Reversibility of chronic cyclosporine nephropathy in ratsafter withdrawal of cy - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：CanLi, Chul WooYang, WanYoungKim, Ju YoungJung, Jung HoCha, Yong SooKim, JinKim, William M.Bennett,  Byung KeeBang作者单位：Departments of Internal Medicine and Anatomy, The Catholic University of Korea, Seoul,Korea 137-701; Nephrology and DialysisUnit, Department of Internal Medicine, Affiliated Hospital, YanBianUniversity Medical College, 133000 YanJi, JiLin, China;and Cellular Transplantation Division, Legacy GoodSama


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      【摘要】
   Renal interstitial inflammation isan important factor in the pathogenesis of chronic cyclosporin A(CsA) nephropathy. We studied the expression of the chemoattractantosteopontin (OPN) and the relationship between OPN expression andtubulointerstitial injury in a rat model of chronic CsA nephropathy.Chronic CsA nephropathy was induced in Sprague-Dawley rats byadministering CsA (15 mg/kg sc) for 5 wk and then withdrawing it for 5 or 10 wk. Renal function, histopathology (arteriolopathy, ED-1-positive cells, and tubulointerstitial fibrosis), renin-angiotensin system (RAS)activity, and OPN expression were observed during the follow-up period.Renal function deteriorated in CsA-treated rats, with the developmentof typical histopathology and activation of RAS. After CsA withdrawal,these parameters were significantly reversed (all P seen inCsA-treated rat kidneys was decreased 5 wk after CsA withdrawal and wasfurther decreased after 10 wk. Of note, OPN mRNA expression correlatedwith the number of infiltrating macrophage ( r = 0.651, P r = 0.729, P These findingssuggest that OPN expression and macrophage infiltration decrease afterlong-term CsA withdrawal in rats with established chronic CsAnephropathy, and this is closely associated with recovery from renal injury. 3 U- s3 s7 [9 @2 U/ A. f! M8 Q
      【关键词】 cyclosporin withdrawal osteopontin macrophage tubulointerstitialfibrosis reninangiotensin system- i+ c2 T) Y. \3 ]+ Y: H& ~
   INTRODUCTION  d+ t6 N( S0 {" |0 J+ b( ^
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CYCLOSPORIN A (CsA) isone of the most potent immunosuppressants used for the management ofmultiple-organ transplantation. However, the clinical utility of CsA asan immunosuppressive agent has been significantly limited by thefrequent occurrence of chronic CsA nephropathy, characterized byprogressive renal insufficiency, arteriolopathy, interstitialinflammation, and striped fibrosis ( 22 ). The exactmechanism underlying chronic CsA nephropathy is poorly understood.However, using a well-established animal model, a complex networkincluding the renin-angiotensin system (RAS) ( 44 ), nitricoxide (NO) ( 1 ), apoptotic cell death( 18 ), transforming growth factor (TGF)- 1( 33 ), and chemoattractant molecules ( 29 ) hasbeen shown to be involved. Of these, chemoattractants, and theresultant inflammatory cell infiltration, have been proposed to beimportant players, because interstitial inflammatory events precedeongoing fibrosis in chronic CsA nephropathy ( 29, 45 ).
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Osteopontin (OPN) is a highly acidic phosphoprotein containing anarginine-glycine-aspartic acid (RGD) motif. It is involved in celladhesion and migration ( 4 ) and is expressed by several cell types in a constitutive or inducible fashion. These include osteoclasts, some epithelial cells, macrophages, T cells, smooth musclecells, and some tumor cells ( 2, 7, 8, 11, 19, 26 ). OPNacts as a chemotactic factor for macrophages and monocytes by bindingto ligands including v 3 -integrin, CD44,collagen type I, and fibronectin ( 10, 39 ). In the kidney,OPN is expressed constitutively in the renal medulla, in the loop ofHenle, and in the distal convoluted tubules. It is absent from thenormal renal cortex, with the exception of the parietal epithelium of Bowman's capsule ( 11, 19 ). Upregulation of OPN expression correlates with macrophage infiltration and the development of tubulointerstitial injury in several models of kidney disease ( 13, 15, 28, 49 ). This relationship is supported bystudies using OPN knockout mice or an anti-OPN antibody ( 9, 24, 25, 48 ).

