Departments of 1Internal Medicine and 2 Pathology, Korea University Hospital, and 3 The Institute of Renal Disease, Seoul, Korea
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. After unilateral nephrectomy in female SpragueDawley rats, the renal artery of the contralateral kidney was clamped for 40 min and reperfused. -MSH or vehicle was injected intraperitoneally immediately after reperfusion and at 1, 6, or 24 h after reperfusion. The expression of Fas and Fas ligand was studied by western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick-end labelling (TUNEL) method, and neutrophil infiltration by naphthol AS-D chloracetate staining. The degree of apoptosis, neutrophil infiltration, and Fas and Fas ligand, and ICAM-1 expression, as well as biochemical and histological data were compared between the
-MSH and the vehicle-treated groups.
Results. Intraperitoneally administered -MSH significantly reduced renal injury, measured by blood urea nitrogen (BUN) and creatinine and by the degree of tubular necrosis (109.6±7.1/54.7±3.1 mg/dl for BUN, and 1.6±0.2/1.03±0.06 mg/dl for creatinine 24 h after ischaemia) (5.4±0.8/2.6±0.3 for injury score 24 h after ischaemia). Ischaemia caused an increase in Fas and Fas ligand expression and was accompanied by morphological evidence of apoptosis.
-MSH significantly reduced the degree of apoptosis, as well as Fas and Fas ligand expression (mean apoptotic cell number, 41.7±3.5/14.2±2.2 per x200 field at 24 h after ischaemia. Fas protein expression: sham, 1409±159 DI (densitometric index); vehicle/
-MSH, 2818±635/1306±321 DI at 24 h and 5542±799/2867±455 DI at 72 h after ischaemia. Fas ligand protein expression: sham, 1221±181 DI; vehicle/
-MSH, 2590±85/1279±169 DI at 4 h, 4376±268/2432±369 DI at 24 h and 5200±648/2253.7±1104 DI at 72 h after ischaemia). Neutrophil infiltration and ICAM-1 expression were also significantly reduced in
-MSH group (neutrophil infiltration: vehicle/
-MSH, 5.05±1.8/1.59±0.4) (ICAM-1 expression, vehicle/
-MSH 0.46±0.21/0.29±0.19).
Conclusion. These results suggest that apoptosis clearly contributes to tubular cell loss in ischaemia/reperfusion (I/R) injury possibly by neutrophil-mediated pathways or an increase in FasFas ligand expression. The observed beneficial effect of -MSH could be related to these mechanisms.
Keywords: acute renal failure; apoptosis; ICAM-1; -MSH; Fas; neutrophil
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis, a particular form of cell death, has been frequently observed in kidney ischaemia/reperfusion (I/R) injury animal model and human ATN [3,4] and is thought to be an important mechanism of renal dysfunction in ARF. During the last decade, there has been much progress in understanding the role of apoptosis and also its triggering factors. Relative deficiencies in survival signals, various cytotoxic stimuli like reactive oxygen species, nitric oxide (NO) and receptor-mediated mechanisms can trigger apoptosis in the setting of ARF [5].
Fas, a 45-kDa transmembrane glycoprotein, is the best characterized receptor that can trigger apoptosis in various cells [6,7], and is also expressed in renal tubular epithelial cells [8]. The ligand for Fas belongs to the tumour necrosis factor (TNF) family and the FasFas ligand system plays an important role in B and T lymphocyte development and maturation and in T cell cytotoxicity [9]. Recently Lorz et al. [10] demonstrated that Fas ligand is also expressed in normal renal tubular epithelial cells, suggesting that FasFas ligand system may have some role in renal tubular cell biology. Although evidence for the involvement of this receptor mediated apoptosis in ARF is still lacking, recent reports by Feldenberg et al. [11] suggested that a Fas-mediated pathway may play a critical role in ischaemia, by demonstrating that increased apoptosis of MDCK cells was accompanied by increased Fas protein expression with partial ATP depletion. In addition, Ortiz-Arduan et al. [12] reported that lipopolysaccharide (LPS) can induce Fas and Fas ligand transcripts in cultured murine renal cells and suggested that they may play some role in endotoxaemia-induced ARF. These results suggest that FasFas ligand interaction-induced apoptosis can be a major contributing factor to apoptosis in ARF.
