**1. Introduction**

414 Chronic Kidney Disease

Rush D, Nickerson P, Gough J et al. (1998). Beneficial effects of treatment of early subclinical rejection: a randomized study. *J Am SocNephrol*; Vol. 9, pp. 2129–2134 Safinia N, Afzali B, Atalar K, Lombardi G, Lechler RI. (2010). T-cell alloimmunity and chronic allograft dysfunction. *Kidney Int;* Vol. 78 (Suppl 119) pp. S2–S12 Salama AD, Delikouras A, Pusey CD et al. (2001). Transplantaccommodation in highly

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Chronic kidney disease (CKD) is a disease which is characterized by the presence of renal damage or decreased GFR for at least 3 months. The prevalence of CKD in the US has been reported to be 3.3% (stage 1), 3.0% (stage 2), 4.3% (stage 3), 0.2% (stage 4), and 0.1% (stage 5) (Levey et al., 2003; 2002). Because of the increasing elderly population in industrial countries, the development of new strategies for the prevention and regression of CKD is important.

Clinical studies have suggested that renin-angiotensin system (RAS) inhibitors can exert a renoprotective effect independent of blood pressure, and attenuate the progression of renal dysfunction (Bakris, 2010; Berl, 2009; Stojiljkovic et al., 2007). Recent studies have suggested that the use of RAS inhibitors, when combined with other treatment modalities such as aggressive blood pressure control, lowering of blood lipids, tight glucose control for diabetics, and lifestyle changes may cause remission of albuminuria, and stablization or even reversal of the decline in GFR, i.e. regression of CKD in some patients (Aros et al., 2002; Macconi, ; Ruggenenti et al., 2008).

These early clinical findings are important, because they suggest that appropriate interventions may be effective for causing an improvement in renal function, which raises the hope that a 'cure' for CKD may eventually be found in the future. In our laboratory, we have been examining the molecular mechanisms involved in the pathogenesis of CKD and hypertension. Our underlying concept is that both these diseases are highly related, and share common pathophysiological mechanisms, including the abnormal accumulation of extracellular matrix proteins in the kidney. The result is glomerulosclerosis, when the matrix accumulates in the renal glomeruli, and renal arteriolosclerosis, when the matrix is deposited in the renal arterioles and small vessels. In this chapter, we will review the evidence from our and other laboratories that these processes may be reversed in animal models, and possibly in humans.

#### **2.1 Studies on CKD prevention**

Regardless of the initial injury, most causes of CKD (including diabetic nephropathy, and chronic glomerulonephritis) share several common pathological features, one of which is the development of glomerular scarring or glomerulosclerosis.

Prevention and Regression of Chronic Kidney Disease and Hypertension 417

Although it has been widely accepted that established sclerosis is irreversible, recent studies have emerged to challenge this concept and to focus on developing new therapies to cause regression or reversal of established glomerulosclerosis (Ma et al., 2007), (Ruggenenti et al., 2001). In particular, studies have suggested that treatment with high-dose RAS inhibitor may be effective in causing regression of glomerular lesions in animal models (Ma et al.,

Recently, we reported that transient treatment with an angiotensin receptor blocker (ARB) at a 50-100 times the normal dose in rodents causes regression of glomerulosclerosis in mice (Hayashi et al., 2010). In this study, the effects of treatment with different doses of ARB on established lesions of glomerulosclerosis were examined in the adriamycin nephropathy model, with a focus on whether the regression was sustained after cessation of the ARB treatment. Furthermore, the involvement of matrix metalloproteinase (MMP)-2 in the mechanism of glomerulosclerosis regression were examined both in vitro and in vivo, using a non-specific MMP inhibitor (doxycycline), and knockout (KO) mice with targeted deletion

The principal findings of the study are shown in Fig. 2. and Fig. 3. It was found that transient treatment for two weeks with the ARB candesartan caused a regression of established glomerulosclerosis, which was clearly evident with the high doses of ARB and was sustained 6 months after cessation of all treatments. Moreover, the ARB treatment

Fig. 2. Effects of different doses of ARB (candesartan) on regression of glomerulosclerosis in the adriamycin-nephropathy model. (a) Experimental protocol (b) Effects on glomerular sclerosis (c) Representative photomicrographs. Reproduced with permission from Hayashi

**2.2 Studies on CKD regression** 

of MMP-2.

et al. Kidney Int 78:69-78, 2010.

2005), (Teles et al., 2009), (Macconi et al., 2009).

Fig. 1. Relationship between progression and regression of chronic kidney disease.

