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Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder
Life Sciences 75 (2004) 1879 – 1886 www.elsevier.com/locate/lifescie
Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder Motoaki Saito a,b,*, Lika Tominaga c, Eiji Nanba c, Yukako Kinoshita b, Daisuke Houri b, Ikuo Miyagawa a, Keisuke Satoh b a
Department of Surgery, Division of Urology, Tottori University Faculty of Medicine, Yonago, Japan Department of Pathophysiological and Therapeutic Science, Division of Molecular Pharmacology, Tottori University Faculty of Medicine, Yonago, Japan c Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago, Japan b
Received 23 March 2004; accepted 24 May 2004
Abstract HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 F 1.3, 7.9 F 1.7, 4.3 F 0.8, 4.2 F 0.7, 4.5 F 0.6, and 8.1 F 1.2 g/mm2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder
* Corresponding author. Department of Pathophysiological and Therapeutic Science, Division of Molecular Pharmacology, Tottori University Faculty of Medicine, 36-1 Nishimachi, Yonago 683-0826, Japan. Tel.: +81-859-34-8119; fax: +81-859-348435. E-mail address: [email protected] (M. Saito). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.05.010
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were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult. D 2004 Elsevier Inc. All rights reserved. Keywords: Bladder; Ischemia-reperfusion injury; HSP 70; Real-time PCR; mRNA; Rat
Introduction The functions of the urinary bladder include urine storage and subsequent micturition. The urinary bladder requires an adequate supply of oxygen and nutrients via the circulation system in order to maintain homeostasis and proper functioning (Levin et al., 1998; Parekh et al., 2001). Ischemia and the following reperfusion of the bladder are observed in age-related disorders such as urinary retention, atherosclerosis, vasospasm, embolization, and thrombosis (Levin et al., 1998; Parekh et al., 2001). Recently, both clinical and experimental evidence of ischemia and subsequent reperfusion injury have been reported in many tissues, including the kidney, liver, stomach, and heart (Muijsers et al., 1997; Rauen et al., 1999). We and other investigators have recently reported that, after overdistension, catheterization/decompression induce reperfusion injury in the bladder and that reactive oxygen species are one of the main contributing factors in this injury (Lin et al., 2000; Saito and Miyagawa, 2001). Ischemia-reperfusion injury may cause dysfunction of the urinary bladder, which results in instability and impairment of detrusor contractility during urination (Greenland and Brading, 2001). Although the mechanism of ischemia-reperfusion injury is complicated, the main etiology of this injury is thought to involve free radicals, which attack the cell membrane, proteins, RNAs, and DNAs (Muijsers et al., 1997; Rauen et al., 1999). We have previously reported that ischemia and subsequent reperfusion significantly damages the bladder function measured by organ bath techniques and histological studies (Saito et al., 1998, 2002; Saito and Miyagawa, 1999). We demonstrated that peroxynitrite, reacting with NO and superoxide radicals, plays an important role as a cell/tissue-damaging agent during ischemia-reperfusion in the rat bladder (Saito et al., 1998; Saito and Miyagawa, 1999, 2002; Bratslavsky et al., 2003). We also reported that apoptosis plays an important role in the recovery of ischemia-reperfusion injury (Saito and Miyagawa, 2002). For the process of recovery from this injury, knowledge of bladder alteration and dysfunction after rescue of the urinary retention patients is important. Heat shock proteins (HSP), a diverse family of inducible and constitutive stress proteins, are thought to limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interactions (Hutter et al., 1994; Walter et al., 1994). In mammalian cells, the inducible 70-kDa heat shock protein (HSP 70) is the most abundant HSP and the HSP most closely linked to cytoprotection from a variety of dangerous events such as thermal injury or ischemia-reperfusion injury (Hutter et al., 1994; Walter et al., 1994). In both rats and humans, HSP 70 is encoded by two genes that differ only in the 3V-untranslated region. The fact that two genes encode identical proteins may be due to a need to express the same protein under different circumstances (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000). In this study, we attempted to investigate the expression of HSP 70-1/2 mRNAs, and HSP 70 proteins during ischemia-reperfusion in the rat bladder.
