Melatonin reduces renal interstitial inflammation andimproves hypertension in sp - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：MayerlyNava, YasmirQuiroz, NosratolaVaziri,  BernardoRodríguez-Iturbe作者单位：1 Renal Service and Laboratory, HospitalUniversitario, Instituto de Investigaciones Biomédicas(Fundacite-Zulia), Universidad del Zulia, Maracaibo 4001-A,Venezuela; and Division of Nephrologyand Hypertension, Department of Medicine, Physiology, andBiophysics, University of California, Irvine,    A* i1 K" c, ?
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      【摘要】. h* X7 O* i% G: p. s1 `
   Severalstudies have demonstrated that treatment with antioxidants improveshypertension in spontaneously hypertensive rats (SHR). Because ourlaboratory has shown that renal infiltration of immune cells plays arole in the development of hypertension (Rodriguez-Iturbe B, Quiroz Y,Nava M, Bonet L, Chavez M, Herrera-Acosta J, Johnson RJ, and Pons HA. Am J Physiol Renal Physiol 282: F191-F201, 2002), we did the present studies to define whether theantihypertensive effect of antioxidants was associated with animprovement in renal inflammation. Melatonin was administered as anantioxidant. For 6 wk, melatonin was added to the drinking water(10 mg/100 ml) given to a group of SHR (SHR-Mel; n = 10), and we compared them with groups of untreated SHR( n = 10) and Wistar-Kyoto (WKY) control rats( n = 10). Hypertension became increasingly severe inthe SHR group [195 ± 14.3 (SD) mmHg at the end of theexperiment] and improved in the SHR-Mel group (149 ± 20.4 mmHg, P in association with a 40-60%reduction in the renal infiltration of lymphocytes, macrophages, andangiotensin II-positive cells. Intracellular superoxide and renalmalondialdehyde content were reduced by melatonin treatment as was theimmunohistological expression of the 65-kDA DNA-binding subunit ofNF- B. We conclude that melatonin treatment ameliorates hypertensionin SHR in association with a reduction in interstitial renalinflammation. Decreased activation of NF- B, likely resulting from areduction in local oxidative stress, may play a role in the suppressionof renal immune infiltration and, thereby, in the antihypertensiveeffects of melatonin. : J0 l, z  m! A: F) ~$ ?6 {* E
      【关键词】 oxidative stress saltsensitive hypertension immune infiltration interstitial nephritis nuclear factor B angiotensin II) T1 D0 @( s- R. j- u, [3 T
   INTRODUCTION7 V+ p- [& i$ z0 b/ l
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WE HAVE POSTULATED THAT THE renal infiltration of immunocompetent cells plays a role inthe pathogenesis of salt-sensitive hypertension ( 20, 44 ). The most compelling evidence in favor of ourpostulate comes from studies demonstrating that the salt-driven hypertension that follows angiotensin II infusion ( 43 ),nitric oxide synthesis inhibition ( 36 ), and proteinoverload proteinuria ( 2 ) is prevented or markedlyameliorated when the renal immune infiltrate is reduced by theadministration of mycofenolate mofetil (MMF), an immunosuppressiveanti-inflammatory drug. Furthermore, blood pressure in the Okamotostrain of spontaneously hypertensive rats (SHR) decreased to nearlynormal levels when lymphocytes and macrophages infiltratingtubulointerstitial areas were reduced with MMF treatment( 45 ). These recent studies from our group follow thepioneering observations by Svendsen ( 52 ), who showed thatathymic nude mice were resistant to the late, salt-dependent phase ofthe DOCA-salt model of hypertension.
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The role of the immune cells infiltrating the kidney would depend onthe close interrelationship among inflammatory infiltration, generationof free oxygen radicals, and local vasoconstrictor activity( 42 ). In fact, in the experimental models listed above ( 2, 36, 43, 44 ), the correction of hypertension isassociated with a concomitant reduction of the renal immune infiltrate,interstitial angiotensin II-positive cells, and oxidative stress.5 `& X$ ]8 J& s. Y6 _1 q
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The importance of reactive oxygen species (ROS) in the development andmaintenance of hypertension has been recognized for some time ( 4, 11, 23, 47, 57 ). Unchecked production of ROS results in theimpairment of vasodilatory systems, particularly nitric oxide, that isdegraded by oxygen free radicals ( 9, 39, 58 ). In additionto shifting the balance toward systemic vasoconstriction, theadditional consequence of chronic oxidative stress can stimulate saltretention. Unchecked angiotensin II activity, which in the renalinterstitium is unresponsive to systemic volume expansion( 31 ), favors sodium retention by the combined effects ofglomerular vasoconstriction, which reduces filtered sodium load,increased sodium reabsorption, and impairment of thepressure-natriuresis mechanism.

