expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis

Kidney International, Vol. 56 (1999), pp. 2147–2159

Expression of apoptosis regulatory genes in chroniccyclosporine nephrotoxicity favors apoptosis

FUAD S. SHIHAB, TAKESHI F. ANDOH, AMIE M. TANNER, HONG YI, and WILLIAM M. BENNETT

Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah, and Oregon Health SciencesUniversity, Portland, Oregon, USA

Expression of apoptosis regulatory genes in chronic cyclosporine ranging from physiological stimuli to infectious and cyto-nephrotoxicity favors apoptosis. toxic events [6–8]. Apoptosis is an important pathogenic

Background. Chronic cyclosporine (CsA) nephrotoxicity is mechanism in renal diseases and has been shown to occurcharacterized by interstitial fibrosis, tubular dropout, and lossduring development and following injury [9–13]. Lethallyof cellularity in areas of fibrosis. Apoptosis was found to playinjured cells may die by either necrosis or apoptosis,a role in CsA-induced fibrosis. We evaluated the role of the

death genes p53, Bax, and Fas-L (ligand), survival gene Bcl-2, depending on the severity of the injury [14, 15]. Althoughinterleukin-converting enzyme (ICE), and caspase-3. severe renal ischemia results in acute tubular necrosis,

Methods. Salt-depleted rats were administered CsA 15 mg/ chronic low-grade ischemia leads to apoptosis in renalkg/day or vehicle (VH) and were sacrificed at 7 or 28 days.tubular cells [16–19]. Although apoptosis is beneficial inApoptosis was detected by TdT-mediated dUTP-biotin nick

end labeling assay. p53 and Bax expressions were evaluated some cases, it can be deleterious at times; apoptosis mayby Northern and Western blot analysis. Fas-L and Bcl-2 expres- result in tissue disruption if enough cells are lost. Mostsions were evaluated by immunofluorescence. In addition to organs undergoing fibrosis will show a paucity of cellsICE mRNA, caspase-3 enzymatic activity was assayed.

surrounded by an excess deposition of extracellular ma-Results. Although no differences were seen at one week,trix, suggesting that apoptosis may be playing an activeapoptosis-positive cells increased with CsA at four weeks (P ,

0.05) and correlated with tubular atrophy and interstitial fibrosis role in the later stages of fibrosis [5].(r 5 0.8, P , 0.05). CsA induced the expression of p53 (P , Cyclosporine A (CsA) remains an important immuno-0.05) and Bax (P , 0.01) and decreased that of Bcl-2 (P , suppressive drug used for the management of organ0.05). CsA up-regulated Fas-L expression (P , 0.001). ICE

transplantation [20]. However, it has severe deleteriousmRNA and caspase-3 activity were also increased (P , 0.01).effects on renal structure and function, making nephro-The changes occurred as early as one week and remained

statistically significant at four weeks. toxicity a major limiting side-effect [21, 22]. Chronic CsAConclusions. Specific apoptotic genes are increased in chronic nephrotoxicity can progress to an irreversible lesion

CsA nephrotoxicity. The balance favors the induction of apop- characterized by striped interstitial fibrosis, tubular atro-tosis. Increased apoptosis could explain the tubular dropoutphy, and hyalinosis of the afferent arterioles [23]. Theand loss of cellularity with fibrosis. This then may impair thepathogenesis of chronic CsA nephrotoxicity is thoughtability of the tubulointerstitium to remodel. Apoptosis could

also contribute to some of CsA immunosuppressive effects on to result from low-grade hypoxic injury to renal tubularactivated lymphocytes. cells [24]. There is a progressive loss of cellularity in

areas of interstitial fibrosis, suggesting an imbalance inwhich cell loss exceeds the proliferative response to re-

Apoptosis is a programmed cell death defined by char- place them. Although the mechanisms leading to extra-acteristic morphological and biochemical changes [1–3]. cellular matrix accumulation in chronic CsA nephrotox-Apoptosis plays an important role in the regulation of icity have been well explored [25–27], the pathogenesisrenal cell number in both health and disease [4, 5]. Most of tubular atrophy and cell loss is poorly understood.cells can be triggered to undergo apoptosis by insults Recently, it was shown that apoptotic cell death occurs

in CsA-associated fibrosis with little evidence of acutetubular necrosis [28].Key words: programmed cell death, immunosuppression, fibrosis,

nephrotoxicity, death genes. The tumor suppressor p53 is a transcriptional factorthat effects cell cycle arrest or apoptosis in responseReceived for publication February 16, 1999to a variety of stresses [29]. The level of p53 increasesand in revised form July 15, 1999

