**Renal Angiotensinogen Gene Expression and Tubular Atrophy in Diabetic Nephropathy**

Brice E. T. Nouthe1, Maya Saleh2, Shao-Ling Zhang1 and John S. D. Chan1,\* *1Université de Montréal, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Hôtel-Dieu Hospital, Pavillon Masson, Montreal, QC 2McGill University, Department of Medicine, Centre for the Study of Host Resistance and Complex Trait Group, Montreal, QC Canada* 

#### **1. Introduction**

30 Diabetic Nephropathy

Wei, L.; Alhenc-Gelas, F.; Corvol, P. & Clauser, E. (1991). The two homologous domains of

Williams, B. (1994). Insulin resistance: the shape of things to come. *The Lancet,* Vol.344,

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Zimpelmann, J.; Kumar, D.; Levine, D.Z.; Wehbi, G.; Imig, J.D.; Navar, L.G. & Burns, K.D.

*Society of Nephrology*, Vol.17, No.11, (November 2006), pp. 3067-3075. Yusuf, S.; Sleight, P.; Pogue, J.; Bosch, J.; Davies, R. & Dagenais, G. (2000). Effects of an

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Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymatic activity. *Biochemical Journal,* Vol.

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angiotensin II in blood pressure control: lessons from tissue-specific expression of angiotensin-converting enzyme (ACE). *Critical Reviews in Eukaryotic Gene* 

V.J.; Horiuchi, M. (1999). AT2 receptor and vascular smooth muscle cell differentiation in vascular development. *Hypertension*. Vol. 33, No. 6, (June 1999),

localization and expression of Angiotensin-converting enzyme 2 and Angiotensinconverting enzyme: implications for albuminuria in diabetes. *Journal of the American* 

angiotensin converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. *New England Journal of Medicine,* Vol.342, No.3, (January 2000), pp. 145-153, ISSN

(2000). Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat. *Kidney International,* Vol.58, No.6, (December 2000), pp. 2320-2330, ISSN The growing incidence of diabetes mellitus, with predicted rises in prevalence from 285 to 380 million cases in 2025, then 438 million by 2030, is a major public health burden in both developing and developed countries. Type 1 and type 2 diabetes increase the risk of microvascular complications, which cause significant morbidity and mortality. Diabetic nephropathy (DN) and retinopathy represent the major causes of end-stage renal disease and blindness (1-2) in developed countries. DN is associated with an increased risk of hypertension, adverse cardiovascular events (3), chronic kidney diseases and haemodialysis (4). Efforts are therefore being made to find ways of preventing and/or slowing down the progression of DN worldwide.

DN is initiated by glomerular changes, namely hypertrophy, then thickening of the basement membrane with subsequent expansion of the mesangial matrix and glomerulosclerosis (5). This is associated not only with microalbuminuria, an early clinically detectable lesion, but also with tubulointerstitial fibrosis and tubular atrophy (5-6). Oxidative stress, hyperglycemia and renin-angiotensin system (RAS) dysfunction have been linked to the development of these lesions (5-6). Although albuminuria is a useful clinical marker, tubulointerstitial fibrosis and tubular atrophy represent a better predictor of nephropathy progression because of their close association with declining renal function (5). Many randomized controlled trials have shown the efficacy of optimal glycemic control and RAS blockade in the primary and secondary prevention of DN (4, 7-9). The former is easily understood, as decreased "glucotoxicity" reduces end-organ damage. However, the mechanisms underlying the protective action of RAS inhibition, notably angiotensin II (Ang II) receptor blockade, are not well understood. In this review, we present the recent results of studies aiming to understand the consequences of RAS blockade at the molecular level, with an emphasis on tubular lesions in DN.

