGGTG-3′ (sense) and 5′GCTGGGGTGTTGAAGGTCTCAAA-3′ (antisense) for β-actin, respectively. Relative mRNA expression was quantified using the ΔCt method. The products were standardized with a housekeeping gene, β-actin, from the same RNA samples. Journal of Gastroenterology and Hepatology 30 (2015) 199–207
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Western blot analysis. Kidneys were dissected and homogenized. Ten microgram of protein aliquots from each sample were separated on 10% SDS-PAGE gels and subsequently transferred to a nitrocellulose filter membrane by electroblotting. The membrane were probed with specific primary antibodies against eNOS (Chemicon Inc., Temecula, CA, USA), iNOS (Chemicon Inc., Temecula, CA, USA), ETA (Abcam, Cambridge, UK), ETB (Abcam, Cambridge, UK), ERK1/2 (Cell Signaling Technology, Beverly, CA, USA), and p-ERK1/2 (Cell Signaling Technology, Beverly, CA, USA) for 90 min at room temperature (25°C) and washed. β-actin (Chemicon Inc., Temecula, CA, USA) was used as a loading control. Antibody binding was detected after secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Chemicon Inc., Temecula, CA, USA) incubation. Subsequent detection of the specific protein was performed by enhanced chemiluminescence. With a computer-assisted video densitometer and digitalized software (Kodak Digital Science ID Image Analysis Software), the blots were scanned, and the signal intensity of appropriate bands was analyzed. Immunohistochemistry stain. Five micrometer-thick sections obtained from paraffin-embedded kidneys were de-waxed with xylene and rehydrated through a series of ethanol solutions. Sections were subjected to microwave irradiation in citrate buffer to enhance antigen retrieval and pre-incubated with 5% normal rabbit serum in Tris-buffered saline. Primary antibodies were incubated for 1 h in a humidity chamber using the following dilutions: antieNOS polyclonal antibody (1 : 100); anti-iNOS polyclonal antibody (1 : 100); anti-ETA polyclonal antibody (1 : 100); anti-ETB polyclonal antibody (1 : 100). After rinsing twice in phosphatebuffered saline, sections were incubated with fluorochromeconjugated secondary antibodies (Alexa Fluor 488 fluorescent, Jackson ImmunoResearch Laboratories, Inc. Baltimore, USA) for 1 h at room temperature. Finally, slides were coverslipped with carbonate-buffered glycerol, and evaluated in an Olympus AX 80 microscope (Olympus; Hamburg, Germany) equipped with epifluorescence illumination and digital cameras.
Drugs. ET-1, LPS, and reagents for Krebs solution were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Statistical analysis. All results are expressed as mean ± SEM. Differences between the concentration–response curves were analyzed by two-way ANOVA for repeated measures using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical analysis for comparison of mRNA and protein expressions was performed using Student’s t-test. Results were considered statistically significant at a two-tailed P-value of less than 0.05.
Results General characteristics of the rats. Comparing with corresponding saline-treated groups, there was significantly decreased mean arterial pressure at 5 h following LPS injection in both SHAM rats (P < 0.01) and PVL rats (P = 0.017) (Table 1). The SHAM rats developed significantly higher portal pressures
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Table 1
Hemodynamic profiles of experimental rats
Group 0.5 h SHAM + saline SHAM + LPS PVL + saline PVL + LPS 5h SHAM + saline SHAM + LPS PVL + saline PVL + LPS
n
7 7 7 8 7 10 8 9
MAP (mmHg)
HR (beats/min)
PP (mmHg)
120 ± 6.0 113 ± 5.0 95 ± 3.5 90 ± 5.6
362 ± 16 375 ± 18 336 ± 19 336 ± 10
9.0 ± 0.2 13.0 ± 1.2* 15.3 ± 1.0 15.0 ± 1.0
117 ± 4.9 83 ± 3.3* 93 ± 4.7 79 ± 3.4**
345 ± 18 356 ± 8 324 ± 13 351 ± 8.6
8.7 ± 0.5 12.3 ± 1.3* 16.3 ± 0.9 12.5 ± 0.7**
*P < 0.05 between time-matched saline- and LPS-injected SHAM group; **P < 0.05 between time-matched saline- and LPS-injected PVL group. Data were expressed as mean ± SEM. HR, heart rate; LPS, lipopolysaccharide; MAP, mean arterial pressure; PP, portal pressure; PVL, partial portal vein ligation; SHAM, sham surgery.
since 0.5 h after endotoxemia (P = 0.015), while PVL rats showed declined portal pressure at 5 h after LPS injection (P < 0.01) (Table 1). Both SHAM rats and PVL rats have significantly higher serum ET-1 (P ≤ 0.01), serum glutamic oxaloacetic transaminase (P ≤ 0.05), and serum glutamic-pyruvic transaminase (P ≤ 0.05) levels at 5 h following LPS injection (Table 2). All the fraction excretion of sodium (FeNa) levels remained within normal range despite higher FeNa in response to LPS injection. Only PVL rats had significantly increased serum creatinine (P = 0.006) at 5 h following LPS injection (Table 2). In vitro renal vascular contractility to ET-1. The addition of ET-1 into perfusion solution led to a concentrationdependent increase of renal perfusion pressures in both SHAM rats and PVL rats (Fig. 1). Without LPS, the PVL rats showed significant renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (P < 0.05, Fig. 1a,b). Compared with corresponding saline-treated groups, the SHAM rats showed significantly attenuated renal vascular contractility to ET-1 at 5 h following LPS injection (P = 0.018, Fig. 1d). However, the blunted renal vascular reactivity to ET-1 in PVL rats was significantly reversed at 5 h following LPS injection (P = 0.008, Fig. 1f). Pretreatment of ETA antagonist abrogated the LPS-enhanced renal vascular response in PVL rats (P ≤ 0.001, Fig. 1f). Expressions of NOS and ET-1 receptors in kidneys. Compared with corresponding saline-treated groups, there was extensive upregulation of iNOS mRNA in kidneys of both SHAM rats and PVL rats since 0.5 h after endotoxemia (P < 0.01, Fig. 2b,f). The expression of renal ETA mRNA was significantly downregulated in SHAM rats (P < 0.01, Fig. 2c) but upregulated in PVL rats (P < 0.05, Fig. 2g) at 5 h following LPS injection. The expression of renal ETB mRNA was only downregulated in PVL rats (P < 0.05, Fig. 2h) at 5 h following LPS injection. There was no difference in the expression of eNOS in response to LPS injection (P > 0.05, Fig. 2a,e).