Accepted for publication August 3, 1999 following DNA damage [30]. Depending on the cell type,elevated p53 can result in either growth arrest or apopto- 1999 by the International Society of Nephrology

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Shihab et al: Induction of apoptotic genes in CsA nephrotoxicity2148

Table 1. Changes in cyclosporine A (CsA) whole blood level, before by the CsA-treated rats. Animals were dividedsystolic blood pressure (BP), serum creatinine, and creatinine

into two groups of 16 CsA-treated and 16 control rats.clearance in the experimental groups at 7 and 28 daysEight rats from each group were studied at days 7 and

CsA whole Serum Creatinine 28. Body weight was recorded daily. At the end of eachblood level Systolic BP creatinine clearancetreatment period, 24-hour urine samples were collectedGroups ng/ml mmHg mg/dl ml /min /100gin metabolic cages (Nalge, Rochester, NY, USA), andVH-7 days 0 60 15262 0.5960.04 0.35 60.01

CsA-7 days 2752 6136 14968 0.6360.02 0.30 60.01a systolic blood pressure was measured by tail plethysmo-VH-28 days 0 60 14362 0.5760.02 0.39 60.02 graphy (Narco Bio-systems, Houston, TX, USA). TheCsA-28 days 5164 6270 14464 1.0260.06a 0.2060.01a

following day, rats were anesthetized with ketamine.Values are expressed as mean 6 sem of eight rats. Abbreviations are: VH, After opening the abdomen through a midline incision,placebo; CsA, cyclosporine; BP, blood pressure.a P , 0.01 vs. VH the aorta was cannulated retrogradely below the renal

arteries with an 18-gauge needle. With the aorta oc-cluded by ligation above the renal arteries and the renalveins opened by a small incision for outflow, the kidneys

sis [31]. p53 is also a transcriptional modulator of the were perfused with 20 ml of cold, heparinized saline.bax gene, which promotes apoptosis [32]. Additionally, The kidneys were removed. The cortex was carefullya p53-negative response element has been identified in dissected from the medulla and was then processed forthe bcl-2 gene, which protects from apoptosis [33]. Bcl-2 evaluation by light microscopy, RNA analysis, and im-itself is a suppressor of apoptosis that homodimerizes or munohistochemistry.heterodimerizes with the homologous protein Bax [34].In contrast to Bcl-2, Bax is a death promoter that is Functional studiesneutralized by heterodimerization with Bcl-2 [35]. Gen- Blood was collected from the jugular vein in plasticerally, one assumes that the ratio of Bcl-2 to Bax deter- syringes transferred to metal-free tubes containing po-mines the cellular susceptibility toward apoptosis. Apop- tassium-ethylenediaminetetraacetic acid (EDTA) andtosis can also be mediated by cross-linking of the Fas was chilled on ice. Plasma was harvested immediatelyreceptor (Fas-R) by either soluble or membrane-bound by centrifugation at 48C and stored at 2708C until deter-Fas ligand (Fas-L) [36]. During apoptosis, there is activa- mined. Urinary and plasma creatinine were measured bytion of an array of cysteine proteases called caspases in a a Cobas autoanalyzer (Roche Diagnostics, Div. Hoffman-cascade-like fashion [37]. The active forms of interleukin- La Roche Inc., Nutley, NJ, USA). Creatinine clearanceconverting enzyme (ICE), caspase-1, and also CPP32 (cas- (CCr) was calculated using a standard formula. The CsApase-3) are involved in the final induction of apoptosis. blood level was also measured by a monoclonal radioim-

Although previous studies have shown that apoptotic munoassay for CsA (Incstar Co., Stillwater, MN, USA).cell death is increased in CsA-treated kidneys [28], theirevidence was based on morphological findings and did Morphologynot define the mechanisms through which it occurs. In Tissue samples were fixed in 10% buffered formalinthis article, we studied the specific role of apoptosis- and embedded in paraffin. Sections 2 to 4 mm were stainedregulatory genes in CsA-associated fibrosis. Our findings with periodic acid-Schiff reagent and Trichrome. Hori-suggest that CsA influences the expression of apoptotic zontal and saggital sections were performed. The findingsgenes in a manner that favors the development of apop- of interstitial fibrosis consisted of matrix-rich expansiontosis. In this model, the caspase system is also activated. of the interstitium with distortion and collapse of the

tubules and thickening of the tubular basement mem-branes (TBM). A minimum of 20 fields at a magnificationMETHODSof 3100 were assessed and graded in each biopsy by an

