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【摘要】 Previously, we demonstrated gender differences in Na-K-ATPase (NKA) expression and function after renal ischemia-reperfusion (I/R) injury (Sex differences in the alterations of Na +, K + -ATPase following ischemia-reperfusion injury in the rat kidney. J Physiol 555: 471-480, 2004). Postischemic membrane destruction causes inhibition of NKA, whereas heat shock protein (HSP) 72 helps to preserve it. We tested the sex differences in postischemic expression of HSP72 and colocalization with NKA. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 (T2), 16 (T16), and 24 h (T24) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls. Postischemic blood urea nitrogen (BUN), serum creatinine, and renal histology were analyzed. HSP72 mRNA expression was detected by RT-PCR, protein levels by Western blot analysis. Fluorescent immunohistochemistry was performed to evaluate the localization of HSP72 and NKA 1 -subunit. Postischemic BUN and creatinine were higher, and renal histology showed more rapid progression in M vs. F ( P < 0.05). HSP72 mRNA expression was higher in F vs. M in control and in all I/R groups ( P < 0.05). Similar changes were observed in HSP72 protein levels (F vs. M, P < 0.05, control, T2, T16, T24, respectively). Immunohistochemical localization of HSP72 and NKA 1 was similar in control F and M. In postischemic F kidneys, the majority of NKA 1 and HSP72 was colocalized on the basolateral membrane of tubular cells, whereas in M prominent staining was observed in the cytosol and apical domain. This study indicates that in female kidneys the higher basal and postischemic levels of HSP72 and different colocalization with NKA might contribute to the gender differences in renal I/R injury.
【关键词】 differences expression localization following ischemiareperfusion
gender
ISCHEMIA - REPERFUSION (I/R) injury-induced acute renal failure remains a significant clinical problem with an unacceptable high mortality and still without any relevant therapy ( 6 ). I/R injury is one of the most important alloantigen-independent risk factor in transplantation, where longer periods of cold and warm ischemia can contribute to kidney graft loss ( 16 ).
I/R injury not only causes renal failure and disrupts cellular homeostasis but also induces cellular stress responses that participate in repair processes and may protect specific cellular structures against subsequent injury ( 10 ). Members of the heat shock protein (HSP) 70 chaperone family, particularly HSP72, contribute to these repair mechanisms. They act by refolding denatured proteins and restore function, limit detrimental peptide interactions, translocate proteins to physiological intracellular location, or degrade irreparably damaged proteins ( 21 ). Recently, the involvement of HSP72 has been demonstrated in the protection against I/R injury of several organs including the liver ( 3 ), heart ( 5 ), and brain ( 24 ). In the postischemic kidney, elevated HSP72 expression has been observed ( 22 ), and it has also been reported that after I/R injury decreased HSP72 synthesis results in diminished renal function and ischemic tolerance ( 13 ).
Recent studies revealed that ischemia-induced expression of HSP72 has beneficial effects on the reintegration of cytoskeletal structure and protection of renal tubular epithelial cell polarity ( 1 ), which is essential for the postischemic recovery and efficient enzyme function of Na-K-ATPase (NKA) ( 20 ). Our previous study reported that female rats have better survival rates and postischemic renal function than males ( 14 ). We also demonstrated that in female kidneys NKA stays in its physiological place, in the membrane fraction, and enzyme activity is preserved. This suggests that the enzyme is more stable and protected from the detrimental effects of postischemic injury than in males; however, the molecular mechanism still remains elusive ( 4 ).
Regarding the importance of HSP72 in the restoration of cytoskeletal anchorage after I/R injury, in this study we tested the hypothesis that different HSP72 expression and localization exist in female and male rats after I/R injury, which might explain the previously observed female/male disparity of NKA.