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Table 2
Biochemistry of experimental rats
Variables
Pre-LPS
LPS-5h
P-value
6 1.1 ± 0.1 64.8 ± 1.8 47.5 ± 1.6 0.19 ± 0.01 11.4 ± 0.1 28.9 ± 1.7 0.08 ± 0.01 0.3 ± 0.04
6 6.6 ± 0.7 111 ± 11.5 82.0 ± 9.8 0.25 ± 0.03 12.3 ± 0.5 32.7 ± 3.8 0.21 ± 0.05 0.25 ± 0.02
< 0.001 0.01 0.017 0.141 0.121 0.492 < 0.001 0.253
6 3.3 ± 0.3 70.8 ± 4.4 55.6 ± 6.3 0.18 ± 0.01 12.3 ± 0.8 27.0 ± 2.4 0.08 ± 0.02 0.12 ± 0.02
8 15.7 ± 2.5 200 ± 41.9 120 ± 26.0 0.33 ± 0.04 13.0 ± 0.7 39.1 ± 5.4 0.23 ± 0.05 0.26 ± 0.05
0.001 0.018 0.043 0.006 0.537 0.087 0.032 0.064
SHAM rats Number ET-1 (pg/ml) SGOT (IU/L) SGPT (IU/L) Cr (mg/dL) PT (second) aPTT (second) FeNa (%) FeCl (%) PVL rats Number ET-1 (pg/ml) SGOT (IU/L) SGPT (IU/L) Cr (mg/dL) PT (second) aPTT (second) FeNa (%) FeCl (%)
Data were expressed as mean ± SEM. aPTT, activated partial thromboplastin time; Cl−, chloride; Cr, creatinine; ET-1, endothelin-1; FeCl, fractional excretion of chloride; FeNa, fractional excretion of sodium; LPS, lipopolysaccharide; Na+, sodium; PT, prothrombin time; PVL, partial portal vein ligation; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; SHAM, sham surgery.
Similarly, there were higher iNOS protein levels in kidneys of both SHAM rats (P < 0.01, Fig. 3b) and PVL rats (P < 0.05, Fig. 3f) at 5 h after LPS injection. The renal ETA protein was downregulated in SHAM rats (P < 0.05, Fig. 3c) but upregulated in PVL rats at 5 h following LPS injection (P < 0.05, Fig. 3g). There was no difference in the expressions of renal eNOS and ETB in response to LPS injection (P > 0.05, Fig. 3a–h). Immunohistochemical staining of NOS and ET-1 receptors in kidneys. Compared with corresponding saline-treated groups, the expressions of iNOS (Fig. 4d) and ETA (Fig. 5b) in interlobular arteries as well as afferent and efferent arterioles were significantly upregulated in PVL rats at 5 h following LPS injection. There was no difference in the expressions of renal eNOS and ETB in response to LPS injection (Figs 4a,b,5c,d). Expressions of ERK1/2 and p-ERK1/2 in kidneys. There was no significant difference of renal phosphorylated ERK1/2 protein between saline-treated and LPS-treated SHAM rats (P > 0.05, supplementary Fig. 1a). However, the phosphorylation ratio of renal ERK1/2 was significantly increased in PVL rats at 5 h following LPS injection (P < 0.05, supplementary Fig. 1b).
Discussion LPS, a component of the outer membrane of Gram-negative bacteria released during septicemia, is a major cause of septic shock. 202
Cumulative evidences have demonstrated that administration of LPS elicited a marked elevation in plasma levels of NO and ET-1.13,14 The present study provides the first comprehensive analysis concerning the time-course and expression pattern of two important endothelium-released vaso-regulatory factors (NO and ET-1) in renal vasculature between SHAM rats and PVL rats following LPS-induced endotoxemia. The principal finding in current study was that renal ETA overexpression rather than iNOS overexpression might be the key event in modulating renal vascular reactivity to endotoxemia during portal hypertension. It is widely accepted that systemic vasodilation during endotoxemia is caused by excessive production of NO, which is mainly synthesized by activation of iNOS.15,16 During endotoxemia, increased renal tissue NO is thought to be largely responsible for increased renal blood flow through its vasodilatory effects on the afferent arteriole during periods of hypoperfusion.17,18 Consistent with previous observations, our results showed decreased mean blood pressures and extensive upregulations of renal iNOS levels in both SHAM rats and PVL rats after LPS administration. However, in contrast to the impaired vascular contractility to ET-1 in endotoxemic SHAM rats, the renal vascular bed of endotoxemic PVL rats displayed significant enhancement of the contractile response to cumulative concentrations of ET-1 despite upregulated iNOS. The reciprocal pattern of renal vascular response to ET-1 between SHAM rats and PVL rats was in parallel with the paradoxical change of renal ETA expression after LPS administration, indicating a clear correlation between renal ETA and the pathogenesis of renal vascular response during LPS-induced systemic hypotension. It is conceivable that, in spite of the large amounts of NO generated by iNOS, the NO system is unable to counteract the renal vasoconstrictor influences of ET-1 in response to endotoxemia during portal hypertension. The renal effects of enhanced ET-1 expression and secretion in endotoxemia are harmful by reducing blood volume and renal cortical blood flow.19,20 The compromised ability of PVL kidneys to counteract the pressor effect of ET-1 during endotoxemia may ultimately be responsible for kidney injury, consistent with our findings. This could, at least partly, explain the increased susceptibility to renal dysfunction in cirrhosis during endotoxemia. ET-1, a potent vasoactive peptide, induces its vascular effects through two G protein-coupled receptors, namely the ETA and ETB. ETA is mainly expressed in vascular smooth muscle cells (VSMCs) throughout and mediates vasoconstriction.21 ETB is predominantly expressed in endothelial cells, and its activation causes a transient vasodilation.22 ET-1 elicits renal cortical vasoconstriction and reduces total renal blood flow in endotoxemia primarily through ETA activation.23 ETA inhibition has been shown to improve medullary blood flow in endotoxemia associated hypotension and reverse LPS-induced mortality.24,25 In agreement, our results revealed that renal ETA expression is the major determinant factor of renal vascular reactivity in response to LPS-induced systemic hypotension, although the possible involvement of some other mediators. As for many G-protein-coupled receptors, the signaling capacity of endothelin receptors is fine-tuned by posttranslational modifications. The binding of ET-1 to ETA results in the contractile effects in VSMCs through activation of diverse signaling molecules such as phospholipase C, protein kinase C, and ERK1/2.26 It has been reported that activation of ERK1/2 is important for ET-1-induced VSMCs contraction,
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Figure 1 Concentration–response curves to ET-1 in isolated perfuse kidneys of SHAM or PVL rats. (a) PVL rats (n = 7) developed renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (n = 7) at 0.5 h following saline. (b) PVL rats (n = 8) developed renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (n = 7) at 5 h following saline. (c) LPS-injected SHAM rats (n = 7) showed unchanged renal vascular reactivity to ET-1 at 0.5 h following LPS in comparison with saline-injected SHAM rats (n = 7). (d) LPS-injected SHAM rats (n = 10) showed attenuated renal vascular contractility to ET-1 at 5 h following LPS in comparison with saline-injected SHAM rats (n = 7). (e) LPS-injected PVL rats (n = 8) showed unchanged renal vascular reactivity to ET-1 at 0.5 h following LPS in comparison with saline-injected PVL rats (n = 7). (f) LPS-injected PVL rats (n = 9) showed enhanced renal vascular contractility to ET-1at 5 h following LPS in comparison with saline-injected PVL rats (n = 8). The renal vascular hyperreactivity in LPS-injected PVL rats was significantly attenuated by pre-incubation of ETA antagonist (n = 9). ET-1, endothelin-1; ETA, endothelin receptor type A; LPS, lipopolysaccharide; PVL, portal vein ligation; SHAM, sham surgery. (a) , SHAM-saline-0.5 h; , PVL-saline-0.5 h; (b) , SHAM-saline-5 h; , PVL-saline-5 h; (c) , SHAM-saline-0.5 h; , SHAM-LPS-0.5 h; (d) , SHAM-saline-5 h; , SHAMLPS-5 h; (e) , PVL-saline-0.5 h; , PVL-LPS-0.5 h; (f) , PVL-saline-5 h; , PVL-LPS-5 h; , PVL-LPS-5 h-BQ-123.
