Kimiko Ishiguro

Developmental activity of the renin-angiotensin system during the "critical period" modulates later L-NAME-induced hypertension and renal injury.

The incidence of hypertension and hypertensive renal disease is increasing worldwide, and new strategies to prevent these diseases need to be investigated. The aims of this study were 1) to examine if transient exposure to an angiotensin receptor blocker (ARB) during an early period in hypertension development confers protection against subsequent worsening of hypertension and renal injury induced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), and 2) conversely, to examine the effects of transient exposure to angiotensin II (Ang II) during the same period. First, spontaneously hypertensive rats (SHR) were treated transiently from age 3 to 10 weeks with an ARB (candesartan cilexetil), a calcium channel antagonist or a vasodilator, then taken off treatment for 2 months. Administration of L-NAME at age 18 weeks caused severe hypertension and renal injury. However, the rats that had been exposed to the ARB not only had a lower blood pressure, but also failed to show signs of renal injury or increase of oxidative stress. Furthermore, the elevation of components of the renin-angiotensin-aldosterone system was also suppressed in these rats. In the second study, Wistar-Kyoto rats (WKY) and SHR were exposed to Ang II from age 4 to 8 weeks. The follow-up showed that the blood pressures in the WKY remained elevated compared to controls, while the SHR had heightened increases in blood pressure, renal renin mRNA, and urinary 8-hyroxydeoxyguanosine after L-NAME administration. Together, these experiments demonstrate that transient treatment of rats during an early phase in the development of hypertension with an ARB suppresses the renin-angiotensin-aldosterone system and confers long-term protection against subsequent L-NAME–induced renal injury and increases in renal oxidative stress. Conversely, developmental exposure to Ang II during this “critical” period had the opposite effect, predisposing rats to higher blood pressure, renal injury, and oxidative stress after L-NAME administration.

Regression of glomerulosclerosis in response to transient treatment with angiotensin II blockers is attenuated by blockade of matrix metalloproteinase-2

Understanding mechanisms that contribute to the regression of glomerulosclerosis is important for developing new strategies to treat chronic kidney disease. We reported that transient high-dose treatment with an angiotensin receptor blocker causes regression of renal arteriolar hypertrophy and hypertension in spontaneously hypertensive rats. To extend those findings to another form of kidney disease, we examined the short- and long-term effects of transient high-dose angiotensin receptor blocker treatment in a mouse model of adriamycin-induced glomerulosclerosis. A 2-week course of candesartan caused a dose-dependent regression of established glomerulosclerotic lesions sustained for over 6 months following cessation of treatment. Highly sensitive in situ zymography and activity assays showed that glomerular matrix metalloproteinase (MMP)-2 activity was increased after high-dose angiotensin blocker therapy. Treatment of cultured podocytes with candesartan resulted in an increase in MMP-2 activity. The regression of glomerulosclerosis was partially attenuated in mice pretreated with the MMP inhibitor doxycycline, as well as in MMP-2 knockout mice. Our results suggest that transient high-dose angiotensin receptor blocker treatment effectively induced sustained regression of glomerulosclerosis by a mechanism mediated, in part, by changes in MMP-2 activity.

“Pulse” Treatment With High-Dose Angiotensin Blocker Reverses Renal Arteriolar Hypertrophy and Regresses Hypertension