MATERIALS AND METHODS
Animal description and care. Experiments were performed using sexually mature female (F; weighing 270 ± 20 g) and male (M; weighing 280 ± 30 g) Wistar rats. Rats were allowed free access to standard chow and water. All experimental protocols were performed in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of the Council on Animal Care at the Semmelweis University of Budapest, Hungary.
Experimental protocol. General anesthesia was induced by intraperitoneal administration of 50 mg/kg pentobarbital sodium (Nembutal, Abbott Laboratories, Budapest, Hungary). Renal ischemia was accomplished by cross-clamping of left renal pedicles for 55 min with an atraumatic vascular clamp. Before the end of ischemic period the clamp was withdrawn, the contralateral kidney was removed, and the abdomen was closed. Uninephrectomized, sham-operated F and M rats served as controls ( n = 6/group).
F and M rats were reanesthetized, blood samples were collected from the abdominal aorta, and the kidney was removed at 2 (T2), 16 (T16), and 24 (T24) h of reperfusion ( n = 6/group). Kidney samples were immediately snap-frozen in liquid nitrogen or fixed in 4% buffered formalin (pH 7.4) for further investigations.
Measurement of blood urea nitrogen and serum creatinine levels. Blood urea nitrogen (BUN) and creatinine (CN) were photometrically determined with commercially available kits (Diagnosticum, Budapest, Hungary) on a Hitachi-712 automated spectrophotometer.
Renal histopathology. Paraffin sections of kidneys fixed in 4% neutral buffered formalin were stained with hematoxylin and eosin and periodic acid-Schiff reagent. Samples were coded and examined in a blinded fashion. Tubular damage and leukocyte infiltration were semiquantitatively evaluated on a scale [0, none (no necrotic tubules, no infiltrating cells); 1, mild (<25%, necrotic tubules, <10 cells); 2, moderate (26-50% necrotic tubules, 11-50 cells); 3, massive (51-75% necrotic tubules, 51-100 cells); 101 cells)]. Nucleus atypia, vacuolization and hyalinization in the tubular cells, and dissolving, sloughing off, and lack of tubular cells such as hyaline in the tubules were used for more detailed quantification as described previously ( 4 ).
RT-PCR. All reagents, enzymes, and isolation kits were purchased from Qiagen (Hilden, Germany). Total RNA extraction and first-strand cDNA synthesis were performed as previously described ( 4 ). PCR was performed in PCR buffer, 2 mM MgCl 2, 2 mM dNTPs, 1.5 U Ampli Taq DNA polymerase, and 0.5 µmol of the following HSP72 primers: F: 5'-cgc cgc tgt cgc tgg gtc tgg ag-3'; R: 5'-ggc ggc cct tgt gtc tgg tga tgg-3'(product length: 327 bp).
The amplification profile consisted of denaturation at 94°C for 15 s, annealing at 66°C for 15 s, and extension at 72°C for 30 s for 35 amplification cycles. PCR products were analyzed on 2.5% agarose gel stained with ethidium bromide.
The complementary DNA samples were also used to generate glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR products, and their amount was considered as internal control. GAPDH forward and reverse primers were as follows: F: 5'-ggt gaa ggt cgg agt caa cg-3'; R: 5'-ctc atc gcg ctt gcc agt g-3' (product length: 496 bp). The amplification profile was the same as by HSP72 with a modification of the annealing temperature to 55°C.
Tissue homogenization and Western blot analysis. One hundred milligrams of renal tissue were homogenized in chilled extraction buffer containing 60 mM HEPES, 1 mM EDTA, 1 mM EGTA, 100 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.75 mg/l leupeptin, and 0.1 mM DTT, using a Potter Elvehjem homogenizer. The total tissue homogenate was centrifuged at 680 g for 5 min at 4°C to pellet cell nuclei and large fragments.
Protein determinations were performed in duplicate by Bradford analysis using bovine serum albumin as a standard. All reagents for PAGE and Western blotting were purchased from Sigma (Budapest, Hungary).