because inhibiting the enzyme attenuates vascular ETA and ETB.27 Consistently, our study showed significantly increased phosphorylation of renal ERK1/2 in PVL rats at 5 h following LPS injection, indicating that LPS-induced renal vascular hyperresponsiveness in PVL rats is associated with the activation of ERK1/2 pathway. The present results raise the question of whether ERK signaling path-
ways represent potential therapeutic targets aimed at attenuating intrarenal vasoconstriction, to prevent renal dysfunction in cirrhotic patients exposed to secondary injuries such as endotoxemia. There remains a limitation in this study. PVL model, mimicking the characteristic features of hyperdynamic splanchnic circulation and porto-systemic collateral vessels in cirrhosis, lacks the
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Figure 2 Lipopolysaccharide upregulated renal iNOS mRNA in both SHAM and PVL rats, but showed a paradoxical change of ETA between SHAM and PVL rats. RT-PCR analysis of (a) eNOS; (b) iNOS; (c) ETA; and (d) ETB in kidneys of SHAM rats (n = 6). RT-PCR analysis of (e) eNOS; (f) iNOS; (g) ETA; and (h) ETB in kidneys of PVL rats (n = 6). *P ≤ 0.05 versus saline-treated group; **P ≤ 0.01 versus saline-treated group. ETA, endothelin receptor type A; ETB, endothelin receptor type B; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; PVL, portal vein ligation; RT-PCR, real-time polymerase chain reaction; SHAM, sham surgery. , SHAM-Saline; , SHAM-LPS; , PVL-Saline; , PVL-LPS.
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Figure 3 Lipopolysaccharide upregulated renal iNOS and ETA proteins in PVL rats, but downregulated renal ETA protein in SHAM rats. Western blot analysis of (a) eNOS; (b) iNOS; (c) ETA; and (d) ETB protein expression in kidneys of SHAM rats (n = 6). Western blot analysis of (e) eNOS; (f) iNOS; (g) ETA; and (h) ETB in kidneys of PVL rats (n = 6). *P ≤ 0.05 versus saline-treated group; **P ≤ 0.01 versus saline-treated group. ETA, endothelin receptor type A; ETB, endothelin receptor type B; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; PVL, portal vein ligation; SHAM, sham surgery. , SHAM-Saline; , SHAM-LPS; , PVL-Saline; , PVL-LPS.
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Figure 4 Immunohistochemical detection of upregulated iNOS proteins in renal arterioles of endotoxemic PVL rats. Expression and localization of eNOS in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (a) saline-treated PVL rat and (b) LPStreated PVL rat; Expression and localization of iNOS in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (c) saline-treated PVL rat and (d) LPS-treated PVL rat. Magnifications: × 200 in a through d. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PVL, portal vein ligation.
Figure 5 Immunohistochemical detection of upregulated ETA proteins in renal arterioles of endotoxemic PVL rats. Expression and localization of ETA in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (a) saline-treated PVL rat and (b) LPS-treated PVL rat; Expression and localization of ETB in interlobular artery (arrow) and afferent/ efferent arteriole (arrowhead) of (c) salinetreated PVL rat and (d) LPS-treated PVL rat. Magnifications: × 200 in a through d. ETA, endothelin receptor type A; ETB, endothelin receptor type B; LPS, lipopolysaccharide; PVL, portal vein ligation.
evidence of significant parenchymal dysfunction. The mechanisms explored from an experimental model of pre-hepatic portal hypertension do not shed light on liver cirrhosis, the most common liver parenchymal disease. However, tremendous high mortality of cirrhotic rats following LPS injection during our preliminary experiment hindered the feasibility of cirrhotic model in this study. 206
Taking the above evidences and the present data together, we speculate that upregulated renal ETA and subsequent activation of the ERK1/2 pathway represents a likely mechanism mediating the renal vascular hyperreactivity to ET-1 that exists in PVL rats challenged with LPS. It may help improve our understanding of the pathophysiology of renal dysfunction in portal hypertensive
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patients during endotoxemia and lead to the development of useful therapeutic strategies.
Acknowledgments This work was supported by the grants from Taipei Veterans General Hospital (V99B2-007) and National Science Council (NSC 97-2314-B-075-015), Taiwan.
