**3.1 Prevention of hypertension**

420 Chronic Kidney Disease

MMP-2 activity decreased to baseline after the ARB treatment was discontinued. The transient increase in MMP-2 was probably sufficient to permanently reverse the

Interestingly, clinical studies using different ARBs (Rossing et al., 2005), (Hollenberg et al., 2007), (Burgess et al., 2009) also suggest that high-dose ARB treatment may have a greater beneficial effect on the kidney compared to standard doses. One potential reason may be that standard doses of ARB do not fully suppress the RAS in the kidneys. Another possibility is that mechanisms unrelated to RAS inhibition may be involved, for example an antioxidant action independent of AT1 receptor blockade (Chen et al., 2008). Currently, we are performing further studies to examine why high-dose ARB is particularly effective in

The clearest clinical demonstration of glomerulosclerosis regression was provided by Fioretto et al., who showed that pancreas transplantation in patient with type 1 diabetes caused regression of established lesions of glomerulosclerosis in patients with type 1

There are also several studies which examined the effect of RAS inhibition on structural changes in diabetic and non-diabetic CKD. In the study on type 1 diabetic patients with microalbuminuria, treatment with enalapril, perindopril, or metoprolol resulted in a decrease in glomerular basement membrane thickness after 3-4 years of follow-up (Nankervis et al., 1998) (Rudberg et al., 1999). Other studies have suggested that glomerular volumes may be reduced by RAS inhibition, however the contribution of changes in blood pressure is unclear (Perrin et al., 2008). On the other hand, a recent study by Mauer et al. did not detect a statistical difference in mesangial fractional volume in patients treated with placebo, ARB, or ACEI (Mauer et al., 2009). In the ESPRIT study, 3-year treatment with enalapril or nifedipine did not

In the case of type 2 diabetes, the study by the Diabiopsies group suggested that treatment with perindopril resulted in stabilization of the percentage of sclerosed glomeruli, but this could not be confirmed by electron microscopy (Cordonnier et al., 1999). In the case of nondiabetic CKD, Ohtake et al. reported that treatment of 15 patients with mild to moderate IgA and non-IgA mesangial proliferative glomerulonephritis with an ARB for an average of 28 months caused a decrease in mesangial matrix expansion and interstitial fibrosis (Ohtake et al., 2008). In summary, although there is encouraging evidence that RAS inhibition can cause regression of glomerular structural changes in humans, the clinical data are not as clear as the data from animal experiments, possibly because the human studies have not

One of the reasons that there are relative few large-scale studies on CKD regression is that demonstration of resolution of glomerular lesions requires repeat kidney biopsies, which may not be feasible in large populations. One potential way to overcome this problem is to find surrogate biomarkers of disease regression in the serum and urine of patients with early (stage 1-2) CKD, using the new science of metabolomics (Hayashi et al., 2011).

cause a significant change in renal structural abnormalities (2001).

**2.4 The search for clinical biomarkers of disease regression** 

focused on the use of high-dose RAS inhibitors.

glomerulosclerosis in that model, but its effect in other disease states is unclear.

ameliorating glomerular injury.

diabetes (Fioretto et al., 1998).

**2.3 Clinical studies of CKD regression** 

It has been recognized that the kidney plays an important role in the control of systemic blood pressure, and is involved in the pathogenesis of hypertension, which is a major risk factor for cardiovascular disorders such as stroke, heart failure, vascular disease, and endstage renal disease, and an important cause of morbidity and mortality. Similar to CKD, the development of hypertension appears to be progressive: the systolic blood pressure of an individual patient rises progressively over time, so that median values of systolic blood pressure in the population increases at every age (Qureshi et al., 2005).

In our laboratory, we have been studying the use of RAS inhibitors to prevent the development of hypertension, using the spontaneously hypertensive rat (SHR) and other animal models of hypertension. Previous studies by Harrap et al. demonstrated that treatment of SHR from age 6 to 10 weeks with an angiotensin-converting enzyme (ACE) inhibitor resulted in the sustained suppression of hypertension at age 25 weeks (Harrap et al., 1986), (Harrap et al., 1990). Studies from the group of Berecek et al. suggested that these results could result from a decrease in arginine vasopressin (AVP) levels (Lee et al., 1991), (Zhang et al., 1996). Similar findings have been reported by other laboratories, using both ACE inhibitors (Giudicelli et al., 1980), (Christensen et al., 1989) and ARBs (Morton et al., 1992), (Gillies et al., 1997).

