Effects of Rosiglitazone on Heat Shock Protein and the Endothelin System in Deoxycorticosterone Acetate-Salt Hypertensive Rats

The deoxycorticosterone acetate (DOCA)-salt rat is known as a model of volume dependent hypertension and characterized by increased cardiac endothelin-1 (ET-1) content. Recently, it has been reported that rosiglitazone (RGT), a peroxisome proliferator-activated subtype gamma receptor agonist, shows blood pressure lowering effect. We investigated whether DOCA-salt hypertension is associated with altered expression of heat shock proteins (HSP) and ET-1 in the heart, aorta, and kidney, and whether RGT changes HSP expression and ET-1 in association with its blood pressure lowering effect. Two weeks after the silastic DOCA (200 mg/kg) strips implantation, DOCA-salt rats were randomly divided to receive control diet with or without RGT (10 mg/kg/day) for another 2 weeks. The mRNA expression of ET-1 was determined by real time polymerase chain reaction. The expression of HSP was determined by semiquantitative immunoblotting. In DOCA-salt rats, systolic blood pressure was markedly increased, while creatinine clearance decreased. RGT treatment attenuated high blood pressure and decreased creatinine clearance in DOCA-salt rats. The mRNA expression of ET-1 was increased in DOCA-salt rats compared to controls, which was counteracted by RGT treatment. The protein expression of HSP70, HSP32, and HSP25 was increased in the kidney and heart in DOCA-salt rats, which was attenuated by RGT treatment in the kidney, but not in the heart. In conclusion, increased expression of ET-1 may play a role in the pathogenesis of hypertension in DOCA-salt rats, which was counteracted by the treatment of RGT. Up-regulation of HSP70, HSP32, and HSP25 in the kidney and heart may play a role in organ protection against a variety of stresses.

constriction" in the pathogenesis of hypertension 1) . The Deoxycorticosterone acetate (DOCA) treated animals had elevated plasma sodium and osmolality levels 2) , which is consistent with the observations that this model of hypertension is associated with increased renal sodium and water reabsorption 3) and with the expansion of extracellular fluid volume 4) . The mechanism by which the abnormality in sodium homeostasis raises blood pressure remains unclear, but must involve an element of vasoconstriction.
There is accumulating evidence indicating that endothelin-1 (ET-1) plays an important role in the development

Effects of Rosiglitazone on Heat Shock Protein and the Endothelin System in Deoxycorticosterone Acetate-Salt Hypertensive Rats
Eun Hui Bae, M.D. 1 , In Jin Kim, M.S. 2 , Jeong Woo Park, M.D. 1 of hypertension. ET-1 is a potent vasoconstrictor, as well as a mediator of smooth muscle mitogenesis, and thus, a determinant of systolic blood pressure 5) . ET-1 content and its mRNA expression are elevated in vascular and renal tissues of DOCA-salt hypertensive rats 6) . These findings suggest that ET-1 contributes to the development of high blood pressure and vascular growth in DOCA-salt hypertensive rats 7) .
Heat shock proteins (HSP) may be induced in response to a variety of stresses, including hypertension. The cellular expression of major HSP gene is increased in response to heat exposure in hypertensive animals and humans 8) , and has been known to possess a protective role in various pathphysiologic states. The expression of HSP32/heme oxygenase (HO)-1 is increased in several tissues in response to angiotensin II infusion, along with increased blood pressure 9,10) . An induction of HSP70 plays a protective role in the vasculature from hemodynamic stress 11) , and HSP25 protect cells from heat shock and oxidative stress when overexpressed 12) . Furthermore, altered regulation of HSP has been implicated in the development and maintenance of hypertension. A prolonged HSP32 induction counteracts the increased blood pressure in renovascular hypertension 13) .
Thiazolidinediones (TZDs) are used to treat type 2 diabetes because of their efficacy in controlling blood glucose secondary to enhancing insulin action through a mechanism that is yet to be completely elucidated 14) .
These compounds are also capable of a rich array of beneficial effects, including reductions of in blood pressure in animal models and humans [15][16][17] . TZD treatment has been shown to attenuate the release of endothelin-1 from bovine vascular cells, which may contribute to the observed blood pressure lowering effect 18) . Thus, it may be possible that antihypertensive effect of rosiglitazone is caused by changes of endothelin system in DOCA-salt hypertensive rats. In addition, rosiglitazone treatment inhibits apoptosis and decreases protein expression of HSP60 in vascular endothelial cells, which may have beneficial vascular effects 19) . In this context, the blood pressure lowering effects of TZD may be associated with the changes of HSP and endothelin system in the vasculature and kidney.
The present study was aimed to investigate whether antihypertensive effects of rosiglitazone in DOCA-salt hypertensive rats is associated with altered regulation of endothelin system and HSP isoforms in the kidney and heart in order to elucidate the underlying molecular mechanisms responsible for the pathogenesis of hypertension.
Materials and Methods

Animals
Male . Melting curve analysis was performed to enhance specificity of the amplification reaction.