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Materials and methods Production of the animal model All animal experiments were performed in accordance with the guidelines set by the Tottori University Committee for Animal Experimentation. Male Wistar rats, 8 weeks old, were divided into six groups: controls (A), 30-min ischemia alone (B), 30-min ischemia plus 30- (C), and 60-minute (D), 1-day (E), and 1-week reperfusion (F). The urinary bladders were subjected to ischemia-reperfusion as described previously (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Briefly, under pentobarbital anesthesia (50 mg/kg, i.p.), the abdominal aorta was clamped just above its bifurcation with a small clip (Sugita standard aneurysm clip, holding force 145 g; Mizuho Ikakogyo, Tokyo) for 30 minutes in the B, C, D, E and F groups. We have previously reported that blood flow in the aorta clamped rat was approximately 10 % of that of controls (Saito and Miyagawa, 1999). Measurement of contractile response The contractile function of the bladder muscles was examined as described previously (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Just after the ischemia-reperfusion, the rat bladder dome was removed to obtain smooth muscle strips, which were mounted in 3.0-ml organ baths and maintained at 37jC in Krebs-Henseleit solution gassed with 95% O2 and 5% CO2. Isometric force was monitored with a TB-612T transducer (Nihonkoden, Tokyo) and processed by an AP-621G (Nihonkoden, Tokyo). Contractile response to carbachol was expressed as force per cross-sectional area of muscle (g/mm2) (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Cumulative doseresponse curves were constructed in a stepwise manner after the response to the previous concentration had reached a plateau. The contractile response of the same muscle strips to 100 mM KCl was also monitored in each group. Measurement of HSP mRNAs in the bladder HSP 70-1/2 mRNAs in the experimental bladder were measured by real-time PCR methods according to our previous report (Saito et al., 2004). The RNA was purified by RNeasy Mini Kit (Quiagen, Valencia, CA) according to the manufacturer’s instructions. The reverse transcriptase (RT) mixture (30 Al) containing 2 ml of total RNA was made and incubated at 37jC for 60 min by a previously reported method (Ueta et al., 2003). One ml of the mixture was used for real-time PCR, which was carried out using a LightCycler thermal cycler system with a Light Cycler SYBR Green I kit according to the manufacturer’s instructions (Roche Diagnostics, Tokyo, Japan) (Wittwer et al., 1997). The following primers were used: 5V-TTTCTGGCTCTCAGGGTGTT-3V(forward) and 5V-CTGTACACAGGGTGGCAGTG-3V(reverse) for HSP 70-1, and 5V-GCTACAAGGCGGAGGACG-3V (forward) and 5V-AGATCACACCTGGAGCGCC-3V (reverse) for HSP 70-2. A total of 10 ml of solution was used for the sample. The PCR products of HSP 70-1 and HSP 70-2 were subcloned into pGEM-T vectors, and the plasmids were amplified, purified, and diluted for the standard curve. The specificity of the reaction was confirmed by a melting curve analysis and 2% agarose gel electrophoresis. The following primers for the beta actin gene were used as the internal standard and analyzed by real-time PCR using the same RT mixture: 5V-CCTCTATGCCAACACAGT-3Vand 5V-AGCCACCAATCCACACAG-3V.
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Measurement of protein level of HSP in the bladder For biochemical studies, the rest of the bladder was minced, frozen in liquid nitrogen, and stored at 80jC until used. The protein levels of HSP-70 were measured as in our previous report (Saito et al., 2004). The expression of HSP-70 proteins was measured using a StressXpress HSP 70 ELISA kit (Stressgen Biotechnologies, Victoria, BC, Canada) according to the manufacturer’s instructions. Protein content was determined using bovine serum albumin as standard (Lowry et al., 1951). Data analysis The data for contractions were calculated as grams of active force per cross-sectional area in square millimeters. The cross-sectional area was calculated using the following equation: cross sectional area ¼ weight=ðlength 1:05Þ; where 1.05 is the assumed density of the muscle (Saito et al., 1998; Saito and Miyagawa, 1999, 2002). ED50 values (the concentration of agonist producing half-maximal contractile responses) were calculated as geometric means, whereas Emax values were calculated as arithmetic means. The expressions of HSP 70-1/-2 mRNAs were quantified according to the expression of beta actin mRNAs in the experimental rat bladder (Saito et al., 2004). The expressions of HSP 70-1/-2 mRNAs in the rat bladder were compared to those of the controls using the following ratio: Ratio of expression of HSP 70 1= 2 mRNAðGroup A FÞ ¼ HSP 70 1= 2 mRNA ðGroup A FÞ=beta actin mRNA ðGroup A FÞ The expression of HSP 70 was quantified to compare the protein concentration. Statistical comparison of differences between groups was performed using analysis of variance and Fisher’s multiple comparison tests. P < 0.05 was regarded as the level of significance. Drugs and chemicals A kit for colorimetric assay of HSP 70 was purchased from Stressgen Biotechnologies (StressXpress HSP 70 ELISA kit, Victoria, BC, Canada). All other chemicals were of reagent grade.
Results Measurement of contractile response In the functional studies, the maximal contractile force produced by carbachol (Emax values) was slightly decreased in 30 min ischemia (alone) (group B). In the 30 min ischemia plus brief reperfusion (30 min, group C), (60 min, group D) and (1day, group E) were markedly decreased. However, in the 30
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Table 1 Functional data in the experimental rat bladder dome Group
Emax g/mm2
% CONTROL
ED50 microM
100mM KClg/mm2
A B C D E F
9.3 7.9 4.3 4.2 4.8 7.8
100 84.9 46.2 45.2 51.6 83.8
1.9 2.4 4.8 5.0 4.6 2.3
7.2 6.4 3.2 3.3 3.5 6.5
F F F F F F
1.3 1.7 0.7* 0.7* 0.6* 1.1
F F F F F F
0.3 0.3 0.9* 0.8* 0.7* 0.9
F F F F F F
0.3 0.2 0.3* 0.5* 0.4* 0.6
Emax and ED50 are for carbachol. Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, B and F.