References 1 Koppel MH, Coburn JW, Mims MM, Goldstein H, Boyle JD, Rubini ME. Transplantation of cadaveric kidneys from patients with hepatorenal syndrome: evidence for functional nature of renal failure in advanced liver disease. N. Engl. J. Med. 1969; 280: 1367–71. 2 Epstein M, Berk DP, Hollenberg NK et al. Renal failure in the patient with cirrhosis. The role of active vasoconstriction. Am. J. Med. 1970; 49: 175–85. 3 Bataller R, Sort P, Gines P, Arroyo V. Hepatorenal syndrome: definition, pathophysiology, clinical features and management. Kidney. Int. 1998; 53 (Suppl. 66): S47–S53. 4 Arroyo V, Guevara M, Gines P. Hepatorenal syndrome in cirrhosis: pathogenesis and treatment. Gastroenterology. 2002; 122: 1658–76. 5 García-Estañ J, Atucha NM, Groszmann RJ. Renal response to methoxamine in portal hypertensive rats: role of prostaglandins and nitric oxide. J. Hepatol. 1996; 25: 206–11. 6 Ortiz MC, Manriquez MC, Nath KA, Lager DJ, Romero JC, Juncos LA. Vitamin E prevents renal dysfunction induced by experimental chronic bile duct ligation. Kidney. Int. 2003; 64: 950–61. 7 Stadlbauer V, Wright GA, Banaji M et al. Relationship between activation of the sympathetic nervous system and renal blood flow autoregulation in cirrhosis. Gastroenterology. 2008; 134: 111–19. 8 Wong F, Blendis L. New challenge of hepatorenal syndrome: prevention and treatment. Hepatology. 2001; 346: 1242–51. 9 Ruiz-del-Arbol L, Monescillo A, Arocena C et al. Circulatory function and hepatorenal syndrome in cirrhosis. Hepatology. 2005; 42: 439–47. 10 Chuang CL, Huang HC, Chang CC, Lee FY, Wu JC, Lee SD. Chronological changes in renal vascular reactivity in portal hypertensive rats. Eur. J. Clin. Invest. 2013; 43: 267–76. 11 Chojkier M, Groszmann RJ. Measurement of the portal-systemic shunting in the rat by using γ-labeled microspheres. Am. J. Physiol. 1981; 240: G371–G375. 12 Lee FY, Colombato LA, Albillos A, Groszmann RJ. Administration of Nω-nitro-L- arginine ameliorates portal-systemic shunting in portal-hypertensive rats. Gastroenterology. 1993; 105: 1464–70. 13 Theimermann C. Nitric oxide and septic shock. Gen. Pharmacol. 1997; 29: 159–66.
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14 Sugiura M, Inagami T, Kon V. Endotoxin stimulates endothelinrelease in vivo and in vitro as determined by radioimmunoassay. Biochem. Biophys. Res. Commun. 1989; 161: 1220–7. 15 Szabo C, Salzmann AL, Ischiropoulos H. Endotoxin triggers the expression of an inducible nitric oxide synthase and the formation of peroxynitrite in the rat aorta in vivo. FEBS. Lett. 1995; 363: 235–8. 16 MacMicking JD, Nathan C, Hom G et al. Altered response to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 1995; 81: 641–50. 17 Cohen RI, Hassell AM, Marzouk K, Marini C, Liu SF, Scharf SM. Renal effects of nitric oxide in endotoxemia. Am. J. Respir. Crit. Care. Med. 2001; 164: 1890–5. 18 Di Giantomasso D, May CN, Bellomo R. Vital organ blood flow during hyperdynamic sepsis. Chest. 2003; 124: 1053–9. 19 Denton KM, Shweta A, Finkelstein L, Flower RL, Evans RG. Effect of endothelin-1 on regional kidney blood flow and renal arteriole calibre in rabbits. Clin. Exp. Pharmacol. Physiol. 2004; 31: 494–501. 20 Filep JG. Role for endogenous endothelin in the regulation of plasma volume and albumin escape during endotoxin shock in conscious rats. Br. J. Pharmacol. 2000; 129: 975–83. 21 de Nucci G, Thomas R, D’Orleans-Juste P et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 9797–800. 22 Seo B, Oemar BS, Siebenmann R, von Segesser L, Luscher TF. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation. 1994; 89: 1203–8. 23 Gurbanov K, Rubinstein I, Hoffman A, Abassi Z, Better OS, Winaver J. Differential regulation of renal regional blood flow by endothelin-1. Am. J. Physiol. Renal. Physiol. 1996; 271: F1166–72. 24 Fenhammar J, Andersson A, Forestier J et al. Endothelin receptor A antagonism attenuates renal medullary blood flow impairment in endotoxemic pigs. PloS. One. 2011; 6: e21534. 25 Albertini M, Clement MG, Hussain SN. Role of endothelin ETA receptors in sepsis- induced mortality, vascular leakage, and tissue injury in rats. Eur. J. Pharmacol. 2003; 474: 129–35. 26 Aramori I, Nakanishi S. Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. J. Biol. Chem. 1992; 267: 12468–74. 27 Henriksson M, Stenman E, Vikman P, Edvinsson L. MEK1/2 inhibition attenuates vascular ETA and ETB receptor alterations after cerebral ischaemia. Exp. Brain. Res. 2007; 178: 470–6.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Lipopolysaccharide increased p-ERK1/2 in kidneys of PVL rats.
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H E PAT O L O G Y
Lipopolysaccharide enhanced renal vascular response to endothelin-1 through ETA overexpression in portal hypertensive rats Chiao-Lin Chuang,*,†,‡ Hui-Chun Huang,†,§ Ching-Chih Chang,*,† Fa-Yauh Lee,†,§,1 Jaw-Ching Wu,‡,¶,1 Jing-Yi Lee,§ Hsian-Guey Hsieh¶ and Shou-Dong Lee†,** *Division of General Medicine, Department of Medicine, §Division of Gastroenterology, Department of Medicine, ¶Department of Medical Research and Education, Taipei Veterans General Hospital, †Department of Medicine, National Yang-Ming University School of Medicine, ‡Institute of Clinical Medicine, National Yang-Ming University School of Medicine, and **Cheng-Hsin General Hospital, Taipei, Taiwan
Key words endothelin receptor type A, lipopolysaccharide, partial portal vein ligation, portal hypertension, renal vascular reactivity. Accepted for publication 26 May 2014. Correspondence Professor Fa-Yauh Lee, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Email: [email protected] 1
These authors contributed equally to this paper. Conflicts of interest: The authors disclose no conflicts
Abstract Background and Aim: Hypo-perfusion resulting from intense renal vasoconstriction is traditionally contributed to renal dysfunction in advanced liver disease, although cumulative studies demonstrated renal vasodilatation with impaired vascular contractility to endogenous vasoconstrictors in portal hypertension and compensated liver cirrhosis. The pathophysiology of altered renal hemodynamics remains unclear. This study, using a rat model of portal hypertension with superimposed endotoxemia, was designed to delineate the evolution of renal vascular reactivity and vaso-regulatory gene expression during liver disease progression. Methods: Rats were randomized into sham surgery (SHAM) or partial portal vein ligation (PVL). Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS) on the seventh day following surgery. Isolated kidney perfusion was performed at 0.5 h or 5 h after LPS to evaluate renal vascular response to endothelin-1. Results: In contrast to impaired vascular contractility of SHAM rats, PVL rats displayed enhanced renal vascular reactivity to endothelin-1 at 5 h following endotoxemia. There were extensive upregulations of inducible nitric oxide synthase in kidney tissues of endotoxemic rats. The changes of renal endothelin receptor type A (ETA) level paralleled with the changes of renal vascular reactivity in LPS-treated rats. Compared with SHAM rats, PVL rats showed increased renal ETA and phosphorylated extracellular-signalregulated kinases 1/2 (p-ERK1/2) at 5 h after LPS. Conclusion: LPS-induced systemic hypotension induces a paradoxical change of renal vascular response to endothelin-1 between SHAM and PVL rats. LPS-induced renal vascular hyperreactivity in PVL rats was associated with upregulation of renal ETA and subsequent activation of ERK1/2 signaling.