While oxidative stress causes hypertension ( 27, 53, 54, 59 ) and a variety of antioxidant strategies improve hypertension in genetically hypertensive rats (reviewed in Refs. 40 and 56 ), it is not clear whether the blood pressure reduction induced by antioxidant therapy is associated with a decrease in renal interstitial inflammation. The confirmation of this prediction is central to ourtheory of the pathogenesis of salt-sensitive hypertension; therefore,the present studies were designed to answer this question.
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Melatonin is a very potent antioxidant, due chiefly to its capacity toact as an electron donor ( 34, 40 ). Therefore, to test ourhypothesis, we administered melatonin in the drinking water to SHR fora period of 6 wk. We found that hypertension was improved inassociation with suppression of oxidative stress, diminished activationof transcriptional NF- B, and reduction in the lymphocyte andmacrophage infiltration of the kidney.
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MATERIALS AND METHODS- x; g  w( c+ d+ W
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Experimental design. Male genetically hypertensive rats from the Okamoto strain (SHR) andcontrol normotensive Wistar-Kyoto rats (WKY) 23-25 wk of age atthe beginning of the experiment were obtained from the InstitutoVenezolano de Investigaciones Científicas (Los Teques, Venezuela). Rats received regular rodent chow with 4% NaCl (Protinal, Purina, Maracay, Venezuela), had free access to food and water, andwere handled according to the institutional guidelines of animal care.Animals were maintained on 12:12-h (7 AM-7 PM) light-dark cyclesthroughout the experiments.+ y5 I# P. T" S: J
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Three animal groups were studied for 6 wk. The SHR-Mel group( n = 10) consisted of SHR that received melatonin(M-5250, Sigma). Because melatonin is water soluble at concentrationsof 0.1 mg/ml (Sigma Technical Library) or more ( 48 ), weadministered in the drinking water (10 mg/100 ml) using containerswrapped in aluminum foil because melatonin is light sensitive. The SHRgroup ( n = 10) consisted of SHR that received nomelatonin. Control WKY rats ( n = 10) received nomelatonin and were similarly studied.

Because the investigation demonstrated (see below) a reduction in thetubulointerstitial infiltrating cells after 6 wk of melatonintreatment, an additional group ( n = 5) of SHR of23-24 wk of age (SHR-23w) was studied to evaluate the spontaneouschanges in renal cellular infiltration occurring in untreated SHR over 6 wk.

Rats are known to drink more water during the night than during the day( 54 ), which was confirmed by measuring water consumption in 12-h cycles (7 AM-7 PM). Melatonin serum levels were determined (seebelow) at three different times during a 24-h period (10-11 AM,5:30-6:30 PM, and 11:30 PM-12:30 AM) in the rats from the SHR-Meland SHR groups.
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Blood pressure was determined weekly. Weight, serum creatinine andproteinuria were determined before and at the end of the experiments.At the end of 6 wk, all rats were anesthetized with ether andeuthanized by aortic desanguination, and the kidneys were harvested forstudies. One kidney was divided into two parts and used forhistological, histochemical, and immunohistological studies. As inprevious investigations ( 2, 36, 43, 45 ), one part wasfixed in methyl-Carnoy's and embedded in Paraplast Plus (Monoject,Sherwood Medical Scientific Division, St. Louis, MO), and the other wassnap-frozen in dry ice and acetone. The contralateral kidney was usedto determine malondialdehyde (MDA), GSH, and catalase content.