Drugs and statistical analysis
Drugs were purchased from Sigma Chemical Co.

Blood Pressure and LV weight
As shown in Fig. 1    affected by RGT treatment. The expression of ETBR was increased in the kidney and aorta of DOCA-salt rats, which was conteracted by RGT treatment.  Table 2 shows the protein expression of HSP70, HSP32, and HSP25 in the kidney and heart. The expression of HSP70, HSP32, and HSP25 was increased in the kidney of DOCA-salt rats, which was counteracted by RGT treatment. The expression of HSP70, HSP32, and HSP25 was also increased in the heart, which was further affected by RGT treatment.

ET-1 not only induced vasoconstriction and has been
demonstrated to play a role in renal and cardiac target organ damage 22) . The present study demonstrated that ET-1 mRNA expression was increased in the aorta and kidney associated with the increased blood pressure and decreased GFR. These findings may suggest that enhanced vascular and renal production of ET-1 play a role in the development of hypertension and renal dysfunction. These results are in line with the previous observations that chronic administration of a selective endothelin ETAR antagonist or nonselective endothelin ETAR/ETBR antagonist to DOCA-salt rats suppress the development of hypertension, vascular hypertrophy and renal injury 23,24) . However, the mechanism by which ET-1 production is enhanced in blood vessels and kidneys in DOCA-salt rats are unknown. Recent study demonstrated that incubation with xanthine/xanthine oxidase or H2O2 augments ET-1 mRNA levels in human mesangial cells 25) . These findings suggest that oxidant stress stimulates endothelin-1 production at a stage of its gene expression.
ET-1 produces its effects by acting on specific subtypes of receptors, ETAR and ETBR, which receptors are ex-  pressed mainly in the vascular tissues, in which it mediates the vasoconstrictor and growth effects 26,27) . The ETBR also exist on endothelial cells, where they mediate vasodilation via the release of endothelium-derived nitric oxide 28) . In the present study, the mRNA expression of Alterations of HSP has been suggested to play a protective role against potential tissue damage. Molecular adaptation of vascular endothelial cells to oxidative stress is accompanied by an induction of HSP. An induction of HSP70 has a protective effect on the vasculature from hemodynamic stress 11) . The inducible form of HSP70 is shown to be regulated in the kidney in response to stimuli such as ischemia 30) and hyperthermia 31) . Overexpression of transfected HSP70 in the renal cells was found to have a protective action against cisplatin toxicity and oxidative injury 32) . Induction of renal HSP32 has been shown to ameliorate renal injury induced by rhabdomyolysis 33) . Gene ablation of HSP32 exacerated cisplatin-induced renal tubular injury 34) . Thus, renal HSP32 is thought to play a role in protecting renal function against renal insults. The small HSP from human HSP27 and mouse HSP25 form large oligomers which can act as molecular chaperones in vitro, and protect cells from heat shock and oxidative stress when overexpresssed 12) . In addition, angiotensin II infusion induces renal HSP70 and HSP25, as well as HSP32 9) .
These findings may suggest the beneficial and compensatory role of HSP70, HSP32 and HSP25. In the present study, expression of HSP isoforms was increased in heart and kidney of DOCA-salt rats, which may play a protective role in the pathogenesis of hypertension and renal damage.
Interestingly, RGT treatment attenuates expression of HSP in kidney, but not in heart. This discrepancy of HSP expression may represent that HSP expresion can be regulated by at the level of post-translation by cell specific cofactors in the vasculature and kidney. This remains to be further elucidated.
In summary, RGT treatment decreased blood pressure and impaired renal function in DOCA-salt hypertensive rats. Up-regulation of mRNA expression of ET-1 in the aorta and kidney play a role in the pathogenesis of development of hypertension in DOCA-salt rats, which was counteracted by the treatment of RGT. Up-regulation of HSP70, HSP32 and HSP25 may play a role in organ protection against to a variety of stress.