min ischemia plus 1 week reperfusion was improved the maximal contractile force (group F). Similar results were obtained by use of 100 mM KCl. The precise data were shown in Table 1. Measurement of HSP mRNAs in the bladder The data of mRNA expression in the bladder are shown in Table 2. In the control group, expression of HSP 70-1/2 mRNA was detected. Expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70-2 mRNAs showed the same manner as the expression of HSP 70-1 mRNAs after 30 min ischemia-1 day or 1 week reperfusion. Measurement of protein level of HSP70 in the bladder The data of expression of HSP 70 in the bladder are shown in Table 3. The expression of HSP 70 proteins was increased after a short period of the expression of their mRNAs (groups C and D), which was normalized in groups E and F. The level of HSP 70 after 30 min of ischemia (21.5 mg/g protein) was
Table 2 Expression of HSP 70-1/2 mRNAs in the Experimental Rat Bladder Group
HSP 70-1/ h-actin
HSP 70-2/ h-actin
A B C D E F
3.26 9.12 5.08 4.30 3.20 3.55
0.74 0.94 1.41 0.40 0.52 0.60
F F F F F F
0.57 3.15* 0.85 0.64 0.35 0.42
Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, D, E and F.
F F F F F F
0.14 0.47 0.16* 0.11 0.13 0.18
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Table 3 Expression of HSP 70 (Protein) in the Experimental Rat Bladder Dome Group
HSP 70 mg/g protein
A B C D E F
20.3 21.5 50.3 49.3 23.0 19.5
F F F F F F
1.3 0.5 0.7* 0.8* 0.6 0.4
Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, B, E and F.
similar to that in the controls. After 30 min of ischemia and 30 min of reperfusion, the levels of HSP 70 in the bladder were significantly increased (50.3 mg/g protein), and was maintained at a similar level after 30 min of ischemia and 60 min of reperfusion (49.3 mg/g protein). The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels (23.0 and 19.5 mg/g protein, respectively). Our data indicated that the protein levels of HSP 70 were increased after a short interval of increase in their mRNAs, and that decreased within one day.
Discussion In this study, we demonstrated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. In functional study, ischemia added bladder dysfunction. Additional reperfusion (30 minutes) caused even more severe dysfunction in the rat bladder response to carbachol and KCl, which were continued more than one week. In the control group, expressions of HSP 70-1/2 mRNAs were detected. Expression of HSP 70-1 mRNA increased in groups B, and decreased in groups D, E and F. Expression of HSP 70-2 mRNA increased in groups C, and decreased in groups D, E and F. The expression of HSP 70 was increased after a short interval of the expression of their mRNAs (Groups C and D). Both the HSP 70 mRNAs and proteins were normalized after 1-day reperfusion. Although bladder dysfunction measured by functional study was continued more than one week, the protein levels of HSP 70 were increased after a short interval of increase in their mRNAs, which was normalized in one day. The increased expression of HSP 70 means the existence of damaged tissue, and playing a role in recovery from stress (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999; Bidmon et al., 2000). It also may be an indicator of stressed tissue/cells. There are increasing evidences that ischemia in the bladder is present in acute/chronic urine retention (Levin et al., 1998; Parekh et al., 2001; Lin et al., 2000; Saito and Miyagawa, 2001). The bladder dysfunction seen after acute overdistension and decompression is likely to be due to ischemiareperfusion. Lin and associates have demonstrated that acute overdistension and the subsequent decompression in the bladder induce bladder dysfunction and enhance lipid peroxidation. This bladder dysfunction can be prevented by mannitol, a free-radical scavenger (Lin et al., 2000). We have also demonstrated that bladder dysfunction is induced by urinary retention and subsequent catheterization, and that bladder dysfunction following catheterization is, in part, caused by free-radicals (Saito and Miyagawa, 2001). Free-radicals caused by ischemia-reperfusion attack and damage the cell membrane,
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and induce bladder dysfunction. HSPs are thought to this limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interaction (Hunt and Morimoto, 1985). Previous reports have indicated that the most important thing in the prevention of bladder dysfunction is to reduce the production of free-radicals in the bladder (Lin et al., 2000; Saito and Miyagawa, 2001). In mammalian cells, HSP 70 is the most abundant HSP and the HSP most closely linked to cytoprotection from a variety of dangerous events, including thermal injury and ischemia-reperfusion injury (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000; Bidmon et al., 2000; Kelly et al., 2001). There is increasing evidence that ischemia-reperfusion induces HSP 70 in some organs, e.g., the kidney and heart (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999). HSPs are thought to limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interactions. In some tissues, expression of HSP 70-1 mRNA is report to be higher than that in HSP 70-2 mRNA (Akcetin et al., 1999, 2000; Saito et al., 2004). In the bladder, we also demonstrated that expression of HSP 70-1 mRNA is higher than that in HSP 70-2 mRNA in this study. In the kidney, it has been shown that there is a significant induction of both HSP 70-1/-2 genes immediately after ischemia. While