The effects of CsA dose reduction, or complete withdrawal of CsA, havebeen previously examined in clinical trials and experimental studies.However, the results obtained are conflicting ( 12, 14, 27, 40 ). The majority of these divergent studies are focused onmorphological changes and lack evidence of a molecular basis. Moreover,most of the clinical trials are based on protocols of CsA dosereduction or complete withdrawal and addition of other nonnephrotoxicimmunosuppressants (e.g., mycophenolate mofetil). Thus it is not clearin the clinical setting whether improved chronic allograft nephropathy(or chronic CsA nephropathy) is due to CsA dose reduction orelimination alone or the beneficial effects of other drugs. Therefore,we designed this study to investigate the progressive nature of chronicCsA nephropathy after long-term drug discontinuation and to determinethe possible molecular mechanism.. x6 O( V2 H9 W* B
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MATERIALS AND METHODS% ^' p; D* O) N# K6 t8 \- b) D
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Animals and drugs. Male Sprague-Dawley rats (Charles River, Wilmington, MA), initiallyweighing 200-220 g, were housed in cages (Nalge, Rochester, NY) ina temperature- and light-controlled environment and allowed free accessto standard laboratory chow and tap water. Rats were placed on asalt-deficient diet (0.05% sodium, Teklad Premier, Madison, WI)throughout the treatment. Cyclosporin (Novartis Pharma, Basel,Switzerland) was diluted in olive oil to a final concentration of 15 mg/ml.
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Experimental design. The experimental protocol was approved by the Animal Care Committee ofthe Catholic University of Korea. Rats were randomized to six groups asdepicted in Fig. 1. Animals received adaily subcutaneous injection of CsA (15 mg/kg) for 5 wk or CsA for 5 wkfollowed by 5 (CsAW5) or 10 wk (CsAW10) of CsA withdrawal. Controlvehicle-treated (VH) groups received a daily subcutaneous injection ofolive oil (1 ml/kg) for 5 (VH5), 10, and 15 wk ( n = 5 rats/group). At each time point, animals were euthanized under ketamineanesthesia and the kidneys were rapidly removed for morphological andmolecular studies.: I4 ?  b, p( i% X. _' T
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Fig. 1. Diagram showing the experimental design. Kept on a salt-deficientdiet, animals were randomized into 6 subgroups and treated daily withvehicle (VH5; olive oil, 1 ml/kg) or cyclosporin A (CsA; 15 mg/kg) for5 wks (CsA5) or CsA for 5 wk followed by 5 and 10 wk of withdrawal(CsAW5 and CsAW10, respectively). Each withdrawal group was paired withvehicle-treated group ( n = 5 rats/group). VH10 andVH15, groups treated with vehicle for 10 and 15 wk, respectively.
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Functional parameters. After the treatment was started, rats were pair-fed and daily bodyweight was monitored. Systolic blood pressure (SBP) was recorded inconscious rats by the tail-cuff method, with plethysmography using atail manometer-tachometer system (BP-2000, Visitech System, Apex, NC);at least three readings for each rat were averaged. Before death, theanimals were individually housed in metabolic cages for 24-h urinecollection, and blood samples were obtained to evaluate serumcreatinine (S Cr ). The creatinine clearance rate (C Cr ) was calculated using a standard formula. Whole bloodCsA levels were measured by monoclonal radioimmunoassay (Incstar, Stillwater, MN). Plasma renin activity (PRA) was measured byradioimmunoassay (angiotensin I-Biotecx radioimmunoassay; Biotecx,Friendswood, TX).1 g) k% a3 T, g. |" D& j
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Histopathology. Harvested rat kidney tissues were fixed inperiodate-lysine-paraformaldehyde (PLP) solution and embedded in wax.After dewaxing, 4-µm sections were processed and stained withperiodic acid-Schiff (PAS) or Masson's trichrome and hematoxylin.Arteriolopathy of the afferent arterioles was manifested by expansionof the cell cytoplasm of terminal arteriolar smooth muscle cells byeosinophilic, granular material and semiquantitatively evaluated bycounting the percentage of juxtaglomerular afferent arterioles witharteriolopathy per total number of juxtaglomerular afferent arteriolesavailable for examination with a ×20 objective; at least 60 glomeruli/specimen were assessed. A finding of tubulointerstitialfibrosis (TIF) was defined as a matrix-rich expansion of theinterstitium with tubular dilatation, tubular atrophy, tubular castformation, sloughing of tubular epithelial cells, or thickening of thetubular basement membrane. A minimum of 20 fields/section was assessedand graded using a color image analyzer (Mustek Paragon 800 SP,Macintosh PowerPC 7100, NIH Image, version 1.5). The extent of TIF wasestimated by counting the percentage of areas injured per field ofcortex using a scoring scale of 0-3 : 0 = normalinterstitium; 0.5 = area; 2 = 26-35% injured area; 2.5 = 36-45% injured area; and 45% injured area, as previously described( 18 ). Histopathological analyses were performed inrandomly selected cortical fields of sections by a pathologist blindedto the identity of the treatment groups.