In our laboratory, it was found that treatment of stroke-prone SHR (SHRSP) with an ACE inhibitor from age 3 to 10 weeks resulted in a sustained suppression of blood pressure, whereas such an effect was not found with the vasodilator hydralazine (Nakaya et al., 2001). The same results were found with an ARB, suggesting that this effect could be explained by the inhibitory actions of ACE inhibitors and ARB on the RAS. Importantly, it was also found that the development of renal injury was also suppressed in this model.

To examine if the effects of RAS inhibitors to suppress the development of hypertension was specific to the SHR and its related strains, studies were performed on the Dahl salt-sensitive

Prevention and Regression of Chronic Kidney Disease and Hypertension 423

This hypothesis was supported by experiments in which the agonist angiotensin II was administered during the 'critical period' from age 4 to 8 weeks, after which all treatments were discontinued. Rats which had been transiently exposed to angiotensin II during this period were found to have elevated values of blood pressure which were 10-20 mmHg higher than rats which had been exposed to saline vehicle. Morever these rats were more susceptible to the subsequent development of renal vascular injury, and increased renin synthesis at a later time point (age 18 weeks), and to have a much higher mortality after L-NAME administration (Ishiguro et al., 2007). Thus, the effects of angiotensin II administration were the opposite of the effects of ARB, and were found to cause an acceleration of the 'reno-vascular amplifier' in

The results of animal studies on hypertension prevention have been supported clinically by the TROPHY study (Julius et al., 2006). In this prospective, randomized, multi-center study designed by Julius et al., patients with prehypertension and systolic blood pressure of 130- 139 mmHg and/or diastolic blood pressure of 85-89 mmHg were randomized to placebo or the ARB candesartan cilexetil (16 mg/day) for two years, then both groups were switched to placebo for the next two years. The primary end-point was the development of hypertension. As in the animal studies, the treatment with ARB caused a suppression of the development of hypertension, not only during the active treatment period (first two years), but even after the active treatment had been discontinued. The absolute risk reduction at the end of two or four years was 26.8 % and 9.8 % respectively, whereas the corresponding values of relative risk reduction (when relative risk is defined as the frequency of events in the treated group divided by the events in the placebo group) were 66.3 % and 15.6 %, respectively. Changes in the systolic blood pressure at the end of the study were small (2

Hypertension is associated with increased peripheral arterial resistance, and most of the resistance develops in the resistance arteries of the microvasculature, which includes both arterioles and small arteries with diameters < 400 um. The importance of the microvasculature in the pathogenesis and maintenance of hypertension was originally proposed by Folkow, who pointed out that a vicious cycle exists between increased blood pressure and vascular hypertrophy (Folkow, 1990). According to this hypothesis, hypertension may be initiated by a specific fast-acting pressor mechanism (e.g. angiotensin II) that increases blood pressure and initiates a positive feedback loop that induces vascular hypertrophy and maintains the hypertension. The hypothesis was later refined by Lever and Harrap, who proposed further elements: an abnormal or 'reinforced' hypertrophic response to pressure, and an increase of a humoral agent that causes hypertrophy directly (Lever et al., 1992). Animal studies have provided evidence to support the hypothesis that arteriolar restructuring may act as a primary accelerator of hypertension and provide a driving force for the progression of hypertension (Feihl et al., 2006; Intengan et al., 2001; Skov et al., 2004)). In particular, increased renal vascular resistance has been well documented in the SHR model of hypertension (Dilley et al., 1984), and morphometric studies on the afferent arteriole of SHR and Wistar-Kyoto rats (WKY) have confirmed that afferent arteriolar diameters are smaller in SHR compared to WKY (Kimura et al., 1989) (Gattone et al., 1983). Importantly, these differences are already seen in the 4-week-old SHR, even before blood pressure is significantly increased compared to WKY controls (Kimura et al., 1989).

this model of accelerated hypertension and renal injury.

mmHg), but statistically significant.