HSP 70-1 mRNA expression constantly increases during reperfusion, HSP 70-2 mRNA is strongly induced (three fold) during reperfusion only after brief periods (10 min) of ischemia in the kidney (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000; Bidmon et al., 2000; Kelly et al., 2001). Akcetin et al reported that the HSP 70-2 gene was far more sensitive with a lower threshold activation and only works in a range of mild injury while the HSP 70-1 gene mediates the big response after severe injury (2000). Many reports indicate that when not induction of ischemia, but induction of reperfusion occures, HSP 70-1/2 mRNA levels are increased (Akcetin et al., 1999; Bidmon et al., 2000; Hutter et al., 1994; Kelly et al., 2001). However, in this experiment, ischemia for 30 min induced an increase of HSP 70-1 mRNA. This may be due to clamping of aorta resulted in partial ischemia (approximately 10 % of basal level) of the bladder and not complete ischemia of the organ (Saito and Miyagawa, 1999). Interestingly, we found that protein levels of HSP 70 were increased after 30 min of ischemia and 30 min of reperfusion. Our data suggest that the expression of HSP 70 proteins is increased after a short interval of the expression of their mRNAs in the bladder, and that this increased expression continues longer than that of mRNA in the bladder. These data are similar to those of the prostate (Saito et al., 2004). In the functional study, we demonstrated that the contractile responses of the rat bladder dome after 30 minutes of ischemia differed slightly, not significantly, from those of controls, and that reperfusion (30 min) produced significant reductions in the contractile responses to carbachol and KCl in the rat bladders, which continued more than one week. In contrast, expression of HSP 70 ( both mRNA and protein) increases early phase of ischemia-reperfusion injury and, is normalized within one day. Thus, our data indicate that HSP 70 and its mRNAs are increased in only early phase of ischemia-reperfusion injury in the bladder. The patho-physiological role of ischemia-reperfusion in the bladder, however, remains unclear and warrants further study.
Acknowledgements This study was supported by a grant from the Ministry of Education, Science, and Culture of Japan (#14704041).
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References Akcetin, Z., Pregla, R., Darme, D., Heynemann, H., Haerting, J., Bromme, H.-J., Holtz, J., 1999. Differential expression of heat shock protein 70-1 and 70-2 mRNA after ischemia-reperfusion injury of rat kidney. Urol. Res. 27, 306 – 311. Akcetin, Z., Pregla, R., Busch, A., Kessler, G., Heynemann, H., Holtz, J., Bromme, H.-J., 2000. Lipid peroxidation and the expressional regulation of the heat-shock response during ischemia-reperfusion of rat kidney. Urol. Int. 65, 32 – 39. Bidmon, B., Endemann, M., Muller, T., Arbeiter, K., Herkner, K., Aufricht, C., 2000. Heat shock protein-70 repairs proximal tubule structure after renal ischemia. Kid. Int. 58, 2400 – 2407. Bratslavsky, G., Kogan, B.A., Matsumoto, S., Aslan, A.R., Levin, R.M., 2003. Reperfusion injury of the rat bladder is worse than ischemia. J. Urol. 170, 2086 – 2090. Greenland, J.E., Brading, A.F., 2001. The effect of bladder outflow obstruction on detrusoblood flow changes during the voiding cycle in conscious pigs. J. Urol. 165, 245 – 248. Hunt, C., Morimoto, R.I., 1985. Conserved features of eukaryotic hsp 70 genes revealed by comparison with the nucleotide sequence of human hsp 70. Proc. Natl. Acad. Sci. USA 82 (19), 6455 – 6459. Hutter, M., Sievers, R., Barbosa, V., Wolf, C., 1994. Heat-shock protein induction in rat hearts: A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 89, 355 – 360. Kelly, K.J., Baird, N.R., Greene, A.L., 2001. Induction of stress response protein and experimental renal ischemia/reperfusion. Kid. Int. 5, 1798 – 1802. Levin, R.M., Leggett, R., Whitbeck, C., Horan, P., 1998. Effect of calcium and calcium chelator on the response on the bladder to in vitro ischemia. Brt. J. Urol. 82, 882 – 887. Lin, A.T.-L., Chen, K.-K., Yang, C.H., Chang, L.S., 2000. Mannitol facilitates rabbit urinary bladder recovery from overdistension injury. Urology 56, 702 – 707. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265 – 275. Muijsers, R.B.R., Folkerrts, G., Henricks, P.A.J., Sadeghi-Hashjin, G., Nijkam, F.P., 1997. Peroxynitrite: A two-faced metabolite of nitric oxide. Life Sci. 60, 1833 – 1845. Parekh, M.H., Lobel, R., O’Connor, L.J., Leggett, R.E., Levin, R.M., 2001. Protective effect of vitamin E on the response of the rabbit bladder to partial outlet obstruction. J. Urol. 166, 341 – 346. Rauen, U., Polzar, B., Stephan, H., Mannherz, H.G., de-Groot, H., 1999. Cold induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB J. 13, 155 – 168. Saito, M., Miyagawa, I., 1999. Direct detection of nitric oxide on rat urinary bladder during ischemia-reperfusion. J. Urol. 162, 1490 – 1495. Saito, M., Miyagawa, I., 2001. Bladder dysfunction after acute urinary retention in the rat. J. Urol. 165, 1745 – 1747. Saito, M., Miyagawa, I., 2002. NG-nitro-L-arginine methylester, a nitric oxide synthase inhibitor, diminishes apoptosis induced by ischemia-reperfusion in the rat bladder. Neurourol. Urodyn. 21, 566 – 571. Saito, M., Suzuki, H., Yamada, M., Miyagawa, I., 2002. Preventive effect of long-chain fatty alcohol on ischemia-reperfusion injury in the rat bladder. Eur. J. Pharmacol. 