Glomerulosclerosis occurs because of the excessive deposition of components of the extracellular matrix (ECM) in the glomeruli, resulting in changes in glomerular integrity and albuminuria. This process is triggered by increased synthesis of ECM components, and decreased degradation of ECM components, resulting in net accumulation of ECM (Ma et al., 2007). It is thought that, once renal function declines below a 'point of no return', the decline in glomerular function continues inexorably due to the continuous accumulation of ECM and progression of glomerulosclerosis. Glomerular hypertension has been suggested to play an important role in this process, because the decrease in glomerular filtration leads to a compensatory increase in glomerular hypertension, resulting in a vicious cycle which causes progression of glomerular injury and loss of renal function (Neuringer et al., 1992).

Because of the progressive nature of CKD, one of the optimum strategies for reducing CKD would be to find interventions to prevent new-onset CKD. Multiple clinical studies have shown that the use of RAS inhibitors in patients with and without diabetes can cause a decline in the progression of CKD, which may be mediated, at least in part, by a blood pressure-independent mechanism (Bakris, 2010; Berl, 2009; Stojiljkovic et al., 2007). More recently, several studies have suggested that the use of RAS inhibitors may also be effective in preventing new-onset CKD, especially in patients with diabetes. In particular, Ruggenenti et al. showed in the BENEDICT trial that, in hypertensive patients with type 2 diabetes and no microalbuminuria at baseline, the angiotensin-converting enzyme (ACE) inhibitor trandolapril significantly decreased the risk of developing microalbuminuria compared with conventional therapy (Ruggenenti et al., 2008) . Similarly, in the recent ROADMAP study, the use of the ARB olmesartan was associated with a delayed onset of microalbuminuria (Haller et al.) . These results are important, because they suggest that diabetic nephropathy can be prevented or at least delayed by appropriate intervention (Remuzzi et al., 2006).

At present, it is unclear from clinical studies whether these measures may be effective for prevention of new-onset CKD in non-diabetic patients. However, the data from animal studies are encouraging, and suggest that early intervention with a RAS inhibitor may be effective for the prevention of renal injury due to hypertension (Ishiguro et al., 2007; Nakaya et al., 2001), salt-loading (Nakaya et al., 2002), or irradiation (Moulder et al., 1996).

#### **2.2 Studies on CKD regression**

416 Chronic Kidney Disease

Fig. 1. Relationship between progression and regression of chronic kidney disease.

Glomerulosclerosis occurs because of the excessive deposition of components of the extracellular matrix (ECM) in the glomeruli, resulting in changes in glomerular integrity and albuminuria. This process is triggered by increased synthesis of ECM components, and decreased degradation of ECM components, resulting in net accumulation of ECM (Ma et al., 2007). It is thought that, once renal function declines below a 'point of no return', the decline in glomerular function continues inexorably due to the continuous accumulation of ECM and progression of glomerulosclerosis. Glomerular hypertension has been suggested to play an important role in this process, because the decrease in glomerular filtration leads to a compensatory increase in glomerular hypertension, resulting in a vicious cycle which causes progression of glomerular injury and loss of renal function (Neuringer et al., 1992). Because of the progressive nature of CKD, one of the optimum strategies for reducing CKD would be to find interventions to prevent new-onset CKD. Multiple clinical studies have shown that the use of RAS inhibitors in patients with and without diabetes can cause a decline in the progression of CKD, which may be mediated, at least in part, by a blood pressure-independent mechanism (Bakris, 2010; Berl, 2009; Stojiljkovic et al., 2007). More recently, several studies have suggested that the use of RAS inhibitors may also be effective in preventing new-onset CKD, especially in patients with diabetes. In particular, Ruggenenti et al. showed in the BENEDICT trial that, in hypertensive patients with type 2 diabetes and no microalbuminuria at baseline, the angiotensin-converting enzyme (ACE) inhibitor trandolapril significantly decreased the risk of developing microalbuminuria compared with conventional therapy (Ruggenenti et al., 2008) . Similarly, in the recent ROADMAP study, the use of the ARB olmesartan was associated with a delayed onset of microalbuminuria (Haller et al.) . These results are important, because they suggest that diabetic nephropathy can be prevented or at least delayed by appropriate intervention (Remuzzi et al., 2006).

At present, it is unclear from clinical studies whether these measures may be effective for prevention of new-onset CKD in non-diabetic patients. However, the data from animal studies are encouraging, and suggest that early intervention with a RAS inhibitor may be effective for the prevention of renal injury due to hypertension (Ishiguro et al., 2007; Nakaya

et al., 2001), salt-loading (Nakaya et al., 2002), or irradiation (Moulder et al., 1996).