One ultimate goal of hypertension therapy is to cause permanent reversal (“regression”) of already established hypertension. Our aim was to examine whether high-dose “pulse” treatment with a renin-angiotensin system inhibitor could cause regression of established hypertension and to link this action to reversal of arteriolar hypertrophy and changes in vascular matrix metalloproteinase activities. First, 16-week-old male spontaneously hypertensive rats (n=60) were pulse treated for 2 weeks with high-dose angiotensin-converting enzyme inhibitor (enalapril), angiotensin receptor blocker (candesartan), calcium channel blocker (nifedipine), or vasodilator (hydralazine) with or without salt restriction, and the long-term effects on blood pressure were examined. Second, spontaneously hypertensive rats were treated with angiotensin receptor blocker or calcium channel blocker, and the effects on renal gene expressions, arteriolar structure, and vascular matrix metalloproteinase were compared. Treatment of spontaneously hypertensive rats with different antihypertensive agents caused apparently similar reductions in blood pressure during the course of the pulse treatment, within the limitations of the tail-cuff method. After cessation of medications, blood pressure in the rats treated with renin-angiotensin system inhibitor remained reduced by >30 to 40 mm Hg for 4 months. No such effect was seen with calcium channel blocker or vasodilator. The 2-week angiotensin receptor blocker treatment induced a marked reversal of the arteriolar hypertrophy specifically in the small (30 to 100 &mgr;m) renal arterioles, together with increased expression and activity of matrix metalloproteinase-13. In conclusion, transient high-dose pulse treatment with angiotensin receptor blocker caused changes in vascular matrix metalloproteinase activity, specific reversal of renal arteriolar hypertrophy, and regression of hypertension in spontaneously hypertensive rats.

Prevention and regression of hypertension: role of renal microvascular protection

Hypertension is a disease which affects over 26.4% of the world adult population, therefore novel approaches to the prevention and treatment of this disease need to be examined. Previous studies from our and other laboratories have shown that treatment of spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats with a renin–angiotensin system (RAS) inhibitor during the ‘critical period’ in hypertension development results in prevention of the later development of hypertension. In humans, Julius et al. reported similar findings in the landmark TROPHY study. Recently, we reported that ‘pulse’ treatment of SHR with high-dose angiotensin receptor blocker (ARB) is effective in causing sustained reduction of already established hypertension, even when the treatment was started after the ‘critical period’. These results suggest the possibility that ‘regression’ of established hypertension may become feasible, and we have started a prospective, multicenter clinical study (STAR CAST study) to examine this possibility. In our animal studies, we found that treatment of rats during the ‘critical period’ with an ARB inhibits the development of renal arteriolar hypertrophy. Moreover, a high-dose angiotensin blocker caused a remarkable reversal of renal arteriolar hypertrophy in SHR, which was associated with changes in microvascular MMP expression. These results suggest that changes in the renal microvasculature may have an important role in the mechanisms of hypertension prevention and regression by ARB.

Absence of Gelatinase (MMP-9) or Collagenase (MMP-13) Attenuates Adriamycin-Induced Albuminuria and Glomerulosclerosis

Background/Aims: The role of matrix metalloproteinases (MMPs) in the pathogenesis of glomerular injury appears to be complex. To investigate the role of individual MMPs, we examined the course of Adriamycin-induced albuminuria and glomerulosclerosis in mice lacking either a gelatinase (MMP-9) or a collagenase (MMP-13). Methods: Adriamycin was administered to MMP-9 or MMP-13 knockout (KO) mice. Glomerular injury was assessed by the quantification of albuminuria, the glomerular injury score and type IV collagen immunostaining. Results: Treatment of mice with Adriamycin (18 mg/kg i.v.) resulted in marked albuminuria and glomerulosclerosis reaching a peak at 4–8 weeks. The albuminuria and glomerulosclerosis were significantly (p < 0.05) attenuated in both the MMP-9 KO and MMP-13 KO mice compared to controls. In contrast, treatment of wild-type mice with the broad-spectrum MMP inhibitor doxycycline did not have a beneficial effect on the albuminuria and glomerulosclerosis. Conclusion: These results support a role for both gelatinase (MMP-9) and collagenase (MMP-13) in the pathogenesis of glomerular injury in the Adriamycin-induced glomerulosclerosis model. MMP inhibitors with high specificity towards MMP-9 and/or MMP-13 may be potential future candidates to provide more effective therapies to inhibit the development of glomerulosclerosis.