Blood pressure determinations. Blood pressure was determined in conscious restrained rats by tail-cuffplethysmography (IITC, Life Scientific Instruments, Woodland Hills, CA)in a quiet, darkened room as previously reported ( 36, 43, 45 ). Before the beginning of the experiments, rats wereconditioned three to four times to the procedure and, when bloodpressure was determined, the value represented the mean of three tofour separate determinations." l; s+ n/ U9 Y  ^- U( i; {/ E' q
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Serum melatonin levels. Serum melatonin levels were determined by ELISA with commerciallyavailable kits (ICN Biomedicals Research Products, Costa Mesa, CA).Melatonin extraction from the samples was done with reverse-phaseextraction columns provided with the kit. The dried extracts werestored at 20°C and processed within 24 h. The samples of theSHR-Mel group were diluted 20 times for the assay, which has asensitivity of 3 pg/ml. Intra-assay variability was 7.3 ± 4.2%.
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Histological studies. Light microcopy was evaluated with hematoxylin and eosin and periodicacid-Schiff stainings. Histological damage was graded using aglomerular injury score described initially by Raij et al.( 38 ) and a tubulointerstitial injury score describedinitially by Pichler et al. ( 33 ), which depends on thepercent extension of the damaged area, as detailed in several previouscommunications from our group ( 36, 43, 45 ): 0 = nochanges present; 1  = of the renal tubulointerstitialarea showing injury. The entire cortical and juxtamedullary areas wereevaluated. All studies were done in a blinded fashion.

Infiltrating cells and antisera. Avidin-biotin-peroxidase methodology was used to identify infiltratingcells and angiotensin II-positive cells. Intraglomerular cells[positive cells/ glomerular cross section (GCS)] andtubulointerstitial cells (positive cells/mm 2 ) were analyzed separately.

Double-staining methodology was used to determine the proportion ofangiotensin II-positive cells that were lymphocytes and macrophages.The technique has been described before in detail ( 41 ) andincludes an initial incubation with monoclonal antibody anti-CD5 oranti-ED1; a second incubation with rhodamine-conjugated, affinity-purified F(ab')2 anti-mouse IgG; a third incubationwith rabbit anti-angiotensin II antibody; and, finally, incubation withflourescein-conjugated affinity-purified donkey anti-rabbit IgG antibody.' }  ~1 c; z& [
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Monoclonal antibodies were used to identify lymphocytes (anti-CD5,clone MRCOX19, Biosource, Camarillo, CA) and macrophages (anti-ED1,Harlan Bioproducts, Indianapolis, IN). Angiotensin II-positive cellswere identified with rabbit anti-angiotensin II human IgG (PeninsulaLaboratories, Belmonit, CA). The secondary antibody for CD5 and ED1stainings was a biotin-conjugated, affinity-purified F(ab')2 fragmentof rat anti-mouse IgG with minimal cross-reactivity to rat serumproteins (dilution 1:30). The secondary antibody for angiotensin II andNF- B stainings was a biotin-conjugated F(ab')2 fragment of donkeyanti-rabbit IgG with minimal cross-reactivity to rat serum proteins(dilution 1:40). Secondary antibodies were purchased from AccurateChemical and Scientific (Westbury, NY).

Oxidative stress. Oxidative stress was studied by determination of the renal content ofMDA, GSH, and catalase in one of the kidneys harvested at the end ofthe experiment and by determination of intracellular superoxide infrozen kidney sections of the contralateral kidney./ L# \: S& O- Q# o- X/ g

Renal homogenates were prepared, and the content of MDA, GSH, andcatalase were measured by the methods of Ohkawa et al.( 32 ), Beutler et al. ( 5 ), and Aebi( 1 ), respectively. MDA and GSH results are given asnanomoles per milligram of homogenate protein. Catalase results aregiven as the rate constant of a first-order reaction ( ) permilligram homogenate protein. Details of these methods in ourlaboratories have been published previously ( 30 ).

The cytochemical method of Briggs et al. ( 7 ) with minormodifications published previously ( 30 ) was applied to 8- to 10-µm cryostat sections of the kidneys to determine intracellular superoxide. Sections were fixed in formalin and counterstained with 1%methyl green. Results are given as positive cells per square millimeter.5 Q$ g4 W2 g+ b( s3 M  t" c+ g
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NF- B. Activated NF- B was identified using polyclonal antibody specific forthe 65-kDA DNA-binding subunit (rabbit anti NF- B, Zymed, SanFrancisco, CA). Cryostat sections (4 µm) in citrate buffer, pH 6.0, were subject to microwave irradiation for 30 s to enhance epitoperetrieval, fixed with 10% formalin, and stained using theavidin-biotin-peroxidase technique. The secondary antibody was a donkeyanti-rabbit biotin-conjugated, affinity-purified F(ab')2 IgG fragmentwith minimal cross-reactivity to rat serum proteins (Accurate Chemicaland Scientific). Hematoxylin and eosin were used as counterstains.7 F% D" J$ t# }* k  e5 d# {% `/ O" e# c