Experimental design observer masked to the treatment groups using a colorAdult male Sprague-Dawley rats (Charles River, Wil- image analyzer (Mustek Pentagon 800 SP, MacIntosh

mington, MA, USA) weighing 225 to 250 g were housed Power PC 7100, NIH Image vs. 1.5). For tubular atrophy,in individual cages in a temperature- and light-controlled the following semiquantitative score was used: 0 5 noenvironment and received a low-salt diet (0.05% sodium; tubular atrophy, 0.5 5 ,5% of tubules atrophied, 1 5Teklad, Premier, WI, USA), with water ad libitum. After 5 to 20%, 1.5 5 21 to 35%, 2 5 36 to 50%, 2.5 5 51 toone week on the low-salt diet, weight-matched pairs of 65%, and 3 5 .65%. Interstitial fibrosis was estimatedrats were randomly assigned to receive daily subcutane- by counting the percentage of injured areas per field andous injections of CsA at 15 mg/kg (Sandoz Research was scored semiquantitatively using the following: 0 5Institute, East Hanover, NJ, USA) or an identical vol- normal interstitium, 0.5 5 ,5% of areas injured, 1 5 5ume of vehicle (VH) olive oil as control. Control rats to 15%, 1.5 5 16 to 25%, 2 5 26 to 35%, 2.5 5 36 to

45%, and 3 5 .45%.were fed the exact amount of food consumed the day

Shihab et al: Induction of apoptotic genes in CsA nephrotoxicity 2149

Fig. 1. Cyclosporine A (CsA)-induced tubu-lointerstitial fibrosis. Photomicrograph show-ing the renal cortex of a salt-depleted rat givenCsA 15 mg/kg/day for four weeks. CsA-treated animals reveal renal tubular atrophy,inflammatory cell infiltration, and extracellu-lar matrix deposition (trichrome, magnifica-tion 3200).

Table 2. Scores of the morphologic changes of apoptosis by the ringer Mannheim) at 378C for 90 minutes. Reaction wasTUNEL method and semiquantitative scores by light microscopy

terminated by transferring slides to a working-strengthof the extent of changes observed for tubular atrophy andtubulointerstitial fibrosis stop/wash buffer (30 mm sodium citrate, 300 mm sodium

chloride) for 30 minutes at 378C. Sections were washedTubular Interstitialwith phosphate-buffered saline (PBS), then incubatedTUNEL atrophy fibrosis

(1 cells/20 fields) (0–31) (0–31) with peroxidase-labeled streptoavidin for 30 minutes inDay 7-CsA 7 68 0.260.1 0.1 60.1 a humidified chamber at room temperature, and thenDay 7-VH 3 62 0.160.1 0.1 60.1 incubated with the peroxidase-substrate solution (0.02%Day 28-CsA 54 610a 2.260.3a 2.060.3a

3.39-diaminobenzidine in 50 mm Tris-HCl buffer con-Day 28-VH 3 62 0.260.2 0.1 60.1taining 0.02% hydrogen peroxide) for five minutes. AfterValues are expressed as mean 6 sem of eight rats. Abbreviations are: TUNEL,

TdT-mediated dUTP-biotin nick end labeling; VH, placebo; CsA, cyclosporine. rinsing with PBS, sections were counterstained with he-a P , 0.01 vs. VH matoxylin and examined by light microscopy. For posi-

tive controls, some slides were heated with DNAse(Sigma) in DNA buffer. For negative control, slides weretreated with buffer lacking TdT.TUNEL assay

DNA fragmentation associated with apoptosis was de- Northern blot analysistected in situ in tissue sections by the addition of biotinyl- Renal tissue was finely minced with a razor blade onated nucleotides to free 39 hydroxyl groups in DNA ice then homogenized in TRIzol reagent (GIBCO BRL,by the TdT-mediated dUTP-biotin nick end labeling Grand Island, NY, USA). RNA extraction was per-(TUNEL) method [38] using the ApopTag in situ apo- formed according to the manufacturer’s protocol. Afterptosis detection kit (GIBCO, Gaithersburg, MD, USA). resuspension in Tris-EDTA buffer, RNA concentrationsFormalin-fixed, paraffin-embedded sections were depar- were determined using spectrophotometric readings ataffinized and stripped of proteins by incubation with Absorbance260. Thirty micrograms of RNA were electro-proteinase K (20 mg/ml; Boehringer Mannheim, Tokyo, phoresed in each lane in 0.9% agarose gels containingJapan) for 20 minutes at room temperature and then 2.2 m formaldehyde and 0.2 m Mops (pH 7.0) and werewashed in distilled water three times for two minutes. transferred to a nylon membrane (ICN Biomedicals,Endogenous peroxidase was inactivated by covering Costa Mesa, CA, USA) overnight by capillary blotting.the sections with 2% hydrogen peroxide for seven min- Nucleic acids were cross-linked by ultraviolet irradiationutes. Sections were rinsed with distilled water, immersed (Stratagene, La Jolla, CA, USA). The membranes werein terminal deoxynucleotidyl transferase (TdT) buffer prehybridized for two hours at 428C with 50% for-(GIBCO), and then incubated with TdT (0.3 e.u./ml; mamide, 10% Denhardt’s solution, 0.1% sodium dodecyl

sulfate (SDS), 5 3 standard saline citrate (SSC), and 200GIBCO) and biotinylated dUTP (0.04 nmol/ml; Boeh-