Samples were solubilized in a buffer of 12.5 mM Tris·HCl, pH 6.7, containing 4.0% SDS, 1 mM EDTA, 15% glycerol, and 0.01% bromophenol blue. Samples (40 µg protein) were electrophoretically resolved on a 12.5% polyacrylamide gel and transferred to nitrocellulose membranes blocked in 5% nonfat dry milk (NFDM) with 0.1% Tween 20 for 1 h at room temperature. The membranes were incubated with monoclonal antibodies to HSP72 (donated by Dr. L. Laszlo, Eötvös University, Budapest, Hungary) ( 9 ) diluted to 1:10,000 in 5% NFDM for 1 h at room temperature. The membranes were washed with Tween-phosphate-buffered saline and incubated in peroxidase-conjugated goat anti-mouse IgG secondary antibody (Sigma) diluted to 1:5,000 in 5% NFDM for 30 min at room temperature.
Blots were developed with enhanced chemiluminescence Western blotting detection (AP-Biotech, Buckinghamshire, UK). Computerized densitometry of the specific bands were analyzed with Gel-Pro Analyzer 3.1 software. The values were normalized to an internal standard and expressed as the relative optical density (OD).
Double labeling using immunofluorescence microscopy. Preembedded tissue sections were cut to 5 µm and dewaxed with xylol and ethanol. After rinsing in PBS, they were treated in methanolic H 2 O 2 for 30 min. Tissue sections were permeabilized in 0.2% Triton X-100 in PBS and then blocked in 0.5% NFDM for 30 min at room temperature. The samples were incubated overnight at 4°C with primary antibodies (same antibodies as in Western blot: NKA -1 diluted to 1:100; HSP72 diluted 1:500), washed with PBS, and then incubated with secondary antibodies (Alexa Fluor 488, Alexa Fluor 546, Invitrogen, Budapest, Hungary) for 30 min at room temperature. DNA was stained with Hoechst 33342 (Sigma) for 10 min at room temperature. Appropriate controls were performed omitting the primary antibodies to ensure their specificity and to avoid autofluorescence.
Statistical analysis. The data were analyzed using STATISTICA.6 software (StatSoft). For power analysis we used the G* Power program. Data are presented as means ± SD and were tested for normal distribution with a Kolmogorov-Smirnov test. Comparisons were evaluated using ANOVA (for parametric data) followed by a Fisher correction or Kruskal-Wallis ANOVA on ranks. Histological changes were analyzed with a Kruskal-Wallis test followed by multiple pairwise comparisons according to Dunn's test. Criterion for significance was P < 0.05 in all experiments.
RESULTS
BUN and serum CN levels increases after I/R injury. BUN and serum CN levels are shown in Fig. 1. Gender difference was observed in both parameters at T2, T16, and T24 of reperfusion (F vs. M; P < 0.05). Serum BUN and also CN levels were continuously increasing during the reperfusion period in F and M rats, respectively, which refers to acute renal failure due to the ischemic insult ( P < 0.05).
Fig. 1. Serum blood urea nitrogen (BUN) and creatinine levels after renal ischemia-reperfusion injury in female (F) and male (M) rats. BUN level was determined in serum samples from control and at 2, 16, 24 h (T2, T16, and T24, respectively) of reperfusion following 55 min of renal ischemia in F and M rats ( n = 6/ group)., F;, M. Values are means ± SD. * P < 0.05 vs. M. + P < 0.05 vs. T2. P < 0.05 vs. T16. x P < 0.05 vs. T24.
Renal histopathological changes after I/R injury. Kidneys from control F and M rats had no apparent morphological changes ( Table 1 ). After the ischemic insult, there was a continuous progression in the extent of tubular necrosis, tubular dissolving, and peritubular leukocyte infiltration until T24 in both genders ( P < 0.05 control vs. T2, T16, and T24 in F and M rats, respectively); however, the postischemic renal tissue damage (tubular necrosis, dissolving, and leukocyte infiltration) after T2 progressed more rapidly in M vs. F rats ( P < 0.05, F vs. M at T16 and T24, respectively).