**3.2 Regression of hypertension** 

rat, which is a model of salt-sensitive hypertension with a low renin profile (Nakaya et al., 2002). These studies revealed that treatment of Dahl salt-sensitive rats with an ARB during the same 'critical period' (age 3 to 10 weeks) prevented the later development of saltinduced hypertension in this model even when the ARB treatment had been discontinued, and also a partial attenuation of renal injury induced by salt loading.

To examine the mechanisms of these long-lasting effects of RAS blockade, further studies were performed using the SHR/L-NAME model, which is a model of accelerated hypertension characterized by marked renal injury (Ishiguro et al., 2007). SHR were treated with a RAS inhibitor (ACE inhibitor or ARB), or a vasodilator (hydralazine), or a calcium chanel blocker (CCB, nitrendipine) during the 'critical period' from age 3 to 10 weeks. Medications were discontinued at age 10 weeks, and the rats observed without treatment for two months. At age 18 weeks, the rats were administered the NO synthase inhibitor L-NAME in the drinking water for 3 weeks to induce renal injury, and sacrificed at age 21 weeks. Interestingly, the rats treated with a RAS inhibitor had reduced vascular injury (arterial hypertrophy, endothelial thickening, and lumen narrowing) compared to vasodilator- or CCB-treated rats, and reduced renin mRNA, probably due to attenuation of the intrarenal vascular injury and renal ischemia induced by L-NAME. To explain all these experimental findings, we proposed a 'reno-vascular amplifier' mechanism for the development of hypertension and renal injury in this model (Fig. 4). High blood pressure is known to cause vascular hypertrophy in the resistance vessels, which consists predominantly of inward 'eutrophic' remodeling. When this remodeling is accentuated, as in the SHR/L-NAME model, glomerular perfusion decreases, which results in increased synthesis of renin and activation of the RAS. These changes cause a further increase in the blood pressure, resulting in a vicious cycle which causes accelerated hypertension. RAS inhibitors can block this vicious cycle by attenuating both the increase in blood pressure, and importantly, by decreasing the vascular hypertrophy of the resistance arteries.

Fig. 4. Inhibition of the 'reno-vascular amplifier' as a proposed mechanism for prevention of hypertension in the SHR/L-NAME model.

rat, which is a model of salt-sensitive hypertension with a low renin profile (Nakaya et al., 2002). These studies revealed that treatment of Dahl salt-sensitive rats with an ARB during the same 'critical period' (age 3 to 10 weeks) prevented the later development of saltinduced hypertension in this model even when the ARB treatment had been discontinued,

To examine the mechanisms of these long-lasting effects of RAS blockade, further studies were performed using the SHR/L-NAME model, which is a model of accelerated hypertension characterized by marked renal injury (Ishiguro et al., 2007). SHR were treated with a RAS inhibitor (ACE inhibitor or ARB), or a vasodilator (hydralazine), or a calcium chanel blocker (CCB, nitrendipine) during the 'critical period' from age 3 to 10 weeks. Medications were discontinued at age 10 weeks, and the rats observed without treatment for two months. At age 18 weeks, the rats were administered the NO synthase inhibitor L-NAME in the drinking water for 3 weeks to induce renal injury, and sacrificed at age 21 weeks. Interestingly, the rats treated with a RAS inhibitor had reduced vascular injury (arterial hypertrophy, endothelial thickening, and lumen narrowing) compared to vasodilator- or CCB-treated rats, and reduced renin mRNA, probably due to attenuation of the intrarenal vascular injury and renal ischemia induced by L-NAME. To explain all these experimental findings, we proposed a 'reno-vascular amplifier' mechanism for the development of hypertension and renal injury in this model (Fig. 4). High blood pressure is known to cause vascular hypertrophy in the resistance vessels, which consists predominantly of inward 'eutrophic' remodeling. When this remodeling is accentuated, as in the SHR/L-NAME model, glomerular perfusion decreases, which results in increased synthesis of renin and activation of the RAS. These changes cause a further increase in the blood pressure, resulting in a vicious cycle which causes accelerated hypertension. RAS inhibitors can block this vicious cycle by attenuating both the increase in blood pressure,

and importantly, by decreasing the vascular hypertrophy of the resistance arteries.