Although it has been widely accepted that established sclerosis is irreversible, recent studies have emerged to challenge this concept and to focus on developing new therapies to cause regression or reversal of established glomerulosclerosis (Ma et al., 2007), (Ruggenenti et al., 2001). In particular, studies have suggested that treatment with high-dose RAS inhibitor may be effective in causing regression of glomerular lesions in animal models (Ma et al., 2005), (Teles et al., 2009), (Macconi et al., 2009).

Recently, we reported that transient treatment with an angiotensin receptor blocker (ARB) at a 50-100 times the normal dose in rodents causes regression of glomerulosclerosis in mice (Hayashi et al., 2010). In this study, the effects of treatment with different doses of ARB on established lesions of glomerulosclerosis were examined in the adriamycin nephropathy model, with a focus on whether the regression was sustained after cessation of the ARB treatment. Furthermore, the involvement of matrix metalloproteinase (MMP)-2 in the mechanism of glomerulosclerosis regression were examined both in vitro and in vivo, using a non-specific MMP inhibitor (doxycycline), and knockout (KO) mice with targeted deletion of MMP-2.

The principal findings of the study are shown in Fig. 2. and Fig. 3. It was found that transient treatment for two weeks with the ARB candesartan caused a regression of established glomerulosclerosis, which was clearly evident with the high doses of ARB and was sustained 6 months after cessation of all treatments. Moreover, the ARB treatment

Fig. 2. Effects of different doses of ARB (candesartan) on regression of glomerulosclerosis in the adriamycin-nephropathy model. (a) Experimental protocol (b) Effects on glomerular sclerosis (c) Representative photomicrographs. Reproduced with permission from Hayashi et al. Kidney Int 78:69-78, 2010.

Prevention and Regression of Chronic Kidney Disease and Hypertension 419

As shown in Fig. 3, the results of highly-sensitive in situ zymography and immunofluorescence suggested that MMP-2 might be upregulated in glomerular podocytes, but this could not be determined accurately because of the relatively low expression of MMP-2 protein. Therefore, to further characterize the mechanisms of the ARB-induced increase in glomerular MMP-2 activity, we examined the effects of ARB treatment in cultured podocytes. These experiments revealed that ARB treatment of podocytes resulted in a dose-dependent increase in MMP-2 activity in the supernatant. Podocytes are known to express components of the RAS, including renin, angiotensinogen, angiotensin-converting enzyme, and AT1 and AT2 receptors (Durvasula et al., 2006), (Durvasula et al., 2008), (Liebau et al., 2006). Morever, functional expression of the renin-angiotensin system has been documented in both mouse and human podocytes (Durvasula et al., 2008), (Liebau et al., 2006). To examine the possibility that the effects of ARB were mediated through inhibition of the RAS, further studies were performed using an ACE inhibitor, and a nonpeptide Ang II antagonist (Saralasin). The use of these different RAS inhibitors yielded similar results, suggesting that the effects of ARB were mediated by inhibition of the

Moreover, it was observed in vitro that the increase in MMP-2 activity was greatest at the high doses of candesartan (greater than 0.1 umol/L), whereas maximum plasma concentrations in humans administered a standard dose of candesartan are below the nanomolar range (Pfister et al., 1999). Assuming that local (glomerular) concentrations of ARB will be greatest with the high-doses of ARB, these in vitro results are consistent with the in vivo observation that the glomerulosclerosis regression was maximal with the high

In humans, it is known that MMP-1 (collagenase-1) also plays a major role in the breakdown of collagens, in particular type I and type III collagen. It has been reported that rodents lack the human MMP-1 gene, and MMP-13 (collagenase-3) is the main collagenase in mice (Henriet et al., 1992), (Parks et al., 2000.). When the possibility that MMP-13 may also contribute to the observed changes was examined, it was found that ARB treatment did not increase glomerular MMP-13 activity, but rather decreased the activity, suggesting that increased MMP-13 activity did not contribute to the observed regression of

We also examined whether the effects of ARB could be attenuated by pretreating the mice with doxycycline, or by performing studies on mice with a deletion of the MMP-2 gene. It was found that neither inhibition of MMP nor deletion of MMP-2 completely abolished the effects of high-dose ARB, suggesting that other mechanisms may be involved, including the involvement of other proteases such as the serine protease plasminogen activator inhibitor-1 (PAI-1) (Ma et al., 2005). Other studies have suggested that regeneration of glomerular podocyte function may also play a role in the regression of glomerulosclerosis by RAS