Introduction In patients with liver parenchymal disease and portal hypertension, renal function deteriorates as liver function worsens. Although a great advance in the researches of portal hypertension and liver cirrhosis has been achieved with highly reproducible experimental models, the mechanisms leading to altered renal blood flow and renal dysfunction in advanced liver cirrhosis are not entirely clear. The critical impediment lies mainly in the paucity of an ideal animal model to correctly mimic human disease and fulfill this complex syndrome. Usually, acute kidney injury secondary to liver dysfunction is functional in nature and occurs in the absence of significant alterations in renal histology.1,2 Traditional consensus defines the renal dysfunction in advanced cirrhotic patients as
kidneys hypo-perfusion resulting from combined intense renal vasoconstriction and decreased renal blood flow in response to generalized systemic arterial vasodilatation.3,4 However, cumulative studies demonstrated that renal vasculature of portal hypertensive and compensated cirrhotic rats had lower perfusion pressure and hypo-responsiveness to endogenous vasoconstrictors, implying renal vasodilatation.5–7 In order to illustrate the evolution of renal vascular reactivity during disease progression of liver cirrhosis, some hypotheses aimed at elucidating the potential mechanisms were proposed.8,9 Usually, the liver dysfunction appears to be an important background factor when the vasodilatation related hypovolemia can be compensated by the development of hyperdynamic circulation, such as increased heart rate, avid renal sodium and water retention, and higher cardiac output.
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Once the auto-regulatory mechanisms are overwhelmed by the precipitating events, extreme effective arterial underfilling will initiate extreme intra-renal vasoconstriction and subsequently renal impairment. Such events may include overzealous use of diuretics, large volume paracentesis, gastrointestinal bleeding, cardiomyopathy, or development of spontaneous bacterial peritonitis in ascitic patients. From our previous study, the evolution of renal vasodilatation in portal hypertensive rats has been well delineated using isolated kidney perfusion experiment.10 This study, furthermore, used a rat model of portal hypertension with superimposed endotoxemia, a common complication of cirrhosis, to mimic the pictures of clinical condition. Renal vascular response to endotoxemia was surveyed by isolated kidney perfusion and two key vaso-regulatory systems, namely nitric oxide (NO) and endothelin-1 (ET-1), were analyzed to delineate the pathogenesis of renal dysfunction in advanced liver disease.
incubation of ETA antagonist at day 7 following PVL. Selective ETA antagonist BQ-123 (10−6 mol/L) was added into the Krebs solution 30 min before the beginning throughout the whole course of the concentration—response relationship study.
Materials and methods
Isolated kidney perfusion. The kidney perfusion system was performed as previously described.10 In brief, right renal artery was cannulated with a 22-gauge needle via superior mesenteric artery. At first, the kidney was perfused in situ with warm oxygenated (95% O2-5% CO2) Krebs solution by means of a roller pump (model 505S; Watson-Marlow Ltd, Falmouth, Cornwall, UK). Right renal vein was cut and ureter was transected to allow exit of perfusate. Finally, right kidney was excised from surrounding tissues, decapsulated, and placed into a chamber containing Krebs solution at 37 ± 0.5°C. Renal vascular responses were recorded as changes in renal perfusion pressure downstream from the pump through a Spectramed DTX transducer attached to the Gould model RS 3400 recorder (Gould Inc., Cupertino, CA, USA). The flow rate of perfusion solution was kept constant (5 ml/min per gm kidney weight), so the changes in perfusion pressure reflected the changes in renal vascular resistance. In each individual preparation, the contracting capability of renal vasculature was challenged with a 125 mmol/L potassium chloride solution at the end of experiments.
Animal model. Male Sprague-Dawley rats weighing 300– 350 g were maintained at 24°C with a 12-h light-dark cycle and allowed free access to food and water. Surgery and hemodynamic studies were performed under ketamine hydrochloride anesthesia (100 mg/kg, intramuscularly). Portal hypertension was induced by partial portal vein ligation (PVL) as previously described.11 Corresponding sham groups (SHAM) received the same abdominal surgery without ligation of portal vein. This study has been approved by Taipei Veterans General Hospital Animal Committee. All the experiments followed the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA and published by the National Institutes of Health (NIH publication no. 86-23, revised 1985). Experimental protocol 1. On the seventh day following surgery, rats were randomly allocated to receive intraperitoneal injection of either normal saline or lipopolysaccharide (LPS, Escherichia coli serotype O111:B4, 3 mg/kg): (i) SHAM + saline (n = 14); (ii) SHAM + LPS (n = 17); (iii) PVL + saline (n = 15); (iv) PVL + LPS (n = 17). After the measurements of body weight, mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP), each group was further randomized for isolated kidney perfusion at 0.5 h, or 5 h following LPS injection. With isolated kidney perfusion technique, cumulative concentration-response curves of renal vascular beds were determined by graded concentrations of endothelin-1 (10−10 to 3 × 10−8 M) in escalation with a constant flow rate of perfusion solution. Each concentration of perfusion solution was allowed to stabilize for 3 min before the next higher concentration was added. Left kidneys were harvested for analysis of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), endothelin receptor type A (ETA) and type B (ETB), extracellularsignal-regulated kinases 1/2 (ERK1/2), and phosphorylated ERK 1/2 (p-ERK1/2). Experimental protocol 2. To evaluate the influence of ETA in renal vascular hyperresponsiveness of endotoxemic PVL rats, another nine rats received isolated kidney perfusion with pre200
Measurement of systemic and portal hemodynamics. Under anaesthesia, right femoral artery was cannulated with polyethylene cannula (PE-50) catheter that was connected to the Spectramed DTX transducer (Spectramed Inc., Oxnard, CA, USA) to measure MAP and HR. Another PE-50 catheter connected to a Spectramed DTX transducer was inserted into the mesenteric vein to access PP. The external zero reference was placed at the level of the mid-portion of rat. Continuous recordings of MAP, HR, and PP were performed through a multi-channel recorder (model RS 3400, Gould Inc., Cupertino, CA, USA).12