Statistical analysis. Comparisons among groups were done with ANOVA and Tukey-Kramerposttests. Repeated-measures ANOVA was used to evaluate changes foundin serial determinations. Results are expressed as means ± SDthroughout the paper, and two-tailed P values considered significant. A commercial statistical package (Instat, GraphPad, San Diego, CA) was used for statistical calculations.1 l" w7 T, G4 V( K+ _& \

RESULTS& {) V, Q* z, g, S* B
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Weight, renal function, and serum melatonin levels. Melatonin was administered in the drinking water. As expected( 54 ), water consumption was higher during the night (7 PM-7 AM), when the animals consumed 70.9 ± 8.6% of the dailytotal. There were no apparent ill effects from melatonin, and SHR-Mel and SHR rats gained weight similarly. At the beginning of the experiments, the weight of the SHR-Mel group was 331.4 ± 18.6 g, and the weight of the SHR group was 338.5 ± 26 g. At the end of the experiments 6 wk later, the corresponding weightswere 380.7 ± 23.4 and 373.0 ± 24.5 g [ P not significant (NS)]. Serum creatinine was similar in the SHR(0.4 ± 0.06 mg/dl) and SHR-Mel (0.4 ± 0.07 mg/dl) groupsand remained unchanged at the end of the experiment. Proteinuria was of the experiments.8 \- h& e$ _8 F! b9 s& V
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Melatonin serum levels are shown in Fig. 1. Endogenous melatonin levels in the SHRrats were 77 ± 13 at 10-11 AM, 56 ± 12 at5:30-6:30, and 202 ± 31 pg/ml at midnight. The melatoninlevels in the SHR-Mel group were more than seven times higher at10-11 AM and 5:30-6:30 PM and reached peak levels of3,510 ± 319 pg/ml at midnight (Fig. 1 ).

Fig. 1. Serum melatonin levels. Endogenous levels [spontaneouslyhypertensive rats (SHR); SHR group; filled bars] are increased7-17 times ( P

Blood pressure. The serial systolic blood pressure (SBP) measurements are shown in Fig. 2. Rats from the SHR group exhibited aprogressive rise in SBP during the 6 wk of experiments. The SBP in themelatonin-treated rats (SHR-Mel group) decreased slowly during theexperiment, and at every interval it was significantly below the valuesin the SHR group.
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Fig. 2. Serial blood pressure in the SHR ( ) andSHR-Mel ( ) groups. The 95% confidence interval ofblood pressure in the Wistar-Kyoto (WKY) control rats is shown as theinterval between the 2 dotted lines. Values are means ± SD. a P b P P8 y* ~4 N/ }0 Q/ e

Histology and infiltrating cells. Light microscopy did not demonstrate significant glomerular sclerosis.Mesangial expansion was observed in some glomeruli of both experimentalgroups. Tubulointerstitial damage score was 2.3 ± 0.8 in theuntreated group and 1.9 ± 0.8 in the melatonin-treated group, andthese differences were not statistically significant. Interstitialfindings consisted of focal areas of widening with mononuclear cell infiltration.

Glomerular infiltration of lymphocytes was scarce and similar in theexperimental groups. Mean values of intraglomerular CD5-positive cellswere 0.4 positive cells/GCS. Intraglomerular macrophages were increasedin the SHR group (1.94 ± 0.92 ED1-positive cells/GCS) and reducedby melatonin treatment (SHR-Mel group = 0.30 ± 0.07, P (0.20 ± 0.10). The SHR-23w group had 1.3 ± 0.90 ED1-positive cells/GCS ( P NS vs. SHR experimental group and P group).
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Tubulointerstitial infiltration of both lymphocytes and macrophages wasdrastically reduced in the melatonin-treated rats, asdemonstrated in Figs. 3 and 4, respectively. SHR-23w rats had 61.8 ± 20.3 CD5-positive cells/mm 2 and 32.6 ± 9.9 ED1-positive cells/mm 2. These values are lower, but notstatistically different from, the corresponding values found in the SHRgroup (corresponding to 6-wk-older SHR) and significantly higher( P cells and P values found inthe SHR-Mel group (Figs. 3 and 4 ).