Fig. 2. Apoptosis in experimental chronic CsA nephrotoxicity by the TUNEL assay. Photomicrographs show apoptosis as detected by the TUNELassay in the kidney of a rat that received vehicle (VH; a) and cyclosporine A (CsA) 15 mg/kg/day (b) at four weeks. Note the marked increasein the number of TUNEL-positive cells (arrows) in the CsA-treated animal (magnification 3200).


[39, 40]. Four micrometer cryostat sections were fixedin acetone and washed in PBS, pH 7.4. The primarypolyclonal antibodies used were rabbit antihuman Bcl-2(DC 21; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA) and rabbit antihuman Fas-L (Q-20; Santa CruzBiotechnology). The secondary antibody was rhodamine-conjugated affiniPure F(ab9)2 fragment goat antirabbitIgG (Jackson Immunoresearch, West Grove, PA, USA)at a 1:100 dilution. A minimum of 20 randomly selectedareas per sample were observed at 3250 magnification.The intensity of staining for the glomerular, tubulointer-stitial, and vascular compartments was evaluated by anobserver blinded to the treatment groups using the fol-lowing semiquantitative scale: 0 5 diffuse, very weak,or absent staining; 1 5 staining involving less than 25%;2 5 staining involving 25 to 50%; 3 5 staining involving50 to 75%; and 4 5 staining involving 75 to 100%. Photo-Fig. 3. Correlation between score of fibrosis and number of apoptosis

positive cells in chronic CsA nephrotoxicity. Fibrosis was scored semi- graphs were obtained at identical exposure and develop-quantitatively using a 0 to 31 scale. The number of apoptosis positive

ment time intervals.cells per 20 fields was determined using the TUNEL assay at a 3200magnification.

Western blot analysis

Frozen (2708C) kidney tissue sections embedded inTissue-Tek O.C.T. compound (Miles Inc. Diagnostics

mg/ml denatured salmon sperm DNA. They were thenDiv., Elkhart, IN, USA) were processed for protein ex-hybridized at 428C for 18 hours with cDNA probes la-traction. Tissue was thawed at 48C in 5 ml lysis bufferbeled with 32P-dCTP by random oligonucleotide priming(GITC/BME) and then rinsed three times in 5 ml cold(Boehringer Mannheim). The blots were washed in 2 3(48C) PBS, pH 7.4. Protein extracts were prepared bySSC, 0.1% SDS at room temperature for 15 minutes andhomogenizing the whole cortex in 1 ml cold PBS con-in 0.1 3 SSC, 0.1% SDS at 508C for 15 minutes. Filmstaining 0.05% Tween 20 (PBS-T) with a glass tissue ho-were exposed at 2708C for different time periods tomogenizer (Kontes Glass, Vineland, NJ, USA). The tissueensure linearity of densitometric values and exposurehomogenates were centrifuged at 48C for 15 minutes attime. Autoradiographs were scanned on an imaging den-15,000 3 g, and the supernatants were collected then cen-sitometer (GS-700; Bio-Rad Laboratories, Hercules, CA,trifuged to remove cellular debris. Protein concentrationUSA). The density of bands for the glyceraldehyde-3-in the supernatant was determined using the Micro bicin-phosphate dehydrogenase (GAPDH) mRNA was usedchoninic acid (BCA) assay (Pierce, Rockford, IL, USA).to control for differences in the total amount of RNA

Equal amounts of protein were resolved on 15% SDS-loaded onto each gel line. For quantitative purposes,polyacrylamide gel electrophoresis (SDS-PAGE; Bio-values were divided by the density of bands for GAPDHRad, Richmond, CA, USA) and then electroblotted toin the same lane.nitrocellulose membranes (Bio-Rad). Equal proteinThe cDNA probes used for Northern were cellularloading was verified by Ponceau S staining. The mem-tumor antigen p53, 630 bp Not I-EcoRI murine cDNAbranes were blocked for one hour in TBS-T (10 mm Tris-fragment of clone pT7T3D-Pac purchased from Ge-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20) containingnomeSystems (St. Louis, MO, USA); Bax-alpha, 670 bp5% nonfat milk. Membranes were then incubated withNot I-EcoRI murine cDNA fragment of clone pT7T3D-mouse monoclonal antihuman p53 antibody (1:250 dilu-Pac (GenomeSystems); interleukin-1b–converting en-tion; PharMingen, San Diego, CA, USA) or mouse mono-zyme, 490 bp EcoRI-XhoI murine cDNA fragment ofclonal anti-rat Bax antibody (1:250 dilution; PharMingen)clone pBluescript SK- (Genome Systems); and GAPDH,diluted in the blocking buffer. The membranes were1272 bp Pst I rat cDNA fragment of clone pBS KSII.washed thoroughly in TBS-T, incubated with antirabbit