Quantitative real-time polymerase chain reaction. Total RNA was extracted from frozen kidneys with SV Total RNA Isolation System (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (RT-PCR) was performed on the LightCycler 480 instrument (Roche Applied Science; Mannheim, Germany). The primer sequences were 5′-GGAAGTAGCCAATGCAGTGAA-3′ (sense) and 5′-GCCA GTCTCAGAGCCATACA-3′ (antisense) for eNOS, 5′-AGGCCA CCTCGGATATCTCT-3′ (sense) and 5′-GCTTGTCTCTGGGT CCTCTG-3′ (antisense) for iNOS, 5′-TTCCCTCTTCACTTAAG CCGAA-3′ (sense) and 5′-GCAACAGAGGCATGACTGAA AA-3′ (antisense) for ETA, 5′-TGTAGTCCAAAACCAGCAAA AA-3′ (sense) and 5′-CTGTTGGCTTCCCCTTCAC3′ (antisense) for ETB, 5′-CGCCCTAGGCACCAGGGTG-3′ (sense) and 5′GCTGGGGTGTTGAAGGTCTCAAA-3′ (antisense) for β-actin, respectively. Relative mRNA expression was quantified using the ΔCt method. The products were standardized with a housekeeping gene, β-actin, from the same RNA samples. Journal of Gastroenterology and Hepatology 30 (2015) 199–207
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Western blot analysis. Kidneys were dissected and homogenized. Ten microgram of protein aliquots from each sample were separated on 10% SDS-PAGE gels and subsequently transferred to a nitrocellulose filter membrane by electroblotting. The membrane were probed with specific primary antibodies against eNOS (Chemicon Inc., Temecula, CA, USA), iNOS (Chemicon Inc., Temecula, CA, USA), ETA (Abcam, Cambridge, UK), ETB (Abcam, Cambridge, UK), ERK1/2 (Cell Signaling Technology, Beverly, CA, USA), and p-ERK1/2 (Cell Signaling Technology, Beverly, CA, USA) for 90 min at room temperature (25°C) and washed. β-actin (Chemicon Inc., Temecula, CA, USA) was used as a loading control. Antibody binding was detected after secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Chemicon Inc., Temecula, CA, USA) incubation. Subsequent detection of the specific protein was performed by enhanced chemiluminescence. With a computer-assisted video densitometer and digitalized software (Kodak Digital Science ID Image Analysis Software), the blots were scanned, and the signal intensity of appropriate bands was analyzed. Immunohistochemistry stain. Five micrometer-thick sections obtained from paraffin-embedded kidneys were de-waxed with xylene and rehydrated through a series of ethanol solutions. Sections were subjected to microwave irradiation in citrate buffer to enhance antigen retrieval and pre-incubated with 5% normal rabbit serum in Tris-buffered saline. Primary antibodies were incubated for 1 h in a humidity chamber using the following dilutions: antieNOS polyclonal antibody (1 : 100); anti-iNOS polyclonal antibody (1 : 100); anti-ETA polyclonal antibody (1 : 100); anti-ETB polyclonal antibody (1 : 100). After rinsing twice in phosphatebuffered saline, sections were incubated with fluorochromeconjugated secondary antibodies (Alexa Fluor 488 fluorescent, Jackson ImmunoResearch Laboratories, Inc. Baltimore, USA) for 1 h at room temperature. Finally, slides were coverslipped with carbonate-buffered glycerol, and evaluated in an Olympus AX 80 microscope (Olympus; Hamburg, Germany) equipped with epifluorescence illumination and digital cameras.
Drugs. ET-1, LPS, and reagents for Krebs solution were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Statistical analysis. All results are expressed as mean ± SEM. Differences between the concentration–response curves were analyzed by two-way ANOVA for repeated measures using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical analysis for comparison of mRNA and protein expressions was performed using Student’s t-test. Results were considered statistically significant at a two-tailed P-value of less than 0.05.
Results General characteristics of the rats. Comparing with corresponding saline-treated groups, there was significantly decreased mean arterial pressure at 5 h following LPS injection in both SHAM rats (P < 0.01) and PVL rats (P = 0.017) (Table 1). The SHAM rats developed significantly higher portal pressures
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Table 1
Hemodynamic profiles of experimental rats
Group 0.5 h SHAM + saline SHAM + LPS PVL + saline PVL + LPS 5h SHAM + saline SHAM + LPS PVL + saline PVL + LPS
n
7 7 7 8 7 10 8 9
MAP (mmHg)
HR (beats/min)
PP (mmHg)
120 ± 6.0 113 ± 5.0 95 ± 3.5 90 ± 5.6
362 ± 16 375 ± 18 336 ± 19 336 ± 10
9.0 ± 0.2 13.0 ± 1.2* 15.3 ± 1.0 15.0 ± 1.0
117 ± 4.9 83 ± 3.3* 93 ± 4.7 79 ± 3.4**
345 ± 18 356 ± 8 324 ± 13 351 ± 8.6
8.7 ± 0.5 12.3 ± 1.3* 16.3 ± 0.9 12.5 ± 0.7**
*P < 0.05 between time-matched saline- and LPS-injected SHAM group; **P < 0.05 between time-matched saline- and LPS-injected PVL group. Data were expressed as mean ± SEM. HR, heart rate; LPS, lipopolysaccharide; MAP, mean arterial pressure; PP, portal pressure; PVL, partial portal vein ligation; SHAM, sham surgery.
since 0.5 h after endotoxemia (P = 0.015), while PVL rats showed declined portal pressure at 5 h after LPS injection (P < 0.01) (Table 1). Both SHAM rats and PVL rats have significantly higher serum ET-1 (P ≤ 0.01), serum glutamic oxaloacetic transaminase (P ≤ 0.05), and serum glutamic-pyruvic transaminase (P ≤ 0.05) levels at 5 h following LPS injection (Table 2). All the fraction excretion of sodium (FeNa) levels remained within normal range despite higher FeNa in response to LPS injection. Only PVL rats had significantly increased serum creatinine (P = 0.006) at 5 h following LPS injection (Table 2). In vitro renal vascular contractility to ET-1. The addition of ET-1 into perfusion solution led to a concentrationdependent increase of renal perfusion pressures in both SHAM rats and PVL rats (Fig. 1). Without LPS, the PVL rats showed significant renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (P < 0.05, Fig. 1a,b). Compared with corresponding saline-treated groups, the SHAM rats showed significantly attenuated renal vascular contractility to ET-1 at 5 h following LPS injection (P = 0.018, Fig. 1d). However, the blunted renal vascular reactivity to ET-1 in PVL rats was significantly reversed at 5 h following LPS injection (P = 0.008, Fig. 1f). Pretreatment of ETA antagonist abrogated the LPS-enhanced renal vascular response in PVL rats (P ≤ 0.001, Fig. 1f). Expressions of NOS and ET-1 receptors in kidneys. Compared with corresponding saline-treated groups, there was extensive upregulation of iNOS mRNA in kidneys of both SHAM rats and PVL rats since 0.5 h after endotoxemia (P < 0.01, Fig. 2b,f). The expression of renal ETA mRNA was significantly downregulated in SHAM rats (P < 0.01, Fig. 2c) but upregulated in PVL rats (P < 0.05, Fig. 2g) at 5 h following LPS injection. The expression of renal ETB mRNA was only downregulated in PVL rats (P < 0.05, Fig. 2h) at 5 h following LPS injection. There was no difference in the expression of eNOS in response to LPS injection (P > 0.05, Fig. 2a,e).