-Melanocyte stimulating hormone (
-MSH) is a pro-opiomelanocortin derivative and is an endogenous cytokine that suppresses the inflammation in various animal models by way of its inhibitory action on proinflammatory cytokines and chemo-attractive chemokines [13,14]. Recently Chiao et al. [15] reported the beneficial effects of
-MSH in ischaemic ARF rat models. The mechanisms of action of
-MSH in I/R injury are thought to be its inhibitory effect on neutrophil accumulation by down-regulating neutrophil chemokine and adhesion molecules like ICAM-1, as well as on maladaptive cytotoxic response during reperfusion period mediated by excessive production of NO [15,16].
Because activated neutrophils release various enzymes, inflammatory cytokines, and cytotoxic reactive oxygen species, and because these are also well-known apoptosis-inducing factors, we hypothesized that beneficial effects of -MSH could be partly due to its possible inhibitory effect on apoptotic cell death.
In this study we examined the effects of -MSH on renal injury and also on the occurrence of apoptosis in a model of ischaemic ARF. The degree of renal damage was assessed by biochemical and histological studies and apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick-end labelling (TUNEL) method. ICAM-1 expression and neutrophil infiltration into injured tissue were also examined. Finally, to clarify the effect of
-MSH on the Fas system, we examined Fas and Fas ligand expression in our rat model of ischaemic ARF.
![]() |
Subjects and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biochemical analysis
BUN and plasma creatinine levels were evaluated using Hitachi 747 automatic analyser.
RNA extraction and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) for Fas
Renal tissue was finely minced with a razor blade on ice and then homogenized in Trizol reagent (Gibco BRL, Grand Island, NY, USA). RNA extraction was performed according to the manufacturer's protocol. After resuspension in a Tris-EDTA buffer, RNA concentrations were determined using spectrophotometric readings at absorbance 260 nm. One microgram of RNA was reverse transcribed at 42°C for 60 min in the presence of 5x first-strand buffer, 10 mmol/l dNTP, 20 U Rnasin, and 500 U of Moloney murine leukaemia virus reverse transcriptase (Superscripts, Gibco BRL) in a 25-µl reaction volume. First-strand cDNA (1 µl) was amplified using 2.5 U Taq polymerase (Perkin Elmer, Foster city, CA, USA) in a 50-µl reaction volume containing 0.4 µmol/l primer pair, 200 µmol/l dNTP, 10 mmol/l TrisHCl, 1.5 mmol/l MgCl2, and 50 mmol/l KCl. The sequences of primer for rat Fas were: sense primer, 5'-GACCCAGAATACCAAGTGCA-3', antisense primer, 5'-TGTGTTCTGCTGTGTCTTGG-3' and for rat ribosomal protein L-19, as an internal standard, sense primer, 5'-AGCCTGTGACTGTCCATTCC-3' and antisense primer, 5'-TTGGTCTTAGACCTGCGAGC-3'. The amplifying conditions were 36 cycles of the following: denaturation for 60 s at 94°C, annealing for 60 s at 58°C, and extension for 60 s at 72°C. The PCR products were analysed in a 1% agarose gel stained with ethidium bromide and band densities were estimated using a Digital Imaging & Analysis System (Alpha Innotech Corp., San Leandro, CA, USA). The ratio of Fas PCR product to L-19 were compared.