Northern blot analysis. A 1-kb cRNA probe was generated from a 2B7 cDNA clone of rat smoothmuscle OPN ( 7 ). Sense and antisense cRNA probes were labeled with digoxigenin (DIG)-UTP using a T7 RNA polymerase kit (Boehringer Mannheim, Mannheim, Germany). Probes were precipitated, andincorporation of DIG was determined by dot blotting. Northern blottingwas performed as previously reported by others ( 49 ). Briefly, kidney cortex was homogenized. Total RNA was extracted usingthe RNAzol reagent (TEL-TEST), and 20-µg samples were denatured withglyoxal and dimethylsulfoxide, size fractionated on 1.2% agarose gels,and capillary blotted onto positively charged nylon membranes(Boehringer Mannheim). Membranes were hybridized overnight at 68 or42°C with DIG-labeled cRNA (or 32 P-labeled cDNA probesfor GAPDH) in a DIG wash and Block Buffer Set solution (BoehringerMannheim). After hybridization, membranes were washed finally in 0.1×SSC/0.1% SDS at 68°C or 0.2× SSC/0.1% SDS at 42°C. Bound probeswere detected using sheet anti-DIG antibody (Fab) conjugated withalkaline phosphatase (Boehringer Mannheim) and development withCSPD-star-enhanced chemiluminescence (Boehringer Mannheim).Densitometry analysis was performed using NIH Image software. Threedeterminations for each band were averaged and referenced to GAPDH.' |- _5 L. |6 }

In situ hybridization. In situ hybridization was performed on wax-embedded sections using amicrowave-based protocol as previously described by others ( 49 ). After dewaxing, sections were treated with amicrowave oven twice for 5 min, incubated with 0.2 mol/l HCI for 15 min, followed by 1% Triton X-100 for 15 min, and then digested for 20 min with 10 µg/ml proteinase K at 37°C (Boehringer Mannheim). Sections were washed in 2× SSC, prehybridized, and then hybridized with 0.3 ng/µl DIG-labeled sense or antisense OPN cRNA probeovernight at 37°C in a hybridization buffer containing 50% deionizedformamide, 4× SSC, 2× Denhardt's solution, 1 mg/ml salmon sperm DNA,and 1 mg/ml yeast tRNA. Sections were finally washed in 0.1× SSC at 37°C, and the hybridized probe was detected using sheep anti-DIG antibody (Fab) conjugated with alkaline phosphatase and color development with nitro blue tetrazolium/X-phosphate (Boehringer Mannheim). No signal was seen with the sense riboprobe labeled to thesame specific activity.

Immunohistochemistry. After dewaxing, sections were incubated with 0.5% Triton X-100-PBSsolution for 30 min and washed three times with PBS. Nonspecific binding sites were blocked with normal horse serum diluted 1:10 in0.3% BSA for 30-60 min and then incubated for 2 h at 4°Cin mouse antiserum against OPN (MPIIIB10; obtained from theDevelopmental Studies Hybridoma Bank, University of Iowa, Iowa City,IA) diluted 1:1,000 in a humid environment. After a rinsing inTris-buffered saline (TBS), sections were incubated inperoxidase-conjugated rabbit anti-mouse IgG (Amersham PharmaciaBiotech, Piscataway, NJ) for 30 min. For coloration, sections wereincubated with a mixture of 0.05% 3,3'-diaminobenzidine containing0.01% H 2 O 2 at room temperature until a browncolor was visible, washed with TBS, counterstained with hematoxylin,and examined under light microscopy. The procedure of immunostainingfor ED-1 (Serotec) and renin was similar to that for OPN. The antibodyagainst renin was kindly provided by Dr. Kirsten Madsen (Dept. ofMedicine, University of Florida, Gainesville, FL). This antibody isknown to recognize rat renin and prorenin ( 23 ). The numberof ED-1-positive cells was counted in at least 20 fields ofcortex/section under ×200 magnification, and renin-positive glomeruliwere evaluated by counting a minimum of 50 glomeruli/specimen( 43 ).