Fig. 3. Tubulointerstitial infiltration of lymphocytes(CD5-positive cells). The increased values in the SHR group are reducedby melatonin administration in the SHR-Mel group. The control valuescorrespond to the WKY rats. Values are means ± SD.* P P P
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Fig. 4. Tubulointerstitial macrophage infiltration. ED1-positivecells in WKY control, SHR, and SHR-Mel groups. Values are means ± SD. ** P P& `9 [! A$ p6 @4 {. M

Angiotensin II-positive cells in tubulointerstitium were increased inSHR, and treatment with melatonin resulted in an ~50% reduction inthe number of angiotensin II-positive cells in the SHR-Mel group (Fig. 5 ). Double-staining studies (Fig. 6 A ) revealed that 18-25%of the angiotensin II-positive cells were lymphocytes and 12-18%were macrophages. Nearly 6 of 10 angiotensin II-positive cells weretubular epithelial cells (Fig. 6 B ).

Fig. 5. Angiotensin II-positive cells in the tubulointerstitiumof SHR are reduced by melatonin treatment in the SHR-Mel group. Valuesare means ± SD. *** P
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Fig. 6. Double staining of a biopsy section of rat from the SHRgroup showing several tubulointerstitial angiotensin II-positive cells[fluorescein staining ( A )], 1 of which (arrow) is atubular epithelial cell and 3 are CD5-positive lymphocytes [rhodaminestaining ( B )].0 N; h9 d* E! E- _- h1 X7 Q
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Oxidative stress. Intracellular superoxide was found in tubular epithelial cells as wellas in infiltrating mononuclear cells. Increased numbers ofsuperoxide-positive cells were found in SHR and were reduced bymelatonin treatment to values similar to those found in WKY rats (Fig. 7 ).
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Fig. 7. Superoxide-positive cells are increased in the SHR groupand reduced by melatonin treatment in the SHR-Mel group to levelssimilar to those in the WKY control group. Values are means ± SD.*** P
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Table 1 shows the results of the MDA,GSH, and catalase content of the kidney in SHR and SHR-Mel groups andin WKY rats. The SHR group had increased MDA content with respect toWKY, and melatonin treatment resulted in a highly significant reduction of renal MDA.8 l0 o+ V" I- `* |

Table 1. Renal content of malondialdehyde, gluthatione, and catalase4 |0 D  Z1 s. H

NF- B. Kidney sections of the SHR group demonstrated increased expression ofthe 65-kDA DNA-binding subunit of transcription factor NF- B.Positive cells included tubular epithelial cells and infiltrating mononuclear cells. A reduction in the immunohistological expression ofactivated NF- B was observed in the SHR-Mel group without reaching the low levels present in the WKY rats (Fig. 8 ). Representative microphotographs areshown in Fig. 9, A and B.
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Fig. 8. Immunohistological expression of activated NF- B shownas the no. of cells staining positive for the 65-kDA DNA-bindingsubunit of transcription factor NF- B/mm 2 oftubulointerstitial area. Values are means ± SD.* P P
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Fig. 9. Cells staining positive for the 65-kDA DNA-bindingsubunit of transcription factor NF- B in the tubulointerstitium of arat from the SHR group ( A ) and a rat from the SHR-Mel group( B ). Avidin-biotin-peroxidase methodology was used withhematoxylin and eosin as a counterstain./ Z- f2 J1 S  \0 X7 f& y4 P& Q* K