454, 81 – 84. Saito, M., Tominaga, L., Nanba, E., Miyagawa, I., 2004. Expression of heat shock protein 70 and its mRNAs during ischemiareperfusion in the rat prostate. Eur. J. Pharmacol. 487, 199 – 203. Saito, M., Wad, K., Kamisaki, Y., Miyagawa, I., 1998. Effect of ischemia reperfusion on contractile function of rat urinary bladder: Possible role of nitric oxide. Life Sci. 62, PL149 – PL156. Ueta, E., Tadokoro, Y., Yamamoto, T., Yamane, C., Suzuki, E., Nanba, E., Otsuka, Y., Kurata, T., 2003. The effect of cigarette smoke exposure and ascorbic acid intake on gene expression of antioxidant enzymes and other related enzymes in the livers and lungs of shionogi rats with osteogenic disorders. Toxicol. Sci. 73, 339 – 347. Walter, L., Rauh, F., Gunther, E., 1994. Comparative analysis of the three major histocompatibility complexed-linked heat shock protein 70 genes of the rat. Immunogenetics 40, 325 – 330. Wittwer, C.T., Ririe, K.M., Andrew, R.V., David, D.A., Gundry, R.A., Balis, U.J., 1997. The LightCycler: a micro volume multi sample fluorometer with rapid temperature control. Biotechniques 22, 176 – 181.
Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder Motoaki Saito a,b,*, Lika Tominaga c, Eiji Nanba c, Yukako Kinoshita b, Daisuke Houri b, Ikuo Miyagawa a, Keisuke Satoh b a
Department of Surgery, Division of Urology, Tottori University Faculty of Medicine, Yonago, Japan Department of Pathophysiological and Therapeutic Science, Division of Molecular Pharmacology, Tottori University Faculty of Medicine, Yonago, Japan c Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago, Japan b
Received 23 March 2004; accepted 24 May 2004
Abstract HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 F 1.3, 7.9 F 1.7, 4.3 F 0.8, 4.2 F 0.7, 4.5 F 0.6, and 8.1 F 1.2 g/mm2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder
* Corresponding author. Department of Pathophysiological and Therapeutic Science, Division of Molecular Pharmacology, Tottori University Faculty of Medicine, 36-1 Nishimachi, Yonago 683-0826, Japan. Tel.: +81-859-34-8119; fax: +81-859-348435. E-mail address: [email protected] (M. Saito). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.05.010
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were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult. D 2004 Elsevier Inc. All rights reserved. Keywords: Bladder; Ischemia-reperfusion injury; HSP 70; Real-time PCR; mRNA; Rat
Introduction The functions of the urinary bladder include urine storage and subsequent micturition. The urinary bladder requires an adequate supply of oxygen and nutrients via the circulation system in order to maintain homeostasis and proper functioning (Levin et al., 1998; Parekh et al., 2001). Ischemia and the following reperfusion of the bladder are observed in age-related disorders such as urinary retention, atherosclerosis, vasospasm, embolization, and thrombosis (Levin et al., 1998; Parekh et al., 2001). Recently, both clinical and experimental evidence of ischemia and subsequent reperfusion injury have been reported in many tissues, including the kidney, liver, stomach, and heart (Muijsers et al., 1997; Rauen et al., 1999). We and other investigators have recently reported that, after overdistension, catheterization/decompression induce reperfusion injury in the bladder and that reactive oxygen species are one of the main contributing factors in this injury (Lin et al., 2000; Saito and Miyagawa, 2001). Ischemia-reperfusion injury may cause dysfunction of the urinary bladder, which results in instability and impairment of detrusor contractility during urination (Greenland and Brading, 2001). Although the mechanism of ischemia-reperfusion injury is complicated, the main etiology of this injury is thought to involve free radicals, which attack the cell membrane, proteins, RNAs, and DNAs (Muijsers et al., 1997; Rauen et al., 1999). We have previously reported that ischemia and subsequent reperfusion significantly damages the bladder function measured by organ bath techniques and histological studies (Saito et al., 1998, 2002; Saito and Miyagawa, 1999). We demonstrated that peroxynitrite, reacting with NO and superoxide radicals, plays an important role as a cell/tissue-damaging agent during ischemia-reperfusion in the rat bladder (Saito et al., 1998; Saito and Miyagawa, 1999, 2002; Bratslavsky et al., 2003). We also reported that apoptosis plays an important role in the recovery of ischemia-reperfusion injury (Saito and Miyagawa, 2002). For the process of recovery from this injury, knowledge of bladder alteration and dysfunction after rescue of the urinary retention patients is important. Heat shock proteins (HSP), a diverse family of inducible and constitutive stress proteins, are thought to limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interactions (Hutter et al., 1994; Walter et al., 1994). In mammalian cells, the inducible 70-kDa heat shock protein (HSP 70) is the most abundant HSP and the HSP most closely linked to cytoprotection from a variety of dangerous events such as thermal injury or ischemia-reperfusion injury (Hutter et al., 1994; Walter et al., 1994). In both rats and humans, HSP 70 is encoded by two genes that differ only in the 3V-untranslated region. The fact that two genes encode identical proteins may be due to a need to express the same protein under different circumstances (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000). In this study, we attempted to investigate the expression of HSP 70-1/2 mRNAs, and HSP 70 proteins during ischemia-reperfusion in the rat bladder.