Statistical analysis. Data are expressed as means ± SE. All parameters were comparedwith the VH5 group ( n = 5), because there were nosignificant differences in the control vehicle-treated rats at any timepoint. Multiple comparisons among groups were performed by one-wayANOVA with the post hoc Bonferroni test (SPSS software, version 9.0, Microsoft). The Pearson single-correlation coefficient was used tocompare osteopontin mRNA expression with renal injury (ED-1-positive cell and the TIF score) and plasma renin activity. Statistical significance was assumed as P" g/ T7 y4 D% P1 w, k

RESULTS0 a9 ?- H" A& N3 P

Effects of cyclosporine withdrawal on functional parameters in ratswith established chronic CsA nephrotoxicity. Table 1 summarizes the functionalparameters in each group. In the present study, SBP and renal functionin the VH rat kidneys on a low-salt diet were slightly different fromthat in rats on a normal-salt diet (SBP: 138 ± 11 vs. 117 ± 6 mmHg; S Cr : 0.55 ± 0.02 vs. 0.65 ± 0.02 mg/dl;C Cr : 0.55 ± 0.02 vs. 0.47 ± 0.02 ml · min 1 · 100 g 1, respectively), as previously reported by ourlaboratory ( 44 ). This may be associated with the stresscondition, considering a low-salt diet for a long-term period. SBPmeasured by tail-cuff pressure was not significantly different betweenthe treatment groups throughout the study. Whole blood CsAconcentrations were dramatically decreased in the CsA-treated ratsduring the period of CsA withdrawal. The mean body weight ofCsA-treated rats was significantly lower than that of the VH rats(234 ± 5 vs. 285 ± 4 g, P increasedcompared with CsA-treated rats (CsAW5, 288 ± 8 g; CsAW10, 305 ± 6 g; P rats with CsA for 5 wk caused higherlevels of S Cr (1.14 ± 0.11 vs. 0.65 ± 0.02 mg/dl, P = 0.001) and lower levels of C Cr (0.14 ± 0.01 vs. 0.47 ± 0.02 ml · min 1 · 100 g 1, P withdrawal normalized these parameters.+ k  V) l: B: u: p/ Y
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Table 1. Changes in renal function, systolic blood pressure, body weight,and CsA concentration3 Y, z! M& h/ U. ~6 I8 U& w

Effects of cyclosporine withdrawal on histopathology in rats withestablished chronic CsA nephrotoxicity. CsA-treated rats showed the typical histopathological features ofchronic CsA nephrotoxicity (afferent arteriolopathy, striped interstitial fibrosis, and tubular atrophy with focal inflammatory cellaccumulation). Afferent arteriolopathy was manifested as thereplacement of smooth muscle cells of the afferent glomerular arteriole by a PAS-positive material, resulting in a typicalcircumferential appearance of the lesion (Fig. 2 ). On our semiquantitative scoring system, the arteriolopathy was significantly higher in the CsA groupthan the VH group (26 ± 2 vs. 11 ± 1, P this was markedly decreased after 5 or 10 wk of CsAwashout compared with the CsA group (CsAW5, 13 ± 1; CsAW10,12 ± 1; P CsA, respectively). Asignificant increase in TIF score (Fig. 3 ) was also observed in the CsA groupcompared with the VH group (1.8 ± 0.20 vs. 0.22 ± 0.04, P withdrawalsignificantly decreased the TIF score compared with the CsA group(1.0 ± 0.07, P was found after 10 wk of withdrawal compared with the CsAW5 group (0.5 ± 0.07, P = 0.034).3 k; X* p) U) |

Fig. 2. Photomicrographs and semiquantitative analysis of arteriolopathy inrat kidneys with chronic cyclosporine nephrotoxicity. CsA treatment for5 wk induces a typical afferent arteriolopathy. The micrograph shows aglomerulus with expansion of the cell cytoplasm of terminal arteriolarsmooth muscle cells by eosinophilic, granular material (arrow). Using asemiquantitative score, the increased arteriolopathy seen inCsA-treated rat kidneys was significantly reduced by 5 and 10 wk of CsAwithdrawal. Periodic-Schiff staining; original magnification, ×400.# P P