DISCUSSION+ `3 u! Q. V  ~( P; Z2 l5 f, Z

Melatonin is a hormone that functions as a biological modulator ofsleep, reproduction and sexual behavior, immunological functions, andcircadian rhythms. There are high-affinity melatonin receptors locatedprimarily in the kidney, blood vessels, eye, brain, andgastrointestinal tract ( 6 ). The administration of melatonin has been reported to decrease arterial blood pressure whenadministered intravenously ( 24 ) as well as byintraperitoneal minipumps ( 21 ) as a consequence of variousmechanisms, including a direct hypothalamic effect, a decrease incatecholamine levels, relaxation of the aortic smooth muscle wall, and,most importantly, as a result of its antioxidant properties( 49 ). Antioxidant activity of melatonin results mainlyfrom electron donation and its easy access to intracellularcompartments ( 40 ). The antioxidant properties of melatoninare likely to be the most critical in the results obtained in ourexperiments because they involve the chronic oral administration ofmelatonin, and accumulating evidence gives support to the role ofoxidative stress in the pathogenesis of hypertension ( 4, 11, 47, 56 )." Z2 j- A7 w4 @# q$ x- Q0 d7 K' m
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To our knowledge, the oral administration of melatonin for thetreatment of hypertension has not been tried previously. One of thereasons may be that when melatonin is given by mouth, plasma melatoninlevels increase rapidly but return to baseline levels after only2-3 h. Such was the case in previous studies from our group inwhich melatonin was given orally to humans ( 18 ) as well asby gastric gavage to rats ( 31 ) in the treatment ofconditions characterized by an acute increase in oxidative stress. Infact, in preliminary pilot studies, we found that a single daily dose of melatonin given by gastric gavage had no effect on the hypertension of SHR. Therefore, we gave the drug in the drinking water in containers protected from light, and this method resulted in drug concentrations in serum over 24 h that were 7 (in the morning) to 17 times (at midnight) higher than the endogenous melatonin concentration (Fig. 1 ).The endogenous melatonin levels in the SHR group were similar to thosereported by others ( 19 ) as well as ourselves( 30 ), and there were no apparent ill effects related tothe pharmacologically high circulating levels of melatonin in theSHR-Mel group, which is not surprising because no ill effects have beenreported when the plasma levels are increased 10,000 times( 8 ). The rats gained weight normally, and they appeared tobe as active as the untreated rats. No attempt was made to compareendogenous melatonin levels of the SHR group with the WKY controlsbecause other investigations have shown that young SHR have higher,whereas adult SHR have lower midnight levels of melatonin than the WKYrats ( 22 ).6 j4 {9 x$ ]5 q& K$ F
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As expected, melatonin treatment for 6 wk reduced oxidative stress.Both intracellular superoxide production (Fig. 6 ) and renal MDA content(Table 1 ) were drastically reduced in rats from the SHR-Mel group.Previous studies from our group have also found reduction of theseparameters of oxidative stress in SHR treated with an immnosuppressordrug, mycophenolate mofetil ( 45 ).  \" o* B% Z8 A3 i1 w0 K! T. Y' f
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Rats from the SHR group developed an increasingly severe hypertensionwhen given a regular diet with 4% NaCl. In contrast, themelatonin-treated rats exhibited a significant reduction of SBP withoutreaching normal levels (Fig. 2 ). These findings were associated with asignificant reduction of the renal infiltration of lymphocytes (Fig. 3 ), macrophages (Fig. 4 ), and angiotensin II-positive cells (Fig. 5 ).Double-staining studies revealed that about one-fifth of theangiotensin II-positive cells were lymphocytes and an almost similarproportion were macrophages. The remaining 60% were tubular epithelialcells; these findings are similar to those reported in previous studies( 43, 45 ).: R/ g& h7 x3 y, H( l

The reduction of interstitial inflammation resulting from theadministration of a potent antioxidant such as melatonin is the mirrorimage of the reduction of renal oxidative stress resulting from theadministration of a drug with anti-inflammatory properties, such asmycophenolate mofetil ( 36, 43, 45 )., |+ q4 N, w6 Q; V) H