Table 1. Histological evaluation of kidney damage in tissue samples from control and at 2, 16, and 24 h of reperfusion following 55 min of renal ischemia in female and male rats
There were no significant differences with regard to the other histological parameters investigated. None of the kidney samples presented glomerular or relevant interstitial changes in any of the groups.
Gender differences in HSP72 mRNA expression after I/R injury. Figure 2 shows mRNA expression of HSP72 and GAPDH (as an internal control) as detected in control, T2, T16, and T24 kidneys of F and M animals. HSP72 mRNA expression was higher in F vs. M already in control and also at every investigated time point, i.e., T2, T16, and T24, respectively ( P < 0.05). While in F rats the expression of HSP72 mRNA promptly increased after the ischemic insult and reached its maximum level already at T2 (control vs. T2, T16, and T24, P < 0.05), in M rats there was a slower and protracted increment. In M rats, the HSP72 mRNA level was higher vs. control only at T24 ( P < 0.05).
Fig. 2. Effect of renal ischemia-reperfusion injury on mRNA expression of heat shock protein (HSP) 72 in F and M rat kidney. mRNA expression of HSP72 was determined in kidney samples from control and at T2, T16, and T24 of reperfusion following 55 min of renal ischemia in F and M rats ( n = 6/group). Top : representative examples of RT-PCR analysis of HSP72 and GAPDH in kidney. Results are given as the ratio of intensity of HSP72 mRNA to GAPDH mRNA., F;, M. Values are means ± SD. * P < 0.05 vs. M. + P < 0.05 vs. T2. P < 0.05 vs. T16. x P < 0.05 vs. T24.
HSP72 protein levels are higher in F kidneys after I/R injury. The changes in HSP72 protein levels followed those of mRNA expression, as demonstrated in Fig. 3. The HSP72 protein level was significantly higher in F vs. M rats in the control, T2, T16, and T24 groups, respectively ( P < 0.05). In F rats, the HSP72 protein level increased quickly after the ischemic insult; at T2 it was 1.5-fold higher vs. control ( P < 0.05). On the contrary, in M animals HSP72 increased much more slowly and it reached its maximum level only at T16, when it was still significantly lower compared with F (control vs. T16, P < 0.05).
Fig. 3. Effect of renal ischemia-reperfusion injury on protein expression of HSP72 in F and M rat kidney. Protein expression of HSP72 was determined in kidney samples from control and at T2, T16, and T24 of reperfusion following 55 min of renal ischemia in F and M rats ( n = 6/group). Top : representative examples of Western blot analysis of HSP72 and GAPDH in kidney. Results for HSP72 protein levels in tissue samples were normalized to an internal standard., F;, M; OD, optical density. Values are means ± SD. * P < 0.05 vs. M. + P < 0.05 vs. T2. P < 0.05 vs. T16. x P < 0.05 vs. T24.
Immunolocalization of HSP72 and NKA 1 -subunit. To investigate a potential relationship between HSP72 and NKA, fluorescent immunohistochemistry was used ( Fig. 4.). In the samples of control F ( Fig. 4 A ) and M ( Fig. 4 B ) rats, HSP72 and NKA were colocalized on the basolateral membrane domain of proximal tubular cells, and only a minor appearance was detected in the cytosol or in the apical domain. No gender difference was observed at this time point. Postischemic F rats ( Fig. 4 C ) still had more pronounced HSP72 and NKA expression on the basolateral membrane, although NKA became more prominent in the cytosol compared with control F rats. On the contrary, in postischemic M rats most of both proteins appeared in the cytosol and on the apical membrane domain of tubular cells ( Fig. 4 D ).