Immunofluorescence horseradish peroxidase-conjugated antibody (1:7500 dilu-tion) for two hours, and then washed again in TBS-T.Immunofluorescence microscopy was performed onThe detection of signal employed an enhanced chemilu-tissues snap frozen in cold isopentane with a cryostatminescence detection kit (NEN Life Science Products,microtome (Leica CM 1800; Leica Instruments GmbH,

Heidelberg Strasse, Germany) as previously described Boston, MA, USA).


Fig. 4. Northern blot of mRNA from rat kid-neys. Representative gel of total RNA isolatedfrom whole cortex (A) and medulla (B) ofkidneys from rats treated with CsA or VH.RNA was hybridized with cDNA probes top53, Bax, ICE, and GAPDH. Molecular sizemarkers shown on the right.

Caspase-3 enzyme activity achieving statistical significance (P , 0.01) at day 28.CsA-induced hypertension was not observed in thisCaspase-3 protease activity was assayed in the wholemodel. There was a progressive increase in CsA wholecortex tissue by following the manufacturer’s instructionsblood level in CsA-treated animals: 2752 6 136 ng/ml(PharMingen). Briefly, protein extracts were preparedat day 7 and 5164 6 270 ng/ml at day 28 (Table 1). CsAfrom tissue homogenates as described earlier in this arti-induced a significant increase in serum creatinine onlycle and were transferred to 96-well plates. For each reac-at day 28 (P , 0.01; Table 1). On the other hand, thetion, 200 ml of 1 3 HEPES buffer (reaction buffer) and 5CCr was already lower in the CsA-treated rats at day 7ml (1 mg/ml) of fluorogenic substrate (Ac-DEVD-AMC)(P , 0.05) and was progressively lower at day 28 (P ,were added and incubated for one hour at 378C. Sub-0.01; Table 1). There was no significant proteinuria instrate cleavage was measured using a spectrofluorometerthe CsA or VH-treated rats.(Cytofluor II) with an excitation wavelength of 380 nm

and an emission wavelength of 420 to 460 nm and wasHistologic changescorrected to the protein content. For each blocking reac-

Rats treated with CsA had characteristic morphologiction, Ac-DEVD-AMC, DEVD-CHO (caspase-3 inhibi-findings that were mostly evident at day 28 and thattor) and reaction buffer were added in parallel in eachwere similar to the human CsA lesion. CsA-treated ratswell. As a negative control, another reaction includingdeveloped striped interstitial fibrosis with progressiveAc-DEVD-CHO only was prepared. Tissue homoge-inflammatory cell infiltrates associated with thickening ofnates incubated with both Ac-DEVD-AMC and Ac-the tubular basement membrane (TBM) and Bowman’sDEVD-CHO did not emit fluorescence above the nega-capsule (Fig. 1). There was focal disruption of the TBMtive control.in areas of interstitial infiltrate with loss of tubular defi-

Statistical analysis nition. When the extent of changes was graded using a0 to 31 semiquantitative score, most differences wereResults are presented as mean 6 se. Comparisonsobserved at 28 days (Table 2). The vascular lesion inbetween the CsA group and the other experimentalCsA-treated rats was characterized by arteriolar hyalino-groups were done by Kruskal–Wallis test followed bysis with hyalin deposition within the tunica media ofTukey test. The level of statistical significance was chosenafferent arterioles and terminal portions of the interlobu-as P , 0.05.lar arteries.

RESULTS Morphologic evidence of apoptosis by theTUNEL methodPhysiologic studies

To determine and quantitate the degree of apoptosis,Although weight gain was progressive in the two treat-we performed the TUNEL method, which detects cleavedment groups, animals treated with CsA failed to gain as

much weight as those receiving VH with the difference DNA that is formed by the activation of the DNA poly-


Fig. 6. Expression of Bax in CsA and VH-treated rats. (A) Total RNAFig. 5. Expression of p53 in CsA and VH-treated rats. (A) Total RNAwas isolated from whole cortex and medulla at days 7 and 28 and waswas isolated from whole cortex and medulla at days 7 and 28 and washybridized with a cDNA probe to Bax. (B) Bax protein expression washybridized with a cDNA probe to p53. (B) p53 protein expression wasdetermined by Western blot analysis from whole cortex homogenates.determined by Western blot analysis from whole cortex homogenates.N 5 6 for each group. **P , 0.01; and ***P , 0.001 compared withN 5 6 for each group. *P , 0.05; **P , 0.01; and ***P , 0.001VH control.compared with VH control.