Fig. 3. Degree of renal tubulointerstitial fibrosis in the experimentalgroups and semiquantitative scoring. Representativephotomicrographs showing that striped tubulointerstitialfibrosis, inflammatory cell infiltration, and extracellular matrixdeposition were produced in the kidneys after CsA treatment for 5 wk,whereas lesions were significantly reduced by 5 wk of CsA withdrawaland further improved during 10 wk. Trichrome staining, originalmagnification, ×40. # P P P' r" z  [6 Z" c( f7 ^: R

Effects of cyclosporine withdrawal on the RAS in rats withestablished chronic CsA nephrotoxicity. We used immunohistochemistry for renin and PRA to evaluate the statusof the RAS. Figure 4 shows the status ofRAS in different experimental groups. Intrarenal expression of reninwas minimal in the VH group (Fig. 4, A and B,10 ± 1), but its immunoreactivity and the number ofrenin-positive glomeruli significantly increased in the CsA groupcompared with the VH group (26 ± 4, P With CsA withdrawal for 5 or 10 wk, immunoreactivity and the number ofrenin-positive glomeruli were significantly reduced and were similar tothe levels in the VH group (CsAW5, 13 ± 2; CsAW10, 12 ± 2;both P CsA-treated rats (35.2 ± 2 ng · ml 1 · h 1, P (Fig. 4 C, CsAW5, 24.9 ± 2 ng · ml 1 · h 1,CsAW10, 23.0 ± 3 ng · ml 1 · h 1;both P) _* Q; l& V& d' C
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Fig. 4. A : immunohistochemistry of intrarenal renin. B : semiquantitative analysis of renin-positive glomeruli. C : plasma renin activity. Increased intrarenalimmunoreactivity of renin, renin-positive glomeruli (arrows), andplasma renin activity seen in CsA-treated rat kidneys was markedlydecreased when CsA was withdrawn. Immunohistochemical staining,original magnification, ×200. # P P8 q* l5 j+ H! ?) M3 G' z, T0 H

Effects of cyclosporine withdrawal on macrophage infiltration inrats with established chronic CsA nephrotoxicity. ED-1-positive cells were detected only infrequently in the kidneys withVH treatment (11 ± 1), but with CsA treatment for 5 wk theirnumbers increased markedly (53 ± 5, P in areas where TIF was observed (Fig. 5 ). After 5 wk of CsA withdrawal, ED-1-positive cells significantly decreased compared with the CsA group(31 ± 2, P in ED-1-positive cells when CsA was withdrawn for 10 wk (19 ± 1, P = 0.038 vs. CsAW5) accompanied by areduction in TIF.1 `0 o% R5 Y% K+ Q% i3 x1 m

Fig. 5. Representative photomicrographs of immunohistochemistryfor ED-1 in experimental groups ( A ) and semiquantitativeanalysis ( B ). CsA treatment induced a significant focalmacrophage influx within the tubular interstitium, where fibroticlesions had developed, but their numbers decreased gradually when CsAwas withdrawn for 5 or 10 wk. Immunohistochemical staining, originalmagnification, ×100. # P P P = 0.038 vs. CsAW5.* M1 u6 _1 N/ P6 J: q9 f

Effects of cyclosporine withdrawal on OPN expression in rats withestablished chronic CsA nephrotoxicity. Northern blot analysis of the kidney cortex revealed a dramaticincrease in OPN mRNA in the CsA group vs. the VH group during the 5-wkperiod (Fig. 6; 8.7-fold, P OPN expression wassignificantly decreased after 5 wk of CsA discontinuation (4.8-foldrelative to the VH group, P decreased by 10 wk (2.6-fold relative to the VH group, P To localize OPN mRNA and protein, in situhybridization and immunohistochemistry were carried out. In theVH-treated rat kidneys, much higher levels of OPN mRNA and protein wereobserved. These were confined to tubular epithelium, collecting ducts,and uroepithelial lining cells. Most cortical structures were negativefor OPN. In contrast, the levels of OPN mRNA and protein weredramatically elevated in tubular epithelium and Bowman's capsule cellsin the CsA-treated rats. The most striking changes were seen in therenal cortex (Fig. 7 ), which normallyexpress very little constitutive OPN. It is notable that the sites ofstrong OPN mRNA and protein expression were in areas of severe TIF.After 5 wk of CsA withdrawal, levels of upregulated OPN mRNA andprotein were significantly decreased compared with the CsA group, and afurther decrease was observed in the CsAW10 group.9 Q, f* ^9 u& V$ l  l2 t& @8 ?