The administration of melatonin induced an 80% normalization in bloodpressure in association with a 50% normalization of lymphocyte andangiotensin II infiltration. A possible explanation could be thatinflammatory infiltration is an epiphenomenon, but this would appearunlikely in view of the consistent improvement in hypertension obtainedwith MMF-induced reduction of interstitial inflammation in severalmodels of acquired ( 2, 36, 43 ) and genetic( 45 ) models of hypertension and the correlations foundbetween the severity of hypertension and the intensity of theinfiltration of lymphocytes and angiotensin II-positive cells inprevious studies in the SHR ( 45 ). Furthermore, thesuppression of the interstitial infiltration obtained by melatonintreatment reduced the number of interstitial lymphocytes, macrophages,and angiotensin II-positive cells below the levels present in the SHR-23w group, and, importantly, blood pressure in the SHR-Mel groupactually improved with respect to baseline values. A more likelyexplanation for the finding of an 80% reduction in blood pressure inassociation with only a 50% reduction in the interstitial infiltrateis that a critical amount of lymphocyte infiltration can be toleratedby the kidney before it results in salt-sensitive hypertension.. S9 x; _; y2 N" D; U
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Melatonin is widely available and free of side effects and has beenused with moderate success in humans for the treatment of insomnia( 8 ) and to reduce oxidative stress and inflammatory response ( 15, 18 ). Relevant to the present work, melatonin has been reported to modify characteristics of the immune system and ofinterstitial inflammation ( 16 ), being capable of inducing Th1 and Th2 cytokine responses ( 13, 25, 37 ) and to have proinflammatory ( 10, 28 ), as well as anti-inflammatoryeffects ( 12, 26 ). These conflicting results may be due, atleast in part, to differences in dose, species, sex, age, and immunematuration, all of which are critical factors in the response tomelatonin ( 51 ). However, there seems to be generalagreement on the fact that melatonin inhibits the activation ofNF- B, an effect that may indirectly contribute to its role as anantioxidant ( 14, 29, 35 ). The participation of NF- B inchronic inflammatory diseases ( 3 ) and, specifically, inrenal disease ( 17 ), is well recognized, and we theorizedthat NF- B activation was probably participating in the renalinterstitial inflammation present in SHR because of overproduction ofoxygen-free radicals in SHR with severe hypertension ( 44 ),which act as second messengers in NF- B activation( 48 ). Angiotensin II, which was found previously ( 43 ) as well as in the present studies (Fig. 6 ) to bepresent in cells in the renal tubulointerstitium, could also contribute to activation of NF- B because several studies have shown this effectin vivo and in vitro ( 46 ).

In fact, we found increased expression of activated NF- B in thekidney of severely hypertensive SHR that was reduced with melatonintreatment. NF- B expression was observed in infiltrating mononuclearcells as well as in tubular epithelial cells (Fig. 9 ). Because NF- Bexpression was not investigated in cells of the peripheral blood, it isnot possible to say whether NF- B activation of immune cellsinfiltrating the renal tubulointerstitium took place inside or outsidethe kidney. Nevertheless, activation of this transcriptional factor inthe tubular epithelial cells suggests the participation of genesactivated within these cells in the phenomena of immune cellrecruitment and inflammation present in the kidney of SHR. It is alsological to assume that the reduction of NF- B in the rats of theSHR-Mel group (Figs. 8 and 9 B ) likely played a significantrole in the observed reduction of the renal inflammatory infiltrate.

It is of note that nearly complete correction of oxidative stressparameters (MDA and superoxide-positive cells) was accompanied bysignificant but incomplete (50%) reduction of inflammatory infiltrateand angiotensin II-positive cells. This phenomenon points to the dualaction of melatonin in alleviating oxidative stress. First, throughmechanisms discussed above, melatonin treatment results in reduction ofthe inflammatory infiltrate, which partially reduces but does notnormalize ROS production. Second, there is the direct scavenging of theresidual ROS by melatonin. Nearly complete inactivation of residual ROScan restore lipid peroxidation and MDA levels to normal.
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In summary, we demonstrated that the progressive hypertension in SHR issignificantly improved with the administration of melatonin in thedrinking water, which resulted in pharmacological elevations in thehormone-circulating levels throughout the 24-h period. Melatoninadministration resulted in reduction of oxidative stress, reduction ofNF- B activation, and reduction of interstitial renal inflammation.These results support the role of immune cells infiltrating the kidneyin the pathogenesis of salt-sensitive hypertension and indicate thatmelatonin is a potential antihypertensive treatment that merits further investigation.  E* f- ~4 X6 a. |; X$ I( m- q
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ACKNOWLEDGEMENTS
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We acknowledge the technical assistance of Maribel Chávez.
      【参考文献】
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2. Alvarez, V,Quiroz Y,Nava M,Pons H,andRodríguez-Iturbe B. Overload proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of immune cells. Am J Physiol Renal Physiol 283:F1132-F1141,2002 .
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3. Barnes, PJ,andKahin M. Nuclear factor- B. A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066-1071,1997 .* z9 f5 l& a- b! f4 n: W- d
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