Northern blot analysis
Samples of total RNA (40 µg) were fractionated by electrophoresis on 1% agaroseformaldehyde gel and transferred to a nylon membrane. The equality of RNA samples was substantiated by UV illumination of ethidium bromide. After fixation in a UV cross-linker, membranes were pre-hybridized at 43°C in a Prehyb/Hyb buffer (Ambion, Austin, Tx, USA) for 3 h and then hybridized with [32P]dCTP labelled cDNA clones. The hybridized membranes were washed twice in 1xSSC and 0.1% SDS at 43°C, and subjected to autoradiography at -70°C. The band intensities were compared using the Digital Imaging & Analysis System (Alpha Innotech Corp., San Leandro, CA, USA). Loading of RNA was normalized by rehybridizing with L-19. 0.4 kB portions of rat ICAM-1 and as an internal standard, 0.35 kB rat L-19 cDNA were generated by PCR and cloning (TA cloning kit, Invitrogen, San Diego, CA, USA) and cloned products were confirmed by sequencing.
Western blot analysis
Rat kidney tissue was minced in the presence of 50 mmol/l Tris, 0.1% NP-40, 0.2 mol/l NaCl, 1 mmol/l EDTA, 50 µg/ml PMSF, 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin. The resultant pellet was subjected to homogenization, and the supernatant was collected and saved. The protein content was measured by absorbance at 540 nm using a BCA protein assay kit (Pierce, Rockford, IL, USA). The samples were initially analysed by 10% SDSpolyacrylamide gel electrophoresis and Coomassie stained. The aliquots of each sample containing 25 µg of protein were resolved using a 10% SDSpolyacrylamide gel and transferred to polyvinylidine difluoride (PVDF) membrane. After incubation in a blocking solution (5% non-fat dry milk in PBST (0.05% Tween 20 in PBS)) at room temperature for 1 h, the membranes were subjected to repeated washing in PBST (4x10 min) and then incubated with primary antibodies in blocking solution at 4°C overnight. After repeated washing, membranes were reacted with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. The primary antibodies used were: (i) 1 : 1000 goat anti-Fas IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and (ii) 1 : 2000 mouse anti Fas ligand IgG (Transduction Laboratories, Lexington, KY, USA). After washing in PBST (8x10 min), the membranes were visualized using enhanced chemiluminescence (ECL: Amersham, Arlington Heights, IL, USA) and exposed to Kodak XAR5 film. The band intensities were compared using the Digital Imaging & Analysis System (Alpha Innotech Corp., San Leandro, CA, USA).
Histological examination
Ten per cent formalin-fixed and paraffin-embedded kidney tissues were stained with haematoxylin and eosin (H&E, 3 µmol/l), periodic acidSchiff (PAS) and naphthol AS-D chloracetate esterase (Sigma Chemical Co., St Louis, MO, USA). The severities of renal injury were quantified using histological scoring system that we developed (Table 1). The number of tubular cell necrosis, intratubular cast formation, apoptosis, and brush-border changes in the outer medulla were examined in 20 randomly selected 200x field sections and according to the scoring system, a mean of the total score was compared between the groups. Neutrophil infiltration was also quantitatively measured by counting 20 randomly selected x400 field sections in the outer medulla, and the mean number of infiltrated cells was compared between the groups.
|
In-situ detection of DNA strand breaks
To identify nuclei with DNA strand breaks, the TUNEL method using Apop tagTM (Oncor Inc., Gaithersburg, MD, USA) was used. Briefly, paraffin-embedded sections were deparaffinized in xylene for 5 min and rehydrated through graded concentrations of ethanol. After washing with PBS twice for 5 min, the sections were treated with 1.0 µg/ml proteinase K in PBS at 37°C for 15 min and washed with deionized water for 10 min. To inactivate endogenous peroxidase, the tissue sections were incubated in 2% H2O2 at 37°C for 15 min and then rinsed with deionized water for 10 min. The slides were then incubated with a TdT buffer (25 mmol/l TrisHCl buffer, pH 6.6, 0.2 mol/l potassium cacodylate, and 0.25 mg/ml BSA) at room temperature for 30 min, and after the incubation the slides were reacted with 0.1 U/µl TdT dissolved in a TdT buffer supplemented with 1.0 nmol/l digoxigenindUTP in a humid chamber at 37°C for 1 h. The signals were detected immunohistochemically with a horseradish peroxidase-conjugated sheep anti-Dig antibody. Quantitative measurement of apoptotic cells was done by examining the 10 randomly selected x200 fields in the outer medulla and counting the mean number of apoptotic cells per field.