It should be noted that the effects of ARB on regression may differ widely in different animal models. In particular, the effects of ARB on regression were less marked in the 5/6 nephrectomy model (Ma et al., 2005). This may be because the adriamycin model relies on a single (acute) injury to the glomeruli, whereas the injury in the 5/6 nephrectomy model is a continuous process. In the studies on the adriamycin nephropathy model, it was found that

glomerulosclerosis in the adriamycin nephropathy model (Hayashi et al., 2010).

intrinsic RAS in podocytes.

inhibitors (Macconi et al., 2009).

doses of ARB.

caused a dose-dependent increase in glomerular MMP-2 activity and decrease in type IV collagen accumulation. The ARB-induced regression of glomerulosclerosis was attenuated by pretreatment with the MMP inhibitor doxycycline, as well as in mice with targeted deletion of the MMP-2 gene, suggesting the possibility that increased expression of MMP-2 may contribute to the regression of glomerulosclerosis and type IV collagen deposition seen in the high-dose ARB-treated groups.

The MMP family constitutes a multigene family of zinc- and calcium-dependent endopeptidases which play a major role in the degradation of collagen and other ECM components (Woessner, 1991), (Baramova et al., 1995), (Sasamura et al., 2005). MMP-2 (also known as gelatinase A) is a MMP which is found in the conditioned media of cultured fibroblasts, and is involved in the cleavage of multiple ECM proteins including type IV collagen (Woessner, 1991), (Baramova et al., 1995). In contrast to gelatinase B (MMP-9), MMP-2 is not highly expressed in normal or diseased glomeruli (Urushihara et al., 2002). However, it has been shown that renal MMP-2 expression and activity are upregulated by ACE inhibitors in rats with diabetes (McLennan et al., 2002), (Sun et al., 2006). Moreover, Turkay et al reported that the ACE inhibitor enalapril also increased hepatic MMP-2 expression in rats with experimental hepatic fibrogenesis (Turkay et al., 2008), while Westermann et al. showed that the ARB irbesartan increased MMP-2 activity in the hearts of mice with cardiomyopathy (Westermann et al., 2007), suggesting that the RAS plays a key role in regulation of MMP-2 expression in the kidney and other tissues.

Fig. 3. Effects of different doses of ARB (candesartan) on glomerular MMP-2 and MMP-9 activity and expression in the adriamycin-nephropathy model. Representative results of (a) highly-sensitive in situ zymography (b) immunofluorescence. Quantification of glomerular MMP activity by (c) in situ zymography (d) ELISA. Reproduced with permission from Hayashi et al. Kidney Int 78:69-78, 2010.

caused a dose-dependent increase in glomerular MMP-2 activity and decrease in type IV collagen accumulation. The ARB-induced regression of glomerulosclerosis was attenuated by pretreatment with the MMP inhibitor doxycycline, as well as in mice with targeted deletion of the MMP-2 gene, suggesting the possibility that increased expression of MMP-2 may contribute to the regression of glomerulosclerosis and type IV collagen deposition seen

The MMP family constitutes a multigene family of zinc- and calcium-dependent endopeptidases which play a major role in the degradation of collagen and other ECM components (Woessner, 1991), (Baramova et al., 1995), (Sasamura et al., 2005). MMP-2 (also known as gelatinase A) is a MMP which is found in the conditioned media of cultured fibroblasts, and is involved in the cleavage of multiple ECM proteins including type IV collagen (Woessner, 1991), (Baramova et al., 1995). In contrast to gelatinase B (MMP-9), MMP-2 is not highly expressed in normal or diseased glomeruli (Urushihara et al., 2002). However, it has been shown that renal MMP-2 expression and activity are upregulated by ACE inhibitors in rats with diabetes (McLennan et al., 2002), (Sun et al., 2006). Moreover, Turkay et al reported that the ACE inhibitor enalapril also increased hepatic MMP-2 expression in rats with experimental hepatic fibrogenesis (Turkay et al., 2008), while Westermann et al. showed that the ARB irbesartan increased MMP-2 activity in the hearts of mice with cardiomyopathy (Westermann et al., 2007), suggesting that the RAS plays a key

role in regulation of MMP-2 expression in the kidney and other tissues.

Fig. 3. Effects of different doses of ARB (candesartan) on glomerular MMP-2 and MMP-9 activity and expression in the adriamycin-nephropathy model. Representative results of (a) highly-sensitive in situ zymography (b) immunofluorescence. Quantification of glomerular MMP activity by (c) in situ zymography (d) ELISA. Reproduced with permission from

in the high-dose ARB-treated groups.

Hayashi et al. Kidney Int 78:69-78, 2010.