Fig. 4. Immunohistochemical localization of HSP72 and Na-K-ATPase (NKA) 1 -subunit in F and M rat kidney after renal ischemia-reperfusion injury. Shown are representative examples of colocalization (yellow) of HSP72 (red) and NKA 1 -subunit (green) in kidney sections of control F ( A ) and M ( B ) and postischemic F ( C ) and M ( D ) rats. Nuclei are stained with blue. Fluorescence signal intensities of HSP72 (red) and NKA 1 -subunit (green) generated from a scanned horizontal line shown as a red arrow in the merged image are shown in the top right of each panel.
DISCUSSION
Renal I/R injury induces the time-dependent translocation of NKA from the basolateral into the apical membrane domain and into the cytosol of proximal tubular cells, which results in the temporary inhibition of enzyme activity ( 12 ). Previously, we have shown that the I/R-induced translocation of the NKA 1 -subunit is less pronounced in F rats than in M. In parallel with this, the enzyme was also more active in F rats, which could possibly contribute to the gender differences in postischemic survival ( 14 ) and renal recovery ( 4 ).
Recent data suggest that the protein stabilization by HSP72 results in improved binding and stabilization of the cytoskeletal anchorage of NKA ( 1, 2 ). In vitro studies also revealed that the overexpression of HSP72 is associated with decreased detachment of NKA from the cytoskeleton following ischemic injury ( 17 ). Based on our and others' previous results, here we hypothesized that the different expression and localization of HSP72 might also be responsible for gender disparity in NKA localization after the ischemic insult.
This experiment demonstrates that F rats have higher basal and postischemic renal HSP72 expression than M. We showed that already under physiological circumstances the mRNA and protein expression of HSP72 are higher in F compared with M animals. Previous studies revealed that tissues expressing more HSP72 are more resistant to cellular stress ( 25 ). Our data also confirmed the results of Voss et al. ( 23 ), who detected 40% higher HSP72 levels in the kidney and heart of F rats; however, they studied only protein expression and not mRNA.
It has also been shown that ovariectomy reduces ( 23 ), treatment with 17- estradiol or progesterone upregulates ( 8 ), whereas 5- -dihydrotestosterone has no effect on the level of HSP72 in different tissues ( 15 ). All of these observations, in line with our results, suggest that the higher level of HSP72 in F rats indicates improved tolerance and resistance against ischemic injury. While the mechanism by which estrogen exerts this effect remains to be elucidated, it is interesting to hypothesize that M rats cope with some forms of cellular stress by synthesizing HSPs, whereas F animals are protected by their naturally higher levels of circulating estrogen.
After the ischemic insult, we detected an abrupt and significant increase in the HSP72 mRNA expression in F rats, whereas in M rats the increase was far smaller and slower. The mRNA increase induced by ischemia was similar in F rats to the previously observed changes after administration of ethanol, a specific inductor of HSP72 ( 11 ). Besides quantitative differences, we also detected marked gender differences in the dynamic of changes in postischemic mRNA expression of HSP72. Whereas in F rats we found higher HSP72 mRNA expression already at T2 compared with control, in M rats the HSP72 mRNA expression increased much more slowly, protractedly, and reached its maximum level later. HSP72 protein levels correlated well with HSP72 mRNA expression, being higher in F compared with M rats, even in control, and later, irrespective of the investigated time points.
After an ischemic insult, the synthesis of most proteins is inhibited, despite that of HSP72, whose mRNA and protein expression measurably (15-25%) and quickly increases ( 21 ). HSP72 synthesis is regulated by heat shock factor (HSF)-1, which is phosphorylated during stress and binds to the promoter of the HSP72 gene and thereby induces HPS72 synthesis ( 18 ). It has also been shown that 17-beta estradiol or progesterone treatment activates HSF-1 and increases HSP72 expression in rat cardiac myocytes ( 8 ). Based on these data, it can be postulated that after the ischemic insult estrogen also activates HSF-1 phosphorylation and HSP72 synthesis in the kidney, which could explain our results: the rapid increase in HSP72 mRNA and protein levels in F animals. This extensive increment in HSP72 expression further helps the restoration of protein and membrane structures.