Statistical analysis
Results were presented as means±SEM. Comparison between the ischaemia/vehicle group and ischaemia/-MSH group was done using an independent sample t-test and a P value <0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Histology
Kidneys were processed for histological examination 24 and 72 h after reperfusion, and in the vehicle-treated ischaemia group the most severe and pronounced injuries were seen in the outer stripes of outer medulla at 24 h after reperfusion, with typical acute tubular necrosis pattern; namely, widespread tubular-cell necrosis, intratubular cast formation and flattening of brush borders. However, in the -MSH treated group, these areas of tubular necrosis and tubule obstruction with casts were more focal and mild (Figure 2
). In the histological grading system, the mean score of injury in the vehicle group 24 h after reperfusion was 5.4±0.8 and in the
-MSH group, 2.6±0.3 (P=0.008) (Figure 2
).
|
|
Detection of apoptosis
TUNEL-positive apoptotic cells were observed as early as 4 h after reperfusion and the number of apoptotic cells peaked 24 h after renal ischaemia. There were more apoptotic cells in the outer medulla than in the cortex and most apoptotic cells were detached from tubular basement membrane and located in the tubular lumen (Figure 4). In the
-MSH group, the mean number of apoptotic cells in the outer medulla 24 h after reperfusion decreased significantly compared with the vehicle group (41.7±3.5/14.2±2.2 per x200 field) (P=0.004) (Figure 4
).
|
Effects of -MSH on ICAM-1 mRNA expression
ICAM-1 mRNA expression showed a decrease in the -MSH group at 24 h reperfusion (0.46±0.21/0.29±0.19) and significantly decreased at 72 h reperfusion (0.49±0.01 and 0.31±0.17) (P=0.11 and P=0.04 respectively) (Figure 5
).
|
Fas mRNA expression in ischaemia/reperfusion injury and effects of -MSH on Fas expression
Fas mRNA expression was examined using a semi-quantitative RT-PCR technique. Fas/L-19 ratio increased from baseline values of 0.86±0.2 in the sham-operated group to 1.45±0.4 and 1.14±0.2 at 4 and 24 h after reperfusion. -MSH decreased Fas/ L-19 ratio to 1.18±0.1 and 0.62±0.1 respectively (Figure 6
).
|
Fas and Fas ligand protein expression in ischaemia/reperfusion injury and effects of -MSH on Fas and Fas ligand protein expression
Fas protein expression was detectable at low levels in the sham-operated group (1409±159 DI) and increased in the vehicle group to 2818±635 DI at 24 h, and further increased to 5542±799 DI at 72 h after reperfusion. -MSH markedly inhibited Fas protein expressions to 1306±321 DI and 2867±455 DI at 24 and 72 h respectively (P=0.07 and 0.047) (Figure 7
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Feldenberg et al. [11] reported that partial ATP depletion induced apoptosis in MDCK cells and it was accompanied by Fas upregulation. Nogae et al. [3] also suggested possible involvement of Fas in ischaemia/reperfusion models of mouse kidneys by demonstrating increased Fas mRNA expression.