Fig. 6. Northern blotting and quantitative analysis ofosteopontin (OPN) mRNA. This Northern blot shows that the expression ofOPN mRNA was significantly upregulated in the CsA-treated group,whereas their expression was markedly decreased after 5 wk of drugwithdrawal and further decreased by 10 wk. Relative OPN mRNAs arereferenced to GAPDH; data are expressed as relative-fold increases.# P P P5 [8 F& N0 J0 L* Q3 w" y

Fig. 7. Representative photomicrographs of in situ hybridization( A ) and immunohistochemistry ( B ) for OPN in ratkidneys. Vehicle-treated rat kidney showing that no OPN expression wasdetectable in cortical tubules. However, OPN expression dramaticallyincreased in the cortical tubules of kidneys with CsA treatment for 5 wk, where severe tubulointerstitial fibrosis was observed. IncreasedOPN mRNA and protein expression were significantly decreased after 5 wkof CsA withdrawal, and a further decrease was observed by 10 wk of drugwithdrawal. A : original magnification, ×200; B

riginal magnification, ×100.
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Correlations between OPN expression, renal injury, and plasma reninactivity. To study the relationships between OPN expression and renal injury andthe RAS, correlation analysis was performed in the experimental groups(Fig. 8, n = 29). Thedecrease in OPN mRNA expression correlated well with the reduction inthe number of ED-1-positive cells ( r = 0.651, P fibrosis ( r = 0.729, P was positively correlated with OPNmRNA expression ( r = 0.645, P r = 0.762, P
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Fig. 8. Relationships between OPN mRNA expression and renalinjury and plasma renin activity. To assess the relationship betweenOPN mRNA expression and renal injury (macrophage infiltration andtubulointerstitial fibrosis) and plasma renin activity, correlationanalysis was performed in the experimental groups ( n = 29). OPN mRNA expression is represented as the ratio of OPN to GAPDHmRNA.

DISCUSSION
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This study clearly demonstrated the evolution of chronic CsAnephropathy after CsA withdrawal. Both renal function andhistopathology (arteriolopathy, interstitial inflammatory cellinfiltration, and fibrosis) were significantly improved with CsAwithdrawal. The upregulation of OPN expression in CsA-treated ratkidneys was dramatically decreased after discontinuation of CsA, andthis decrease correlated well with reductions in macrophageinfiltration and tubulointerstitial fibrosis. Thus one of themechanisms by which long-term CsA withdrawal effectively reverseschronic CsA nephropathy may be via decreased expression of theproinflammatory cytokine OPN.0 a5 W: {! J+ }& d6 s- R0 t
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OPN has a number of functions in diseased kidneys, including inhibitinginducible NO synthase ( 32 ), protecting cells from apoptosis ( 24 ), promoting cell proliferation andregeneration in the recovery process after ischemic injury( 41 ), and acting as a chemoattractant formonocyte/macrophages ( 4, 35 ). The diverse functions of OPNare thought to be mediated via distinct receptors ( 42 ). Inthe present study, we found that OPN mRNA and protein expression weremarkedly upregulated in cortical tubules after 5 wk of CsA treatment,along with macrophage infiltration into the tubulointerstitium, wheresevere fibrosis was observed. This finding is consistent with data onchronic CsA nephropathy showing an association between the degree oftubular OPN expression and the severity of interstitial macrophageaccumulation and fibrosis ( 29, 45 ). Furthermore, micelacking OPN (OPN / ) showed less chronic CsA nephrotoxicity than OPNwild-type (OPN  / ) mice ( 21 ), indicating a pathologicalrole for OPN in this model.5 Q6 Q( s9 }! a
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An interesting finding in this study is that CsA withdrawal decreasedOPN mRNA and protein expression in a time-dependent manner. Thedecrease in OPN expression was accompanied by improved renal functionand correlated well with the reduction in the number of macrophages( r = 0.651, P fibrosis ( r = 0.729, P that upregulation of OPN expression was associated with loss of renalfunction, macrophage accumulation, and severe histological damage.Suppression of its expression reversed these abnormalities ( 47, 49 ). Thus we propose that chronic CsA nephropathy may bereversible with long-term drug discontinuation and that this is relatedto decreased OPN expression.9 y+ K. Z' [; O3 r