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Materials and methods Production of the animal model All animal experiments were performed in accordance with the guidelines set by the Tottori University Committee for Animal Experimentation. Male Wistar rats, 8 weeks old, were divided into six groups: controls (A), 30-min ischemia alone (B), 30-min ischemia plus 30- (C), and 60-minute (D), 1-day (E), and 1-week reperfusion (F). The urinary bladders were subjected to ischemia-reperfusion as described previously (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Briefly, under pentobarbital anesthesia (50 mg/kg, i.p.), the abdominal aorta was clamped just above its bifurcation with a small clip (Sugita standard aneurysm clip, holding force 145 g; Mizuho Ikakogyo, Tokyo) for 30 minutes in the B, C, D, E and F groups. We have previously reported that blood flow in the aorta clamped rat was approximately 10 % of that of controls (Saito and Miyagawa, 1999). Measurement of contractile response The contractile function of the bladder muscles was examined as described previously (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Just after the ischemia-reperfusion, the rat bladder dome was removed to obtain smooth muscle strips, which were mounted in 3.0-ml organ baths and maintained at 37jC in Krebs-Henseleit solution gassed with 95% O2 and 5% CO2. Isometric force was monitored with a TB-612T transducer (Nihonkoden, Tokyo) and processed by an AP-621G (Nihonkoden, Tokyo). Contractile response to carbachol was expressed as force per cross-sectional area of muscle (g/mm2) (Saito et al., 1998, 2002; Saito and Miyagawa, 1999, 2002). Cumulative doseresponse curves were constructed in a stepwise manner after the response to the previous concentration had reached a plateau. The contractile response of the same muscle strips to 100 mM KCl was also monitored in each group. Measurement of HSP mRNAs in the bladder HSP 70-1/2 mRNAs in the experimental bladder were measured by real-time PCR methods according to our previous report (Saito et al., 2004). The RNA was purified by RNeasy Mini Kit (Quiagen, Valencia, CA) according to the manufacturer’s instructions. The reverse transcriptase (RT) mixture (30 Al) containing 2 ml of total RNA was made and incubated at 37jC for 60 min by a previously reported method (Ueta et al., 2003). One ml of the mixture was used for real-time PCR, which was carried out using a LightCycler thermal cycler system with a Light Cycler SYBR Green I kit according to the manufacturer’s instructions (Roche Diagnostics, Tokyo, Japan) (Wittwer et al., 1997). The following primers were used: 5V-TTTCTGGCTCTCAGGGTGTT-3V(forward) and 5V-CTGTACACAGGGTGGCAGTG-3V(reverse) for HSP 70-1, and 5V-GCTACAAGGCGGAGGACG-3V (forward) and 5V-AGATCACACCTGGAGCGCC-3V (reverse) for HSP 70-2. A total of 10 ml of solution was used for the sample. The PCR products of HSP 70-1 and HSP 70-2 were subcloned into pGEM-T vectors, and the plasmids were amplified, purified, and diluted for the standard curve. The specificity of the reaction was confirmed by a melting curve analysis and 2% agarose gel electrophoresis. The following primers for the beta actin gene were used as the internal standard and analyzed by real-time PCR using the same RT mixture: 5V-CCTCTATGCCAACACAGT-3Vand 5V-AGCCACCAATCCACACAG-3V.