Expression of p53 and Bax by Northern and Westernmerase during apoptotic cell death (Fig. 2). As shown blot analysesin Table 2, there were occasional apoptotic cells present

Northern blot analysis. The mRNA expression of p53in the VH-treated groups. There were no significant dif-was clearly up-regulated in kidneys of CsA-treated asferences in apoptosis-positive cells between the CsA-compared with VH-treated rats, indicating that CsA ex-and VH-treated groups at one week (7 6 8 vs. 3 6 2,posure activated an intracellular death signal (Figs. 4

P 5 NS). However, there was a remarkably significantand 5A). This effect was seen very early at seven days

increase in the number of apoptosis-positive cells in the (P , 0.05 cortex and , 0.001 medulla) but was alsoCsA- vs. VH-treated groups at four weeks (54 6 10 vs. significant in CsA-treated kidneys at 28 days (P , 0.01).3 6 2, P , 0.05). In the CsA-treated group, the apoptotic Apoptosis was also promoted by the up-regulated ex-cells were mostly present in the tubules and interstitium, pression of the Bax gene in CsA-treated rat kidneyswhereas the number of glomerular TUNEL-positive cells (Figs. 4 and 6A). The mRNA expression of Bax waswas not significant (Fig. 2). The majority of the apoptotic high with CsA at 7 days in the cortex (P , 0.001) andcells were seen at the corticomedullary junction where remained elevated relative to VH-treated kidneys at 28severe tubular atrophy and tubulointerstitial fibrosis days in both cortex (P , 0.01) and medulla (P , 0.001).were also observed. The number of apoptotic cells sig- Interestingly, Bax mRNA expression was unexpectedlynificantly correlated with the degree of tubular atrophy lower in the medulla at seven days in CsA-treated asand fibrosis (both, r 5 0.8, P , 0.05) and is shown in compared with VH-treated kidneys. This does not corre-

spond to the observed findings in the other time pointsFigure 3.


though it was high in the medulla at both 7 and 28 days(P , 0.001; Fig. 10). Overall, the mRNA expressions ofp53, Bax, and ICE were more significant in the cortexwhen compared with the medulla (P , 0.001). In orderto provide evidence for caspase activation, we looked atthe proteolytic activity of caspase-3 (Fig. 11). We founda significantly increased caspase-3 activity in the CsArats at both 7 (P , 0.01) and 28 days (P , 0.001) com-pared with VH rats. Similar to p53 and Bax proteins, theactivity of caspase-3 progressively increased with time inthe CsA-treated rats and was significantly higher at 28days (P , 0.001) when compared with 7 days. On theother hand, caspase-3 activity remained unchanged inFig. 7. Western blot from the cortex of rat kidneys. Representative

gel of protein isolated from whole cortex of kidneys from rats treated the VH-treated rats.with CsA or VH. Membranes were incubated with mouse monoclonalantihuman p53 antibody or mouse monoclonal anti-rat Bax antibody.Molecular size markers shown on the right. DISCUSSION

We examined an experimental model of chronic CsAnephrotoxicity with pathologic features similar to thein the cortex and medulla. There is no clear explanationhuman lesion and that is characterized by tubular atro-for this finding except that it may correspond to differentphy and progressive interstitial fibrosis with loss of cellu-stages of activation of the bax gene.larity in areas of fibrosis. Our first finding was that theWestern blot analysis. To assess whether the changesnumber of apoptotic cells, as discerned by the TUNELin mRNA expression were translated into changes in theassay, was increased. Apoptosis was concentrated in re-synthesis of p53 and Bax proteins, Western blots weregions of fibrosis and affected mostly the tubular and inter-performed on tissue homogenates derived from thestitial compartments, which are also the major areas ofwhole cortex. The expression of p53 protein was similarCsA structural injury. Of interest is that the apoptotic cellsto its mRNA expression (Figs. 5B and 7). It increasedwere more prominent in the corticomedullary area whereat day 7 (P , 0.001) and more so at day 28 (P , 0.001)severe tubulointerstitial injury was also observed. In addi-in CsA-treated rats when compared with VH rats. Thetion, the number of apoptosis-positive cells correlatedexpression of Bax protein by Western blot is shown intightly with scores of tubular atrophy and interstitial fi-Figures 6B and 7 and was similar to its mRNA expres-brosis. These findings are consistent with previous reportssion. Although Bax protein expression was significantlysuggesting that apoptotic cell death is time dependenthigher (P , 0.01) in the CsA group at 28 days, Bax protein

expression tended to be higher in the CsA rats at seven and associated with interstitial fibrosis in chronic CsAdays, although it did not reach statistical significance. nephrotoxicity [28]. Other studies in models of tubuloin-

terstitial injury and progressive glomerulosclerosis haveExpression by immunofluorescence of Bcl-2 and Fas-L also found increased apoptosis in association with fibro-