It is generally accepted that renal function impairment induced by CsAimproves after CsA withdrawal (6, 12, 14, 27, 40, 43; Wei C, Song H,Seta K, Kinjo M, Lau P, Fink JC, Drachenberg C, Papadimitriou J,Bartlett ST, and Weir MR, unpublished observations). However, theefficacy of CsA withdrawal on the progression of chronic CsA-inducedfibrosis remains controversial. The reasons for this discrepancyare unknown. In previous studies, our laboratory has reported thatCsA withdrawal for 8 wk did not reverse tubulointerstitial fibrosis( 6 ). However, 5 wk of CsA withdrawal was effective inreversing the progression of tubulointerstitial fibrosis when chronicCsA nephrotoxicity was induced by administering a relatively low doseof CsA (7.5 mg · kg 1 · day 1 )( 43 ). These findings raise the possibility that theeffectiveness of CsA withdrawal on chronic CsA nephropathy is dependenton the CsA dose used and the timing of drug elimination.

How CsA withdrawal abolishes renal tubular OPN expression in this modelis unclear, but it may be related to the decreased RAS activity seen inCsA-withdrawn rat kidneys. It is well known that the activation of RAScaused by CsA may stimulate OPN expression ( 29, 44 ), andblocking RAS with either an angiotensin-converting enzyme (ACE)inhibitor or angiotensin II receptor type I antagonist could reduce OPNexpression ( 5, 31, 49 ). In the present study, we examinedrenin activity using plasma renin activity and immunohistochemistry,because both parameters have been shown to increase in chronic CsAnephropathy ( 3, 44, 46 ). The results of this studyrevealed that CsA discontinuation significantly decreased plasma reninactivity, renin immunoreactivity, and the number of renin-positiveglomeruli. Furthermore, plasma renin activity was positively correlatedwith OPN mRNA expression ( r = 0.645, P r = 0.762, P report from our laboratory ( 43 ) and a human study(Wei C, Song H, Drachenberg CI, Papadimitriou JC, Bartlett ST, FinkJ, and Weir MR, unpublished observations) describing the efficacy of CsA withdrawal on RAS activity." }8 Y& }* O& m8 i+ W# C8 m, r1 @
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It is also possible that CsA withdrawal decreases OPN expression byreducing CsA-induced hypoxia. Chronic CsA nephrotoxicity is associatedwith afferent arteriolopathy, which ultimately leads to a reduction inrenal blood flow and thus may exert hypoxic injury ( 17, 38, 50 ). Recent in vitro studies in cultured mesangial andLLC-PK 1 tubular epithelial cells exposed to a hypoxic environment showed increased proliferation and collagen synthesis, withstimulation of OPN release ( 36, 37 ). In the present study, CsA withdrawal markedly reversed CsA-induced arteriolopathy to a levelsimilar to that in control animals. This finding is in agreement withrecent reports ( 17, 37, 50 ) implicating a role forCsA-associated hypoxia in the induction of renal OPN expression. Inaddition, CsA is able to induce TGF- 1 production directly orindirectly ( 16, 30, 34 ), which may stimulate OPNexpression ( 20 ). Although we did not examine the levels ofTGF- 1 in this study, a previous study from our laboratory showedthat CsA withdrawal significantly reduced TGF- 1 mRNA expression ( 43 ). Similar results were also reported from a humanstudy (Song H, Seta K, Kinjo M, Lau P, Fink JC, Drachenberg C,Papadimitriou J, Bartlett ST, Weir MR, and Wei C, unpublishedobservations). Therefore, it is likely that decreased TGF- 1expression after CsA withdrawal may affect OPN expression in this model.7 |3 G: E# G8 Q0 i) k) M( a4 R

In summary, long-term CsA withdrawal is able to decrease interstitialinflammation induced by the proinflammatory cytokine OPN. This isclosely associated with the reversal of chronic CsA nephrotoxicity.This study provides a rationale for how to reduce CsA-associatednephropathy in the clinical setting. Further clinical trials arerequired to determine the appropriate dose of CsA (or CsA withdrawal)that with the addition of nonnephrotoxic immunosuppressants (e.g.,mycophenolate mofetil), will achieve optimal immunosuppression whileavoiding the risk of acute rejection." _* Z* {/ o; X

ACKNOWLEDGEMENTS

This study was supported by grants from the Korean Society ofNephrology in 2001.
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