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Measurement of protein level of HSP in the bladder For biochemical studies, the rest of the bladder was minced, frozen in liquid nitrogen, and stored at 80jC until used. The protein levels of HSP-70 were measured as in our previous report (Saito et al., 2004). The expression of HSP-70 proteins was measured using a StressXpress HSP 70 ELISA kit (Stressgen Biotechnologies, Victoria, BC, Canada) according to the manufacturer’s instructions. Protein content was determined using bovine serum albumin as standard (Lowry et al., 1951). Data analysis The data for contractions were calculated as grams of active force per cross-sectional area in square millimeters. The cross-sectional area was calculated using the following equation: cross sectional area ¼ weight=ðlength 1:05Þ; where 1.05 is the assumed density of the muscle (Saito et al., 1998; Saito and Miyagawa, 1999, 2002). ED50 values (the concentration of agonist producing half-maximal contractile responses) were calculated as geometric means, whereas Emax values were calculated as arithmetic means. The expressions of HSP 70-1/-2 mRNAs were quantified according to the expression of beta actin mRNAs in the experimental rat bladder (Saito et al., 2004). The expressions of HSP 70-1/-2 mRNAs in the rat bladder were compared to those of the controls using the following ratio: Ratio of expression of HSP 70 1= 2 mRNAðGroup A FÞ ¼ HSP 70 1= 2 mRNA ðGroup A FÞ=beta actin mRNA ðGroup A FÞ The expression of HSP 70 was quantified to compare the protein concentration. Statistical comparison of differences between groups was performed using analysis of variance and Fisher’s multiple comparison tests. P < 0.05 was regarded as the level of significance. Drugs and chemicals A kit for colorimetric assay of HSP 70 was purchased from Stressgen Biotechnologies (StressXpress HSP 70 ELISA kit, Victoria, BC, Canada). All other chemicals were of reagent grade.
Results Measurement of contractile response In the functional studies, the maximal contractile force produced by carbachol (Emax values) was slightly decreased in 30 min ischemia (alone) (group B). In the 30 min ischemia plus brief reperfusion (30 min, group C), (60 min, group D) and (1day, group E) were markedly decreased. However, in the 30
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Table 1 Functional data in the experimental rat bladder dome Group
Emax g/mm2
% CONTROL
ED50 microM
100mM KClg/mm2
A B C D E F
9.3 7.9 4.3 4.2 4.8 7.8
100 84.9 46.2 45.2 51.6 83.8
1.9 2.4 4.8 5.0 4.6 2.3
7.2 6.4 3.2 3.3 3.5 6.5
F F F F F F
1.3 1.7 0.7* 0.7* 0.6* 1.1
F F F F F F
0.3 0.3 0.9* 0.8* 0.7* 0.9
F F F F F F
0.3 0.2 0.3* 0.5* 0.4* 0.6
Emax and ED50 are for carbachol. Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, B and F.
min ischemia plus 1 week reperfusion was improved the maximal contractile force (group F). Similar results were obtained by use of 100 mM KCl. The precise data were shown in Table 1. Measurement of HSP mRNAs in the bladder The data of mRNA expression in the bladder are shown in Table 2. In the control group, expression of HSP 70-1/2 mRNA was detected. Expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70-2 mRNAs showed the same manner as the expression of HSP 70-1 mRNAs after 30 min ischemia-1 day or 1 week reperfusion. Measurement of protein level of HSP70 in the bladder The data of expression of HSP 70 in the bladder are shown in Table 3. The expression of HSP 70 proteins was increased after a short period of the expression of their mRNAs (groups C and D), which was normalized in groups E and F. The level of HSP 70 after 30 min of ischemia (21.5 mg/g protein) was
Table 2 Expression of HSP 70-1/2 mRNAs in the Experimental Rat Bladder Group
HSP 70-1/ h-actin
HSP 70-2/ h-actin
A B C D E F
3.26 9.12 5.08 4.30 3.20 3.55
0.74 0.94 1.41 0.40 0.52 0.60
F F F F F F
0.57 3.15* 0.85 0.64 0.35 0.42
Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, D, E and F.
F F F F F F
0.14 0.47 0.16* 0.11 0.13 0.18
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Table 3 Expression of HSP 70 (Protein) in the Experimental Rat Bladder Dome Group
HSP 70 mg/g protein
A B C D E F
20.3 21.5 50.3 49.3 23.0 19.5
F F F F F F
1.3 0.5 0.7* 0.8* 0.6 0.4
Data are shown as mean F SEM of five to seven separated determinations in each group. * Significantly different from groups A, B, E and F.
similar to that in the controls. After 30 min of ischemia and 30 min of reperfusion, the levels of HSP 70 in the bladder were significantly increased (50.3 mg/g protein), and was maintained at a similar level after 30 min of ischemia and 60 min of reperfusion (49.3 mg/g protein). The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels (23.0 and 19.5 mg/g protein, respectively). Our data indicated that the protein levels of HSP 70 were increased after a short interval of increase in their mRNAs, and that decreased within one day.