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Table 2
Biochemistry of experimental rats
Variables
Pre-LPS
LPS-5h
P-value
6 1.1 ± 0.1 64.8 ± 1.8 47.5 ± 1.6 0.19 ± 0.01 11.4 ± 0.1 28.9 ± 1.7 0.08 ± 0.01 0.3 ± 0.04
6 6.6 ± 0.7 111 ± 11.5 82.0 ± 9.8 0.25 ± 0.03 12.3 ± 0.5 32.7 ± 3.8 0.21 ± 0.05 0.25 ± 0.02
< 0.001 0.01 0.017 0.141 0.121 0.492 < 0.001 0.253
6 3.3 ± 0.3 70.8 ± 4.4 55.6 ± 6.3 0.18 ± 0.01 12.3 ± 0.8 27.0 ± 2.4 0.08 ± 0.02 0.12 ± 0.02
8 15.7 ± 2.5 200 ± 41.9 120 ± 26.0 0.33 ± 0.04 13.0 ± 0.7 39.1 ± 5.4 0.23 ± 0.05 0.26 ± 0.05
0.001 0.018 0.043 0.006 0.537 0.087 0.032 0.064
SHAM rats Number ET-1 (pg/ml) SGOT (IU/L) SGPT (IU/L) Cr (mg/dL) PT (second) aPTT (second) FeNa (%) FeCl (%) PVL rats Number ET-1 (pg/ml) SGOT (IU/L) SGPT (IU/L) Cr (mg/dL) PT (second) aPTT (second) FeNa (%) FeCl (%)
Data were expressed as mean ± SEM. aPTT, activated partial thromboplastin time; Cl−, chloride; Cr, creatinine; ET-1, endothelin-1; FeCl, fractional excretion of chloride; FeNa, fractional excretion of sodium; LPS, lipopolysaccharide; Na+, sodium; PT, prothrombin time; PVL, partial portal vein ligation; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; SHAM, sham surgery.
Similarly, there were higher iNOS protein levels in kidneys of both SHAM rats (P < 0.01, Fig. 3b) and PVL rats (P < 0.05, Fig. 3f) at 5 h after LPS injection. The renal ETA protein was downregulated in SHAM rats (P < 0.05, Fig. 3c) but upregulated in PVL rats at 5 h following LPS injection (P < 0.05, Fig. 3g). There was no difference in the expressions of renal eNOS and ETB in response to LPS injection (P > 0.05, Fig. 3a–h). Immunohistochemical staining of NOS and ET-1 receptors in kidneys. Compared with corresponding saline-treated groups, the expressions of iNOS (Fig. 4d) and ETA (Fig. 5b) in interlobular arteries as well as afferent and efferent arterioles were significantly upregulated in PVL rats at 5 h following LPS injection. There was no difference in the expressions of renal eNOS and ETB in response to LPS injection (Figs 4a,b,5c,d). Expressions of ERK1/2 and p-ERK1/2 in kidneys. There was no significant difference of renal phosphorylated ERK1/2 protein between saline-treated and LPS-treated SHAM rats (P > 0.05, supplementary Fig. 1a). However, the phosphorylation ratio of renal ERK1/2 was significantly increased in PVL rats at 5 h following LPS injection (P < 0.05, supplementary Fig. 1b).
Discussion LPS, a component of the outer membrane of Gram-negative bacteria released during septicemia, is a major cause of septic shock. 202
Cumulative evidences have demonstrated that administration of LPS elicited a marked elevation in plasma levels of NO and ET-1.13,14 The present study provides the first comprehensive analysis concerning the time-course and expression pattern of two important endothelium-released vaso-regulatory factors (NO and ET-1) in renal vasculature between SHAM rats and PVL rats following LPS-induced endotoxemia. The principal finding in current study was that renal ETA overexpression rather than iNOS overexpression might be the key event in modulating renal vascular reactivity to endotoxemia during portal hypertension. It is widely accepted that systemic vasodilation during endotoxemia is caused by excessive production of NO, which is mainly synthesized by activation of iNOS.15,16 During endotoxemia, increased renal tissue NO is thought to be largely responsible for increased renal blood flow through its vasodilatory effects on the afferent arteriole during periods of hypoperfusion.17,18 Consistent with previous observations, our results showed decreased mean blood pressures and extensive upregulations of renal iNOS levels in both SHAM rats and PVL rats after LPS administration. However, in contrast to the impaired vascular contractility to ET-1 in endotoxemic SHAM rats, the renal vascular bed of endotoxemic PVL rats displayed significant enhancement of the contractile response to cumulative concentrations of ET-1 despite upregulated iNOS. The reciprocal pattern of renal vascular response to ET-1 between SHAM rats and PVL rats was in parallel with the paradoxical change of renal ETA expression after LPS administration, indicating a clear correlation between renal ETA and the pathogenesis of renal vascular response during LPS-induced systemic hypotension. It is conceivable that, in spite of the large amounts of NO generated by iNOS, the NO system is unable to counteract the renal vasoconstrictor influences of ET-1 in response to endotoxemia during portal hypertension. The renal effects of enhanced ET-1 expression and secretion in endotoxemia are harmful by reducing blood volume and renal cortical blood flow.19,20 The compromised ability of PVL kidneys to counteract the pressor effect of ET-1 during endotoxemia may ultimately be responsible for kidney injury, consistent with our findings. This could, at least partly, explain the increased susceptibility to renal dysfunction in cirrhosis during endotoxemia. ET-1, a potent vasoactive peptide, induces its vascular effects through two G protein-coupled receptors, namely the ETA and ETB. ETA is mainly expressed in vascular smooth muscle cells (VSMCs) throughout and mediates vasoconstriction.21 ETB is predominantly expressed in endothelial cells, and its activation causes a transient vasodilation.22 ET-1 elicits renal cortical vasoconstriction and reduces total renal blood flow in endotoxemia primarily through ETA activation.23 ETA inhibition has been shown to improve medullary blood flow in endotoxemia associated hypotension and reverse LPS-induced mortality.24,25 In agreement, our results revealed that renal ETA expression is the major determinant factor of renal vascular reactivity in response to LPS-induced systemic hypotension, although the possible involvement of some other mediators. As for many G-protein-coupled receptors, the signaling capacity of endothelin receptors is fine-tuned by posttranslational modifications. The binding of ET-1 to ETA results in the contractile effects in VSMCs through activation of diverse signaling molecules such as phospholipase C, protein kinase C, and ERK1/2.26 It has been reported that activation of ERK1/2 is important for ET-1-induced VSMCs contraction,