Fig. 4. Inhibition of the 'reno-vascular amplifier' as a proposed mechanism for prevention of

hypertension in the SHR/L-NAME model.

and also a partial attenuation of renal injury induced by salt loading.

This hypothesis was supported by experiments in which the agonist angiotensin II was administered during the 'critical period' from age 4 to 8 weeks, after which all treatments were discontinued. Rats which had been transiently exposed to angiotensin II during this period were found to have elevated values of blood pressure which were 10-20 mmHg higher than rats which had been exposed to saline vehicle. Morever these rats were more susceptible to the subsequent development of renal vascular injury, and increased renin synthesis at a later time point (age 18 weeks), and to have a much higher mortality after L-NAME administration (Ishiguro et al., 2007). Thus, the effects of angiotensin II administration were the opposite of the effects of ARB, and were found to cause an acceleration of the 'reno-vascular amplifier' in this model of accelerated hypertension and renal injury.

The results of animal studies on hypertension prevention have been supported clinically by the TROPHY study (Julius et al., 2006). In this prospective, randomized, multi-center study designed by Julius et al., patients with prehypertension and systolic blood pressure of 130- 139 mmHg and/or diastolic blood pressure of 85-89 mmHg were randomized to placebo or the ARB candesartan cilexetil (16 mg/day) for two years, then both groups were switched to placebo for the next two years. The primary end-point was the development of hypertension. As in the animal studies, the treatment with ARB caused a suppression of the development of hypertension, not only during the active treatment period (first two years), but even after the active treatment had been discontinued. The absolute risk reduction at the end of two or four years was 26.8 % and 9.8 % respectively, whereas the corresponding values of relative risk reduction (when relative risk is defined as the frequency of events in the treated group divided by the events in the placebo group) were 66.3 % and 15.6 %, respectively. Changes in the systolic blood pressure at the end of the study were small (2 mmHg), but statistically significant.

#### **3.2 Regression of hypertension**

Hypertension is associated with increased peripheral arterial resistance, and most of the resistance develops in the resistance arteries of the microvasculature, which includes both arterioles and small arteries with diameters < 400 um. The importance of the microvasculature in the pathogenesis and maintenance of hypertension was originally proposed by Folkow, who pointed out that a vicious cycle exists between increased blood pressure and vascular hypertrophy (Folkow, 1990). According to this hypothesis, hypertension may be initiated by a specific fast-acting pressor mechanism (e.g. angiotensin II) that increases blood pressure and initiates a positive feedback loop that induces vascular hypertrophy and maintains the hypertension. The hypothesis was later refined by Lever and Harrap, who proposed further elements: an abnormal or 'reinforced' hypertrophic response to pressure, and an increase of a humoral agent that causes hypertrophy directly (Lever et al., 1992). Animal studies have provided evidence to support the hypothesis that arteriolar restructuring may act as a primary accelerator of hypertension and provide a driving force for the progression of hypertension (Feihl et al., 2006; Intengan et al., 2001; Skov et al., 2004)). In particular, increased renal vascular resistance has been well documented in the SHR model of hypertension (Dilley et al., 1984), and morphometric studies on the afferent arteriole of SHR and Wistar-Kyoto rats (WKY) have confirmed that afferent arteriolar diameters are smaller in SHR compared to WKY (Kimura et al., 1989) (Gattone et al., 1983). Importantly, these differences are already seen in the 4-week-old SHR, even before blood pressure is significantly increased compared to WKY controls (Kimura et al., 1989).