Discussion In this study, we demonstrated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. In functional study, ischemia added bladder dysfunction. Additional reperfusion (30 minutes) caused even more severe dysfunction in the rat bladder response to carbachol and KCl, which were continued more than one week. In the control group, expressions of HSP 70-1/2 mRNAs were detected. Expression of HSP 70-1 mRNA increased in groups B, and decreased in groups D, E and F. Expression of HSP 70-2 mRNA increased in groups C, and decreased in groups D, E and F. The expression of HSP 70 was increased after a short interval of the expression of their mRNAs (Groups C and D). Both the HSP 70 mRNAs and proteins were normalized after 1-day reperfusion. Although bladder dysfunction measured by functional study was continued more than one week, the protein levels of HSP 70 were increased after a short interval of increase in their mRNAs, which was normalized in one day. The increased expression of HSP 70 means the existence of damaged tissue, and playing a role in recovery from stress (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999; Bidmon et al., 2000). It also may be an indicator of stressed tissue/cells. There are increasing evidences that ischemia in the bladder is present in acute/chronic urine retention (Levin et al., 1998; Parekh et al., 2001; Lin et al., 2000; Saito and Miyagawa, 2001). The bladder dysfunction seen after acute overdistension and decompression is likely to be due to ischemiareperfusion. Lin and associates have demonstrated that acute overdistension and the subsequent decompression in the bladder induce bladder dysfunction and enhance lipid peroxidation. This bladder dysfunction can be prevented by mannitol, a free-radical scavenger (Lin et al., 2000). We have also demonstrated that bladder dysfunction is induced by urinary retention and subsequent catheterization, and that bladder dysfunction following catheterization is, in part, caused by free-radicals (Saito and Miyagawa, 2001). Free-radicals caused by ischemia-reperfusion attack and damage the cell membrane,
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and induce bladder dysfunction. HSPs are thought to this limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interaction (Hunt and Morimoto, 1985). Previous reports have indicated that the most important thing in the prevention of bladder dysfunction is to reduce the production of free-radicals in the bladder (Lin et al., 2000; Saito and Miyagawa, 2001). In mammalian cells, HSP 70 is the most abundant HSP and the HSP most closely linked to cytoprotection from a variety of dangerous events, including thermal injury and ischemia-reperfusion injury (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000; Bidmon et al., 2000; Kelly et al., 2001). There is increasing evidence that ischemia-reperfusion induces HSP 70 in some organs, e.g., the kidney and heart (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999). HSPs are thought to limit injury and accelerate recovery by refolding disrupted proteins and preventing deleterious peptide interactions. In some tissues, expression of HSP 70-1 mRNA is report to be higher than that in HSP 70-2 mRNA (Akcetin et al., 1999, 2000; Saito et al., 2004). In the bladder, we also demonstrated that expression of HSP 70-1 mRNA is higher than that in HSP 70-2 mRNA in this study. In the kidney, it has been shown that there is a significant induction of both HSP 70-1/-2 genes immediately after ischemia. While HSP 70-1 mRNA expression constantly increases during reperfusion, HSP 70-2 mRNA is strongly induced (three fold) during reperfusion only after brief periods (10 min) of ischemia in the kidney (Hutter et al., 1994; Walter et al., 1994; Akcetin et al., 1999, 2000; Bidmon et al., 2000; Kelly et al., 2001). Akcetin et al reported that the HSP 70-2 gene was far more sensitive with a lower threshold activation and only works in a range of mild injury while the HSP 70-1 gene mediates the big response after severe injury (2000). Many reports indicate that when not induction of ischemia, but induction of reperfusion occures, HSP 70-1/2 mRNA levels are increased (Akcetin et al., 1999; Bidmon et al., 2000; Hutter et al., 1994; Kelly et al., 2001). However, in this experiment, ischemia for 30 min induced an increase of HSP 70-1 mRNA. This may be due to clamping of aorta resulted in partial ischemia (approximately 10 % of basal level) of the bladder and not complete ischemia of the organ (Saito and Miyagawa, 1999). Interestingly, we found that protein levels of HSP 70 were increased after 30 min of ischemia and 30 min of reperfusion. Our data suggest that the expression of HSP 70 proteins is increased after a short interval of the expression of their mRNAs in the bladder, and that this increased expression continues longer than that of mRNA in the bladder. These data are similar to those of the prostate (Saito et al., 2004). In the functional study, we demonstrated that the contractile responses of the rat bladder dome after 30 minutes of ischemia differed slightly, not significantly, from those of controls, and that reperfusion (30 min) produced significant reductions in the contractile responses to carbachol and KCl in the rat bladders, which continued more than one week. In contrast, expression of HSP 70 ( both mRNA and protein) increases early phase of ischemia-reperfusion injury and, is normalized within one day. Thus, our data indicate that HSP 70 and its mRNAs are increased in only early phase of ischemia-reperfusion injury in the bladder. The patho-physiological role of ischemia-reperfusion in the bladder, however, remains unclear and warrants further study.
Acknowledgements This study was supported by a grant from the Ministry of Education, Science, and Culture of Japan (#14704041).
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