Differential Effects of Transient Treatment of Spontaneously Hypertensive Rats with Various Antihypertensive Agents on the Subsequent Development of Diabetic Nephropathy

Background/Aims: We have previously shown that treatment of spontaneously hypertensive rats (SHR) with an angiotensin receptor blocker (ARB) during the ‘critical period’ from age 3 to 10 weeks confers protection against L-NAME-induced renal injury later in life. The aim of this study was to examine the effects of transient prepubertal exposure to ARB on the development of nephropathy in streptozotocin-induced diabetic SHR and to compare the results with other antihypertensive agents including a mineralocorticoid receptor antagonist (MR-ant). Methods: Male SHR (n = 43) were transiently treated with candesartan (ARB), potassium canreonate (the active metabolite of the MR-ant spironolactone) or hydralazine (vasodilator) between 3 and 10 weeks of age with untreated rats serving as controls. An additional group was treated continuously with candesartan throughout the study. Rats were injected with streptozotocin to induce diabetes at age 16 weeks and followed until age 8 months. Results: Diabetic control rats showed signs of diabetic nephropathy including albuminuria and mesangial expansion. These changes were significantly suppressed in rats exposed to ARB or MR-ant. Systolic blood pressure was significantly reduced compared to controls in the ARB (transient) and ARB (sustained) groups, but not in the MR-ant or vasodilator groups. Conclusion: Transient prepubertal exposure to ARB or MR-ant, but not vasodilator, confers protection against the later development of diabetic nephropathy and involves blood pressure-independent protective mechanisms.

Renal Arteriolar Injury by Salt Intake Contributes to Salt Memory for the Development of Hypertension

The role of salt intake in the development of hypertension is prominent, but its mechanism has not been fully elucidated. Our aim was to examine the effect of transient salt intake during the prehypertensive period in hypertensive model animals. Dahl salt-sensitive rats and spontaneously hypertensive rats were fed from 6 to 14 weeks with low-salt (0.12% NaCl), normal-salt (0.8% NaCl), high-salt (7% NaCl), or high-sodium/normal-chloride diet and returned to normal-salt diet for 3 months. Rats in the high-salt group saw elevations in blood pressure (BP) not only during the treatment period but also for the 3 months after returning to normal-salt diet. We named this phenomenon salt memory. Renal arteriolar injury was found in the high-salt group at the end of experiment. Dahl salt-sensitive rats were fed from 6 to 14 weeks with high-salt diet with angiotensin receptor blocker, vasodilator, calcium channel blocker, and calcium channel blocker+angiotensin receptor blocker and returned to normal-salt diet. Although BP was suppressed to control levels by vasodilator or calcium channel blocker, elevated renal angiotensin II and renal arteriolar injury were observed, and salt memory did not disappear because of sustained renal arteriolar injury. Calcium channel blocker+angiotensin receptor blocker suppressed renal arteriolar injury, resulting in the disappearance of salt memory. Cross-transplantation of kidneys from Dahl salt-sensitive rats on high salt to control rats caused increase of BP, whereas control kidneys caused reduction in BP of hypertensive rats, inducing the central role of the kidney. These results suggest that renal arteriolar injury through BP and renal angiotensin II elevation plays important roles in the development of salt memory for hypertension.

399 VACCINATION AGAINST THE ANGIOTENSIN II TYPE 1 RECEPTOR PREVENTS L-NAME-INDUCED NEPHROPATHY IN SHR

Objectives: Previous studies have shown that renin–angiotensin (Ang) system vaccines may be effective for the treatment of hypertension, but their efficacy for the prevention of renal disease is unclear. The aim of this study was to examine the effects of an Ang II type 1 (AT1) receptor vaccine on blood pressure (BP) and renal injury in the L-NAME nephropathy model. Methods: Male spontaneously hypertensive rats (SHRs) were divided into six groups and treated transiently with three injections of vehicle or AT1 receptor vaccine (0.1 mg) at age 4, 6 and 8 weeks, or continuously with candesartan cilexetil (0.1 mg/kg/day) or hydralazine hydrochloride (5 mg/kg/day), then administered NG-nitro-Larginine methyl ester (L-NAME) from age 18 to 21 weeks to induce renal injury. Results: Vaccination against the AT1 receptor caused a significant increase in AT1 receptor titers, and a sustained decrease in BP. L-NAME treatment resulted in a marked increase in proteinuria in the control groups, which was markedly suppressed in the AT1 vaccine-treated group, and glomerular injury scores were also significantly decreased. Real-time RT-PCR and immunofluorescence studies revealed increased renin mRNA, and increased glomerular expression of nephrin. Comparable results were seen in rats treated continuously with the ARB candesartan, but not with hydralazine. The elevated titers and hypotensive effect continued for approximately 6 months after the final vaccination. Conclusion: These results suggest that transient AT1 vaccination is as effective as continuous treatment with ARB, not only for the attenuation of hypertension, but also for the prevention of L-NAME-induced nephropathy in SHR.