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Figure 1 Concentration–response curves to ET-1 in isolated perfuse kidneys of SHAM or PVL rats. (a) PVL rats (n = 7) developed renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (n = 7) at 0.5 h following saline. (b) PVL rats (n = 8) developed renal vascular hypo-responsiveness to ET-1 in comparison with SHAM rats (n = 7) at 5 h following saline. (c) LPS-injected SHAM rats (n = 7) showed unchanged renal vascular reactivity to ET-1 at 0.5 h following LPS in comparison with saline-injected SHAM rats (n = 7). (d) LPS-injected SHAM rats (n = 10) showed attenuated renal vascular contractility to ET-1 at 5 h following LPS in comparison with saline-injected SHAM rats (n = 7). (e) LPS-injected PVL rats (n = 8) showed unchanged renal vascular reactivity to ET-1 at 0.5 h following LPS in comparison with saline-injected PVL rats (n = 7). (f) LPS-injected PVL rats (n = 9) showed enhanced renal vascular contractility to ET-1at 5 h following LPS in comparison with saline-injected PVL rats (n = 8). The renal vascular hyperreactivity in LPS-injected PVL rats was significantly attenuated by pre-incubation of ETA antagonist (n = 9). ET-1, endothelin-1; ETA, endothelin receptor type A; LPS, lipopolysaccharide; PVL, portal vein ligation; SHAM, sham surgery. (a) , SHAM-saline-0.5 h; , PVL-saline-0.5 h; (b) , SHAM-saline-5 h; , PVL-saline-5 h; (c) , SHAM-saline-0.5 h; , SHAM-LPS-0.5 h; (d) , SHAM-saline-5 h; , SHAMLPS-5 h; (e) , PVL-saline-0.5 h; , PVL-LPS-0.5 h; (f) , PVL-saline-5 h; , PVL-LPS-5 h; , PVL-LPS-5 h-BQ-123.
because inhibiting the enzyme attenuates vascular ETA and ETB.27 Consistently, our study showed significantly increased phosphorylation of renal ERK1/2 in PVL rats at 5 h following LPS injection, indicating that LPS-induced renal vascular hyperresponsiveness in PVL rats is associated with the activation of ERK1/2 pathway. The present results raise the question of whether ERK signaling path-
ways represent potential therapeutic targets aimed at attenuating intrarenal vasoconstriction, to prevent renal dysfunction in cirrhotic patients exposed to secondary injuries such as endotoxemia. There remains a limitation in this study. PVL model, mimicking the characteristic features of hyperdynamic splanchnic circulation and porto-systemic collateral vessels in cirrhosis, lacks the
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Figure 2 Lipopolysaccharide upregulated renal iNOS mRNA in both SHAM and PVL rats, but showed a paradoxical change of ETA between SHAM and PVL rats. RT-PCR analysis of (a) eNOS; (b) iNOS; (c) ETA; and (d) ETB in kidneys of SHAM rats (n = 6). RT-PCR analysis of (e) eNOS; (f) iNOS; (g) ETA; and (h) ETB in kidneys of PVL rats (n = 6). *P ≤ 0.05 versus saline-treated group; **P ≤ 0.01 versus saline-treated group. ETA, endothelin receptor type A; ETB, endothelin receptor type B; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; PVL, portal vein ligation; RT-PCR, real-time polymerase chain reaction; SHAM, sham surgery. , SHAM-Saline; , SHAM-LPS; , PVL-Saline; , PVL-LPS.
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Figure 3 Lipopolysaccharide upregulated renal iNOS and ETA proteins in PVL rats, but downregulated renal ETA protein in SHAM rats. Western blot analysis of (a) eNOS; (b) iNOS; (c) ETA; and (d) ETB protein expression in kidneys of SHAM rats (n = 6). Western blot analysis of (e) eNOS; (f) iNOS; (g) ETA; and (h) ETB in kidneys of PVL rats (n = 6). *P ≤ 0.05 versus saline-treated group; **P ≤ 0.01 versus saline-treated group. ETA, endothelin receptor type A; ETB, endothelin receptor type B; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; PVL, portal vein ligation; SHAM, sham surgery. , SHAM-Saline; , SHAM-LPS; , PVL-Saline; , PVL-LPS.
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Figure 4 Immunohistochemical detection of upregulated iNOS proteins in renal arterioles of endotoxemic PVL rats. Expression and localization of eNOS in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (a) saline-treated PVL rat and (b) LPStreated PVL rat; Expression and localization of iNOS in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (c) saline-treated PVL rat and (d) LPS-treated PVL rat. Magnifications: × 200 in a through d. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PVL, portal vein ligation.
Figure 5 Immunohistochemical detection of upregulated ETA proteins in renal arterioles of endotoxemic PVL rats. Expression and localization of ETA in interlobular artery (arrow) and afferent/efferent arteriole (arrowhead) of (a) saline-treated PVL rat and (b) LPS-treated PVL rat; Expression and localization of ETB in interlobular artery (arrow) and afferent/ efferent arteriole (arrowhead) of (c) salinetreated PVL rat and (d) LPS-treated PVL rat. Magnifications: × 200 in a through d. ETA, endothelin receptor type A; ETB, endothelin receptor type B; LPS, lipopolysaccharide; PVL, portal vein ligation.
evidence of significant parenchymal dysfunction. The mechanisms explored from an experimental model of pre-hepatic portal hypertension do not shed light on liver cirrhosis, the most common liver parenchymal disease. However, tremendous high mortality of cirrhotic rats following LPS injection during our preliminary experiment hindered the feasibility of cirrhotic model in this study. 206
Taking the above evidences and the present data together, we speculate that upregulated renal ETA and subsequent activation of the ERK1/2 pathway represents a likely mechanism mediating the renal vascular hyperreactivity to ET-1 that exists in PVL rats challenged with LPS. It may help improve our understanding of the pathophysiology of renal dysfunction in portal hypertensive
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patients during endotoxemia and lead to the development of useful therapeutic strategies.
Acknowledgments This work was supported by the grants from Taipei Veterans General Hospital (V99B2-007) and National Science Council (NSC 97-2314-B-075-015), Taiwan.
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Lipopolysaccharide increased p-ERK1/2 in kidneys of PVL rats.
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