Prevention and Regression of Chronic Kidney Disease and Hypertension 425

treated rats compared to CCB-treated rats, while 5,671 were reduced. Several ECM-related genes were elevated in the ARB-treated rats, while MMP-9, TIMP-2, and TIMP-3 gene expressions were decreased in the ARB-treated group. These differences were also confirmed by real time RT-PCR. To examine if these changes in MMP expression could be involved in the observed reversal of renal arteriolar hypertrophy by ARB, the activities of different MMPs in the renal microvasculature were examined using a highly sensitive in situ zymography method. It was found that MMP-13 activity was markedly increased by ARB but not by CCB (Ishiguro et al., 2009). These results are compatible with a role for MMPs in the actions of ARB to cause reversal of renal arteriolar hypertrophy, and subsequent

Fig. 6. Proposed hypothesis for the mechanism of regression of hypertension by high-dose

To our knowledge, there have been no clinical studies which were specifically designed to address the question whether regression of hypertension (i.e. reversal of Grade 1 hypertension to high-normal blood pressure) is feasible in humans. For this reason, we are currently performing a prospective, multi-center study (STAR CAST) study to examine the effects of one-year treatment with an ARB or CCB on regression of hypertension (Sasamura et al., 2008). In this study, patients aged 30-59 with newly diagnosed hypertension and a positive family history of hypertension are randomized to treatment for one year with either an ARB (candesartan) or CCB (nifedipine XL). After one year, the patient's antihypertensive drug dose will be reduced, then withdrawn. The antihypertensive drug withdrawal success rate will be compared between the two antihypertensive agents, as an index of the regression of hypertension in the two groups. Because of safety concerns, the patients' home blood pressure will be monitored in real time using a home blood pressure monitoring system (i-TECHO). Although this study is being performed using standard doses of ARB, it is hoped that this trial will provide information concerning whether RAS inhibitors are indeed different from other antihypertensive agents in terms of long-term effects on blood pressure. If the results are encouraging, we hope to perform further clinical studies on CKD

and hypertension regression, using high or even ultrahigh doses of ARB.

remodeling of the renal microvasculature (Fig.6).

renin-angiotensin inhibitors.

Moreover, when SHR and normotensive rats were crossbred to form second generation hybrids, a narrowed afferent arteriole lumen diameter at 7 weeks was found to be a predictor of the later development of hypertension (Skov et al., 2004).

In our laboratory, the morphological effects of treatment with an ARB or CCB during the 'critical period' on renal small artery structure were examined in SHR. SHR were treated with an ARB or CCB from age 3 to 10 weeks, and sacrificed at age 10 weeks. The arteriolar hypertrophy was significantly reduced in the ARB-treated rats compared to the CCB-treated rats, despite similar reductions in blood pressure. These results were consistent with reports from other groups using RAS inhibitors in both animal models (Freslon et al., 1983), (Christensen et al., 1989) and humans (Schiffrin et al., 1994), (Thybo et al., 1995).

Recently, we reported that treatment of SHR with established hypertension with high-dose ARB (at 50-100 times the normal dose in rodents) resulted in a sustained decrease in hypertension, suggesting that regression of hypertension is feasible in this model (Ishiguro et al., 2009). Similar results were reported previously by Smallegange et al. using an ACE inhibitor combined with a low-salt diet (Smallegange et al., 2004). Examination of the effects of transient high-dose ARB therapy on renal arteriolar structure revealed a remarkable reversal of the arteriolar hypertrophy found in SHR treated with ARB, whereas this effect was not seen with CCB (Fig. 5). Interestingly, these findings were particularly noticeable in the small arteries (diameter 30-100 um) and arterioles in the kidney, compared to small arteries from other vascular beds, such as the brain, heart, and mesentery.

Fig. 5. Regression of hypertension in the SHR model by transient high-dose ARB treatment. (a) Effects on blood pressure (b) Effects on renal arteriolar media/lumen ratios. Reproduced with permission from Ishiguro/Hayashi et al. Hypertension 53:83-89, 2009.