To determine the activity of apoptosis in the different sis [41–45]. The mechanism for this increase in apoptosiskidney compartments, we performed immunohistochemi- is unclear, but apoptosis is favored in low-grade tissuecal staining (IF) of the “survival” protein Bcl-2 and the ischemia, which is thought to play a role in chronic CsA“death-promoter” protein Fas-L (Figs. 8 and 9). The nephrotoxicity [24]. This finding is consistent with data inexpression of Bcl-2 (Fig. 8) was opposite of the other CsA nephrotoxicity showing that apoptosis is acceleratedpro-apoptotic genes and was significantly lower in the with angiotensin II and decreased with nitric oxide [28].CsA-treated groups at both 7 (P , 0.01) and 28 days In our study, we found that CsA treatment was associ-(P , 0.05). On the other hand, the IF expression of

ated with an increased expression of p53 mRNA andFas-L was significantly up-regulated in the CsA-treatedprotein. Accumulation of p53 is sometimes required forgroups when compared with VH (Fig. 9) at both 7 andthe G1 cell cycle checkpoint after DNA damage, and its28 days (P , 0.001). Most of the changes were seenup-regulation in the case of DNA damage is often relatedin the tubulointerstitium and vascular compartments ofto apoptosis [29–31]. Changes in the p53 level were pre-CsA-treated kidneys as expected from the mostly inter-viously reported in an experimental model of progressivestitial lesion of chronic CsA nephropathy.tubulointerstitial fibrosis [46] where apoptosis is also

Interleukin-converting enzyme mRNA expression and known to occur [18]. Additionally, we found that CsAcaspase-3 enzymatic activity treatment resulted in elevated mRNA and protein expres-

sions of the death promoter gene bax, while the proteinIn the cortex, the mRNA expression of ICE becamesignificant in CsA-treated kidneys only at 28 days, al- expression of the survival gene bcl-2 was down-regulated.


Fig. 8. Immunofluorescence photomicrographs and quantitation of scoring for Bcl-2. Kidney sections from CsA-treated rats for 28 days (a) andfor VH control for 7 days (b) and for 28 days (c) were stained with an antibody to Bcl-2. The photographs were taken under identical conditionswith equal exposures of 60 seconds (magnification 3250). IF scoring for Bcl-2 is also shown (d). N 5 6 for each group. *P , 0.05 and **P , 0.01compared with VH controls.

In some cells, Bax can be expressed in a p53-dependent This ability may relate to antioxidant or other cytopro-tective properties of the Bcl-2 protein, which resides inmanner by radiation, chemotherapeutic drugs, and other

genotoxic stressors [30, 32]. In our study, the expression the nuclear and mitochondrial membranes [48, 49]. Thedeath-repressing activity of Bcl-2 may be counteractedof Bax and Bcl-2 remained diametrically opposite in all

treatment groups; although CsA treatment favored Bax by dimerization with Bax [34], suggesting that the rela-tive levels of these two proteins may act as some formexpression, VH treatment favored that of Bcl-2. Because

a change in the expressions of Bax and Bcl-2 is indicative of “rheostat” governing cell survival and death.The 45 kDa cell surface receptor Fas (APO-1/CD95)of p53 activation, our study suggests that the regulation

of Bcl-2 and Bax is under the influence of p53 and that and its ligand are important mediators of apoptosis [36].Their involvement in apoptosis is mostly seen in acti-the sensitivity of CsA-treated renal cells to undergo apo-

ptosis was increased. This mechanism is also operating vated T cells but also can be observed outside the im-mune system [36]. Our study suggests that the Fas systemin mesangial and tubular epithelial cells [46, 47]. In addi-

tion, a large body of data from gene transfection experi- is implicated in CsA-induced apoptosis and supports re-cent findings in the LLC-PK1 renal proximal tubular cellments has shown that Bcl-2 can protect cells from a

variety of stimuli inducing apoptosis, such as growth line where CsA induced Fas/Fas-L expression [50]. TheFas system was also implicated in apoptosis in mousefactor deprivation and DNA-damaging drugs [4, 33, 48].