As shown in Fig. 3, the results of highly-sensitive in situ zymography and immunofluorescence suggested that MMP-2 might be upregulated in glomerular podocytes, but this could not be determined accurately because of the relatively low expression of MMP-2 protein. Therefore, to further characterize the mechanisms of the ARB-induced increase in glomerular MMP-2 activity, we examined the effects of ARB treatment in cultured podocytes. These experiments revealed that ARB treatment of podocytes resulted in a dose-dependent increase in MMP-2 activity in the supernatant. Podocytes are known to express components of the RAS, including renin, angiotensinogen, angiotensin-converting enzyme, and AT1 and AT2 receptors (Durvasula et al., 2006), (Durvasula et al., 2008), (Liebau et al., 2006). Morever, functional expression of the renin-angiotensin system has been documented in both mouse and human podocytes (Durvasula et al., 2008), (Liebau et al., 2006). To examine the possibility that the effects of ARB were mediated through inhibition of the RAS, further studies were performed using an ACE inhibitor, and a nonpeptide Ang II antagonist (Saralasin). The use of these different RAS inhibitors yielded similar results, suggesting that the effects of ARB were mediated by inhibition of the intrinsic RAS in podocytes.

Moreover, it was observed in vitro that the increase in MMP-2 activity was greatest at the high doses of candesartan (greater than 0.1 umol/L), whereas maximum plasma concentrations in humans administered a standard dose of candesartan are below the nanomolar range (Pfister et al., 1999). Assuming that local (glomerular) concentrations of ARB will be greatest with the high-doses of ARB, these in vitro results are consistent with the in vivo observation that the glomerulosclerosis regression was maximal with the high doses of ARB.

In humans, it is known that MMP-1 (collagenase-1) also plays a major role in the breakdown of collagens, in particular type I and type III collagen. It has been reported that rodents lack the human MMP-1 gene, and MMP-13 (collagenase-3) is the main collagenase in mice (Henriet et al., 1992), (Parks et al., 2000.). When the possibility that MMP-13 may also contribute to the observed changes was examined, it was found that ARB treatment did not increase glomerular MMP-13 activity, but rather decreased the activity, suggesting that increased MMP-13 activity did not contribute to the observed regression of glomerulosclerosis in the adriamycin nephropathy model (Hayashi et al., 2010).

We also examined whether the effects of ARB could be attenuated by pretreating the mice with doxycycline, or by performing studies on mice with a deletion of the MMP-2 gene. It was found that neither inhibition of MMP nor deletion of MMP-2 completely abolished the effects of high-dose ARB, suggesting that other mechanisms may be involved, including the involvement of other proteases such as the serine protease plasminogen activator inhibitor-1 (PAI-1) (Ma et al., 2005). Other studies have suggested that regeneration of glomerular podocyte function may also play a role in the regression of glomerulosclerosis by RAS inhibitors (Macconi et al., 2009).

It should be noted that the effects of ARB on regression may differ widely in different animal models. In particular, the effects of ARB on regression were less marked in the 5/6 nephrectomy model (Ma et al., 2005). This may be because the adriamycin model relies on a single (acute) injury to the glomeruli, whereas the injury in the 5/6 nephrectomy model is a continuous process. In the studies on the adriamycin nephropathy model, it was found that

Prevention and Regression of Chronic Kidney Disease and Hypertension 421

Metabolomics is a discipline dedicated to the global study of metabolites, their dynamics, composition, interactions, and responses to interventions or to changes in their environment (Oresic, 2009), and the recent development of metabolome analysis technology allows the global 'metabolome' to be assessed comprehensively in individual patients. An important advantage of metabolome analysis is the potential to identify new and unidentified metabolites which could have important pathophysiological functions. In a recent study, we obtained serum and urine samples from 15 patients and 7 healthy volunteers, and compared the metabolome profiles of the two groups. Serum or urine samples (100 ul) were added to methanol (900 ul) containing internal standards, deproteinised, and subjected to anionic and cationic capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) analysis

The results of our metabolome analysis suggested that serum and urine levels of several amino acid, nucleic acid, and carbohydrate metabolites were altered in patients from an early stage of CKD (Hayashi et al., 2011). We also found evidence for the presence of several novel metabolites which were markedly increased or decreased in the patients with CKD compared to controls. We are performing further studies to examine the structure of these unidentified products, with the final aim to find new biomarkers of disease 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

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.,

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

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

pressure in the population increases at every age (Qureshi et al., 2005).

that the development of renal injury was also suppressed in this model.

which may be utilized in clinical studies.

**3.1 Prevention of hypertension** 

1992), (Gillies et al., 1997).

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

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 ameliorating glomerular injury.