To examine potential mechanisms of these changes, the gene expression profile of kidneys treated with ARB were compared with the kidneys treated with CCB. Using the Affymetrix rat 230 2.0 gene expression array, it was found that 1,345 genes were elevated in the ARB-

Moreover, when SHR and normotensive rats were crossbred to form second generation hybrids, a narrowed afferent arteriole lumen diameter at 7 weeks was found to be a

In our laboratory, the morphological effects of treatment with an ARB or CCB during the 'critical period' on renal small artery structure were examined in SHR. SHR were treated with an ARB or CCB from age 3 to 10 weeks, and sacrificed at age 10 weeks. The arteriolar hypertrophy was significantly reduced in the ARB-treated rats compared to the CCB-treated rats, despite similar reductions in blood pressure. These results were consistent with reports from other groups using RAS inhibitors in both animal models (Freslon et al., 1983),

Recently, we reported that treatment of SHR with established hypertension with high-dose ARB (at 50-100 times the normal dose in rodents) resulted in a sustained decrease in hypertension, suggesting that regression of hypertension is feasible in this model (Ishiguro et al., 2009). Similar results were reported previously by Smallegange et al. using an ACE inhibitor combined with a low-salt diet (Smallegange et al., 2004). Examination of the effects of transient high-dose ARB therapy on renal arteriolar structure revealed a remarkable reversal of the arteriolar hypertrophy found in SHR treated with ARB, whereas this effect was not seen with CCB (Fig. 5). Interestingly, these findings were particularly noticeable in the small arteries (diameter 30-100 um) and arterioles in the kidney, compared to small

Fig. 5. Regression of hypertension in the SHR model by transient high-dose ARB treatment. (a) Effects on blood pressure (b) Effects on renal arteriolar media/lumen ratios. Reproduced

To examine potential mechanisms of these changes, the gene expression profile of kidneys treated with ARB were compared with the kidneys treated with CCB. Using the Affymetrix rat 230 2.0 gene expression array, it was found that 1,345 genes were elevated in the ARB-

with permission from Ishiguro/Hayashi et al. Hypertension 53:83-89, 2009.

predictor of the later development of hypertension (Skov et al., 2004).

(Christensen et al., 1989) and humans (Schiffrin et al., 1994), (Thybo et al., 1995).

arteries from other vascular beds, such as the brain, heart, and mesentery.

treated rats compared to CCB-treated rats, while 5,671 were reduced. Several ECM-related genes were elevated in the ARB-treated rats, while MMP-9, TIMP-2, and TIMP-3 gene expressions were decreased in the ARB-treated group. These differences were also confirmed by real time RT-PCR. To examine if these changes in MMP expression could be involved in the observed reversal of renal arteriolar hypertrophy by ARB, the activities of different MMPs in the renal microvasculature were examined using a highly sensitive in situ zymography method. It was found that MMP-13 activity was markedly increased by ARB but not by CCB (Ishiguro et al., 2009). These results are compatible with a role for MMPs in the actions of ARB to cause reversal of renal arteriolar hypertrophy, and subsequent remodeling of the renal microvasculature (Fig.6).

Fig. 6. Proposed hypothesis for the mechanism of regression of hypertension by high-dose renin-angiotensin inhibitors.

To our knowledge, there have been no clinical studies which were specifically designed to address the question whether regression of hypertension (i.e. reversal of Grade 1 hypertension to high-normal blood pressure) is feasible in humans. For this reason, we are currently performing a prospective, multi-center study (STAR CAST) study to examine the effects of one-year treatment with an ARB or CCB on regression of hypertension (Sasamura et al., 2008). In this study, patients aged 30-59 with newly diagnosed hypertension and a positive family history of hypertension are randomized to treatment for one year with either an ARB (candesartan) or CCB (nifedipine XL). After one year, the patient's antihypertensive drug dose will be reduced, then withdrawn. The antihypertensive drug withdrawal success rate will be compared between the two antihypertensive agents, as an index of the regression of hypertension in the two groups. Because of safety concerns, the patients' home blood pressure will be monitored in real time using a home blood pressure monitoring system (i-TECHO). Although this study is being performed using standard doses of ARB, it is hoped that this trial will provide information concerning whether RAS inhibitors are indeed different from other antihypertensive agents in terms of long-term effects on blood pressure. If the results are encouraging, we hope to perform further clinical studies on CKD and hypertension regression, using high or even ultrahigh doses of ARB.

Prevention and Regression of Chronic Kidney Disease and Hypertension 427

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