Fas ligand, a 40-kDa type II transmembrane protein, was thought to be primarily restricted to immune privileged sites, but is reported to be constitutively expressed in kidney tubular cells and upregulated upon TNF- stimulation, with a resultant increase in apoptosis [10,12,19]. Ortiz-Arduan et al. [12] demonstrated increased Fas and Fas ligand expression accompanied by morphological evidence of apoptosis in LPS-stimulated renal cells as well as in endotoxaemia rat models induced by LPS injection. All these results suggest that Fas and Fas ligand upregulation-induced apoptosis can be an another pathogenetic mechanism of renal dysfunction in ischaemic ARF. However, Fas dependent apoptosis has not always been demonstrated evenly in all experiments, Boonstra et al. [8] reported that despite constitutive expressions of Fas on tubular epithelial cells, they appeared to be resistant to Fas-mediated apoptosis when treated with a stimulatory anti-Fas antibody. So whether Fas-mediated apoptosis of tubular cells contributes to tubular cell loss or to renal dysfunction in ARF still remains unclear.
In this study, apoptosis, confirmed by DNA fragmentation using the TUNEL method, was observed mainly in the outer medulla, the most susceptible area in ischaemia, as early as 4 h after ischaemia, peaking at 24 h. Most apoptotic cells were detached from the tubular basement membrane and found in the tubular lumen, indicating that most apoptotic cells are tubular cells rather than infiltrated inflammatory cells in the interstitium. At 72 h after ischaemia, there were few apoptotic cells, and this was accompanied by frequent mitosis of tubular cells, suggesting that in ischaemic ARF, apoptosis observed in the first 3 days after I/R injury is responsible for tubular cell loss, not regeneration activity.
Recently neutrophils recruited during reperfusion have been implicated as mediators of renal parenchymal injury, and because of their ability to release a variety of toxic materials such as oxygen free radicals and inflammatory cytokines, infiltrated neutrophils can induce tubular-cell apoptosis [20]. In this study we demonstrated an approximately 10-fold increase in neutrophil infiltration compared to the sham group. Most neutrophils were found at the peritubular space in the outer medullary area in the vehicle group compared with the sham group, and this increase in neutrophil infiltration could be associated with increased tubular-cell apoptosis.
But we also demonstrated a significant increase in renal tissue Fas mRNA and protein expression in the vehicle group, which persisted even at 72 h after reperfusion. Ischaemia also induced an increase in Fas ligand expression and this effect persisted at 24 and 72 h after reperfusion as well.
The observation that tubular cell apoptosis in ischaemic ARF was accompanied by increased Fas and Fas ligand expression suggests that FasFas ligand upregulation-induced apoptotic cell death could also play some role in renal dysfunction in ARF. This is consistent with the observations by Feldenberg et al. [11] and Ortiz-Arduan et al. [12], but the exact mechanisms of Fas and Fas ligand upregulation in ischaemic ARF remains undefined. Direct upregulation by ischaemia itself or induction by pro-inflammatory cytokines such as TNF- or interleukin-1ß (IL-1ß) produced in the reperfusion period might be responsible for them [21]. TNF-
in I/R injury can induce various adhesion molecules, like ICAM-1, E-selectin, and P-selectin on endothelial cells and continue the cascade of events that increase cell adherence, leukocyte activation, and inflammatory processes in injured tissues [22]. In addition, local production of TNF-
after ischaemic injury has been reported to be associated with DNA fragmentation, and treatment of anti-TNF monoclonal antibody reduced the degree of DNA fragmentation as well as the degree of biochemical and histological renal damage, suggesting that TNF-
is a major factor not only in inflammation but also in inducing apoptosis in ischaemic ARF [21,23]. In this study, the discrepancy between the peak occurrence of apoptosis at 24 h and that of Fas and Fas ligand expression at 72 h, when apoptosis was hardly found in this study, also favours the suggestion that increased Fas and Fas ligand expression might be just an epiphenomenon secondary to increased inflammatory cytokine, like TNF-
, playing a relatively minor role in apoptosis of tubular cells in I/R injury.