Tubular epithelial cells are the primary targets of renal ischemic injury. I/R injury is characterized by tubular necrosis and apoptosis, extracellular matrix degradation, and infiltration of monocytes or macrophages. Immunohistochemical studies revealed that renal tubular epithelial cells are the main source of HSP72 after ischemic insult of the kidney ( 7 ). An increasing body of indirect evidence suggests that interaction between HSP72 and NKA occurs in both in vitro and in vivo renal ischemic injury. Van Why et al. ( 19 ) detected a similar localization and granulation pattern between HSP72 and the NKA 1 -subunit in postischemic kidneys. Ischemic preconditioning with HSP72 overexpression prevented NKA 1 -subunit dissociation after repeated ischemic insult ( 1 ). Very recently, a direct interaction has been approved between HSP72 and damaged or displaced NKA 1 in an in vitro model of renal ischemic injury ( 17 ). Although these studies further support the concept that HSP72 and NKA have important interactions, no one has as yet investigated the gender differences in colocalization of these proteins after renal I/R injury.
In the present study, we have shown a marked sexual dimorphism in the postischemic pattern of HSP72 and the NKA 1 -subunit ( Fig. 4 ). We found that in control nonischemic F and M rats HSP72 and NKA were colocalized on the basolateral membrane of proximal tubular cells. In F rats, the basolateral staining was still pronounced in the postischemic kidney, whereas in M rats this polar distribution was observed in neither HSP72 nor NKA staining. Both of these proteins were stained in the cytosol and on the apical membrane. The colocalization staining of HSP72 and NKA provides additional in vivo evidence for the postischemic interaction of these two proteins. These results further support our previous observation that in F rats after I/R injury the majority of NKA remained in a membrane-bound form, which represents the basolateral membrane-associated NKA molecules. To the contrary, a marked dislocation was detected in the supernatant fraction in M rats, representing NKA molecules elsewhere in cells ( 4 ).
Regarding the direct interaction between HSP72 and NKA ( 17 ) and the gender differences in the expression of HSP72, one can postulate that the higher levels of HSP72 in F rats contribute to the postischemic restoration of membrane and protein structure of proximal tubular cells. The costaining of HSP72 and NKA on the basolateral membrane of F postischemic kidneys suggests that HSP72 might directly help to prevent postischemic damage to NKA and to preserve enzyme function.
The present study demonstrates that 1 ) F rats have higher basal and postischemic mRNA and protein levels of HSP72 than M rats and 2 ) the dynamic of postischemic HSP72 expression differs between genders. 3 ) Furthermore, this is the first animal model that shows the different postischemic localization and colocalization of HSP72 and NKA between genders. The interaction and the different localization of HSP72 and NKA further support the hypothesis, that HSP72, as a molecular chaperone, involved in cytoskeletal and membrane stabilization, might have a relevant role in the different NKA activity and in the susceptibility to renal I/R injury between genders.
GRANTS
This work was supported by OTKA Grants F042563 -F048842-T37578 and ETT 208-243/2003 and by the National Office for Research and Technology (NKTH), Szentágothai János Knowledge Centre. A. Fekete, V. Müller, and A. J. Szabó are recipients of Bolyai Scholarships.
ACKNOWLEDGMENTS
We are grateful to Prof. Heymut Omran (Uni-Kinderklinik, Freiburg, Germany) for kind help in establishing immunofluorescence staining and to Mária Bernáth for technical assistance.
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作者单位：1 Research Group for Pediatrics and Nephrology, Hungarian Academy of Sciences, 2 1st Department of Pediatrics, 4 Institute of Medical Chemistry, Molecular Biology, and Pathobiochemistry, and 5 Department of Pulmonology, Semmelweis University, and 3 Szentágothai Knowledge Center, Budapest, Hun