Fig. 9. Immunofluorescence photomicrographs and quantitation of scoring for Fas-L. Kidney sections from CsA-treated rats for 7 days (a) andfor 28 days (b) and VH control (c) were stained with an antibody to Fas-L. The photographs were taken under identical conditions, with equalexposures of 60 seconds (magnification 3250). IF scoring for Fas-L is also shown (d). N 5 6 for each group. ***P , 0.01 compared with VHcontrols.

models of ischemia-reperfusion in tubular epithelial cells caspase-3 has a distinct role in the final induction ofapoptosis. In this model, there was a definite and signifi-and CsA nephrotoxicity [51, 52]. There is also abundant

evidence that inhibition of the Fas system is important cant increase in the enzymatic activity of caspase-3.These results are supportive of recent findings in whichin mediating the immunosuppressive effects of CsA on

activated lymphocytes [53, 54]. As a result, activation of a murine tubular epithelial cell line exposed to CsAunderwent apoptosis with evidence of caspases activa-the Fas system, under the influence of CsA in this model,

could be responsible for not only apoptosis of renal cells tion; the administration of caspase inhibitors preventedthe cells from CsA-induced apoptosis [56]. The involve-with structural damage but also for some of CsA immu-

nosuppressive activities. ment of caspases in the final induction of apoptosis hasalso been identified in many cell systems [37].Active ICE and caspase-3 are oligomeric enzymes

made up of two subunits required for catalytic activity In our study, significant increases in the expression ofthe pro-apoptotic genes and the caspase system were[55]. Our study shows that ICE is involved in CsA-in-

duced apoptotic cell death. Although ICE itself has no evident as early as one week. However, histologic evi-dence of apoptosis, as assessed by the TUNEL assay,obvious role in cell death, it is a good marker of the

overall caspase activity, and it is the first identified mem- did not become apparent until 28 days. This finding con-firms that activation of the apoptosis regulatory genesber of the caspases family [37]. On the other hand,


Fig. 11. Caspase-3 activity from the cortex of rat kidneys. Protein ex-tracts were prepared from tissue homogenates as described in the text.N 5 6 for each group. **P , 0.01; and ***P , 0.001 compared withVH control.

Fig. 10. Quantitation of mRNA expression of ICE in CsA and VH-treated rats relative to GAPDH. Total RNA was isolated from wholecortex and medulla at days 7 and 28 and was hybridized with a cDNAprobe to ICE. N 5 6 for each group. ***P , 0.001 compared with VH activities of p53, Bax, and Fas-L and decreased Bcl-2control. expression in this model. The caspase system was also

activated. These findings suggest that apoptotic cell deathplays an important role in the pathogenesis of chronicCsA nephrotoxicity; apoptosis can also contribute to CsA-and the caspase system preceded DNA fragmentationimmune modulatory effects. To the extent that higherand cell death.CsA levels are needed in this model, more work is neededOur model of chronic CsA nephrotoxicity is character-to characterize the apoptosis pathway in patients treatedized by activation of the renin-angiotensin system (RAS)with CsA. It remains unclear whether the activation ofand by increased expression of transforming growth fac-the apoptosis genes is primary or secondary to injury.tor (TGF)-b1 [25–27]. In this model, RAS blockadeStudies using specific inhibitors to apoptotic genes shouldimproved chronic CsA nephrotoxicity and decreased

TGF-b1 expression [27]. There is also enough evidence help clarify their roles in CsA-induced apoptotic cell death.to suggest that both angiotensin II [57] and TGF-b1 [58],acting either independently or in concert, can induce ACKNOWLEDGMENTSapoptosis. In this setting, p53 is induced with a resultant This work was presented in parts at the Annual Meeting of thechange in Bax and Bcl-2 expressions [59]. Along those American Society of Nephrology, Philadelphia, PA, USA, in October

1998, and at the Annual Meeting of the American Society of Trans-lines, intracellular Ca11 elevation and CsA were shownplantation, Chicago, IL, USA, in May 1999, and was supported byto induce TGF-b1–mediated apoptosis synergistically in grants from the Dialysis Research Foundation (F.S.S.) and from the

lymphocytes [60]. These data suggest that angiotensin II Oregon Health Sciences University Foundation and the Clinical Re-search Group of Oregon (W.M.B. and T.F.A.).and/or TGF-b1 may play a role in CsA-induced apopto-

sis. More experiments are needed to establish this rela-Reprint requests to Fuad S. Shihab, M.D., Division of Nephrology,

tionship using specific TGF-b1 antagonists and pharma- 4R312 Medical Center, University of Utah, 50 North Medical Drive,Salt Lake City, Utah 84132, USA.cologic interventions to block the RAS.E-mail: [email protected] apoptosis is at times a desirable event, its

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