The observation by Donnahoo et al. [24] that early renal tissue TNF- expression contributes to neutrophil infiltration in I/R injury also supports this, and we can suggest that neutrophil-mediated or other cytokine-mediated pathway is more important than increased Fas and Fas ligand expression in tubular cell apoptosis. However, to define the exact role of an individual pathway in inducing apoptosis, the temporal relationship between the expression of various cytokines, Fas and Fas ligand expression and apoptosis should be examined as well as the effects of antagonistic Fas monoclonal antibodies.
-MSH, an endogenous anti-inflammatory cytokine, has been known to reduce cellular infiltrations in inflammatory conditions such as various models of inflammation and liver injury from septic shock [13,14]. In LPS-induced liver inflammation,
-MSH prevented liver damage, and its suggested mechanisms of action included an inhibition of pro-inflammatory cytokine (TNF-
) and chemoattractant chemokine (KC/IL-8 or monocyte chemoattractant protein-1) gene expression, with resultant inhibition of hepatic neutrophil infiltrations, in addition to inhibition of systemic NO production [13]. Recently
-MSH has proved beneficial in ischaemic ARF [15]. Chiao et al. [15,16] demonstrated that
-MSH could attenuate I/R injury in bilateral 40-min renal artery clamping murine models through its inhibitory actions on the mouse chemokine KC and ICAM-1 message, and on the induction of iNOS with a resultant decrease in peroxynitrate production [14 16].
In this study we could also demonstrate that intraperitoneally administered -MSH inhibited the increase in the BUN and plasma creatinine levels 24 h after reperfusion and also the degree of histological damage. ICAM-1 message and polymorphonuclear cell (PMN) infiltrations were significantly reduced in
-MSH-treated groups. Additionally,
-MSH significantly decreased apoptosis in the outer medulla at 4 and 24 h after ischaemia with concomitant decreases in Fas and Fas ligand expression. The effects of
-MSH on apoptosis and the expression of Fas and Fas ligand has not been studied previously, but these data suggest that
-MSH may decrease apoptosis via at least two distinct mechanisms: (i) by inhibiting neutrophil- and inflammatory cytokine-mediated pathways, and (ii) by direct inhibitory effect on renal tissue Fas and Fas ligand expression. Although early renal tissue TNF-
and neutrophil infiltration increase tubular-cell apoptosis and the inhibitory action of
-MSH on the expression of TNF-
, neutrophil chemokines, and adhesion molecules, as demonstrated by Chiao et al. [13,15], whether
-MSH has direct inhibitory effect on Fas and Fas ligand has not been clarified; this needs in vitro experiments to exclude neutrophil and cytokine effects.
In addition, our data do not exclude the possibility that tubular-cell apoptosis occurred by Fas-independent mechanisms, and that Fas and Fas ligand up-regulation was mediating subsequent events such as the removal of damaged tubular cells. But Fas and Fas ligand up-regulation beginning as early as 4 h after reperfusion, together with apoptosis and significant inhibition of their expression, as well as apoptosis in the -MSH group, can suggest that increased Fas and Fas ligand expression may partially mediate tubular-cell losses and subsequent renal dysfunction in ARF.
Future availability of soluble Fas or recombinant Fas ligand will help in identifying their exact role in inducing apoptosis and also the effect of -MSH on apoptosis and Fas and Fas ligand expressions. Additionally, the therapeutic trial of
-MSH in conditions in which apoptotic cell death is predominant, such as drug-induced nephrotoxicity or ureteral obstruction, will be useful.
Our study suggests that tubular-cell apoptosis may play some role in renal dysfunction in ischaemic ARF, and the beneficial effects of -MSH are partially related to the inhibitory action on apoptosis. The suggested mechanisms of
-MSH in decreasing apoptosis may include its inhibitory effect on neutrophil infiltration and inflammatory cytokine production. However, its direct effect on renal tissue Fas and Fas ligand system may also be partially responsible for the beneficial effect on renal dysfunction in ischaemic ARF.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|