1014 Regression of atherosclerosis in apolipoprotein-E deficient mice is feasible using high-dose angiotensin receptor blocker

Objectives: Clinical studies have suggested that renin-angiotensin inhibitors are effective for the prevention of atherosclerosis progression, but the results for regression of established lesions are equivocal. The aim of this study was to examine the effects of different doses of the angiotensin receptor blocker (ARB) candesartan on regression of atherosclerosis and lipid-induced nephropathy in apolipoprotein E (apoE)-deficient spontaneously hyperlipidemic (SHL) mice. Methods and results: Male SHL were given an atherogenic diet together with salt loading to induce atherosclerosis. The mice were then treated with various doses of candesartan (0-50 mg/kg/d) for 12 weeks. Treatment with high-dose ARB caused clear regression of atherosclerotic plaques in the aorta, which was not observed with normal-dose ARB. Biglycan and ACAT1 expression were significantly decreased, and aortic free cholesterol:cholesterol ester ratios were increased in these mice. Treatment of cultured THP-1 macrophages in vitro with candesartan resulted in a similar decrease in ACAT1 expression. In the kidney, glomerular lipid accumulation, mesangial expansion, and albuminuria were significantly regressed after the treatment with high-dose ARB, while biglycan and ACAT1 expressions were decreased. Conclusion: These results suggest that regression of established atherosclerosis lesions in ApoE-deficient mice is feasible using high-dose ARB, by mechanisms involving (i) a decrease in the lipid-retaining proteoglycan biglycan, and (ii) suppression of ACAT1 expression resulting in increased free cholesterol for lipid release.

410 EFFICACY OF VACCINATION AGAINST THE ANGIOTENSIN II TYPE 1 RECEPTOR AND APOB100 FOR THE PREVENTION OF ATHEROSCLEROSIS

Objectives: Atherosclerosis is a major cause of cardiovascular morbidity and mortality, therefore new methods for atherosclerosis prevention are required. The aim of this study was to evaluate the effects of an Ang II type 1 (AT1) receptor vaccine and ApoB100 vaccine on atherosclerosis and hyperlipidemia-related nephropathy. Methods: Peptide sequences from the AT1 receptor or ApoB100, were synthesized and coupled to the carrier protein keyhole limpet haemocyanin (KLH). Male spontaneously hyperlipidemic (SHL) mice (n = 60) were treated transiently with three injections of vehicle, AT1 receptor vaccine (0.02 mg) or ApoB100 vaccine (0.05 mg) at age 4, 6 and 8 weeks, or continuously with candesartan cilexetil (1 mg/kg/day). SHL mice were given a high fat diet and 1% NaCl in the drinking water to induce atherosclerosis and glomerular lipid deposition. Atherosclerotic lesions were measured by Sudan IV stain positive areas of aorta and glomerular lesions by Oil-red-O stain positive area. Results: Vaccination against the AT1 receptor and ApoB100 did not cause a significant change in serum lipids or blood pressure. Atherosclerotic lesions and albuminuria were significantly suppressed by vaccination against the AT1 receptor, as well as by continuous treatment with candesartan, and a similar trend was seen for glomerular lipid deposition. However, vaccination against ApoB100 did not cause a significant suppression of the atherosclerotic area or glomerular lesions. Conclusion: AT1 receptor vaccination may be effective for the attenuation of atherosclerosis and hyperlipidemia-related nephropathy. In contrast, ApoB100 vaccination was not found to be effective for prevention of atherosclerosis in this model of atherosclerosis.