<sup>\*</sup> Corresponding Author

Renal Angiotensinogen Gene Expression and Tubular Atrophy in Diabetic Nephropathy 33

It has been demonstrated that high glucose, together with Ang II, is involved in tubular lesions seen in DN (5, 24). Indeed, high glucose and Ang II enhance angiotensinogen (Agt, the sole precursor of all angiotensins) gene expression, both in vitro in rat immortalized renal proximal tubular cells (iRPTC) (25, 26) and in vivo in streptozotocin-treated mice (a model of diabetic mice) proximal tubules (5, 24). This turns into a vicious circle, increasing tubular atrophy, as Agt is the sole substrate of the RAS and is used for synthesis of Ang II.

The importance of the systemic RAS in blood pressure control and sodium homeostasis has been well accepted and Ang II has been recognized as a cardinal parameter in the development of both hypertension and kidney injury (5, 13, 15, 27). Overactivation of AT1 R by Ang II therefore represents a target for treatment, but as Ang II has many other paracrine effects (induction of inflammation, mitogenesis, cell growth, apoptosis, differentiation, migration, etc.), current therapies are not sufficient to reverse the consequences of Ang II hyperaction. Of note, local RAS have been unravelled in some organs, notably the kidneys, with luminal fluid levels of Ang II being at least 1,000-fold higher than in the plasma (28). This local RAS could also play an important role in sodium retention and blood pressure regulation and hypertension, representing both a cause and a consequence of kidney injury. Complex interactions between diabetes and hypertension due to similar etiologies of both conditions, together with the stimulating effect of hyperglycemia on Ang II production in

The classic components of the RAS are all found in renal proximal tubules, including Agt and the enzymes (prorenin/renin, ACE, angiotensin-converting enzyme 2 (Ace2), aminopeptidases and carboxy peptidases). Upon cleavage of the prorenin into a proteolytic enzyme, renin will cleave Agt into a decapeptide: angiotensin I (Ang I). Then the dipeptidyl peptidase ACE will remove 2 amino acids from the latter and generate Ang II. Ang II is further metabolized into smaller fragments, such as Ang 1-7 and Ang III, Ang IV and Ang V, by various peptidases. Among those peptidases, Ace2 is a human homologue to ACE (42% similarity) that was discovered in 2000; it cleaves Ang I into Ang 1-9 /Ang II to Ang 1- 7, both having hemodynamic properties (29). While ACE is present in most tissues, Ace2 is specifically expressed in the kidney, and less in the testes and heart ,with neither ARB nor

iNOs/Larginine/O2 +NADPH (NO&cGMP) NF-kappa B

Table 1. Signalling pathways stimulated by AT1, AT2 and Mas receptors

Phospholipase A2 Phospholipase C Jak/STAT ITP Ca2+

Adenyl cyclase

**2.3 From angiotensinogen to angiotensin II** 

vitro, point to an important role for local RAS in DN.

**2.3.1 Synthesis and degradation of angiotensin II** 

p21ras, C-Src

PKC (MAPK&TGF-1) NADPH (ROS)

AT1 R AT2 R Mas receptors Activation Inhibition Activation Inhibition Activation Inhibition

> PKC (MAPK)

p38MAPK, SHIP-1 (PI3K) Erk 1/2 Bradykinines

TGF-1

(Prostaglandins),

COX

NO

#### **2. The renin-angiotensin system in diabetic nephropathy**

#### **2.1 Clinical findings on the implications of RAS blockade in diabetic nephropathy**

The benefits of RAS inhibition on end-organ protection in diabetic patients are well established. One of the early clinical trials on diabetic patients was performed with captopril, an angiotensin-converting enzyme inhibitor (ACE-I), and showed a reduction in the risks of death, dialysis and transplantation (10). Other trials initially used monotherapy with ACE-I, but also angiotensin receptor blockers (ARB) (11-12). Dual blockade was proposed after one of the largest clinical trials, the Candesartan And Lisinopril Microalbuminuria (CALM) Study, showed reduced albuminuria with dual therapy compared to monotherapy (13). Further clinical trials with larger sample sizes, however, have failed to confirm the superiority of dual RAS blockade compared to monotherapy; multicentric clinical trials are ongoing to resolve this issue (14).

#### **2.2 Background on the roles of angiotensin II in the kidney**

Despite controversies over the efficacy of dual or simple RAS blockade, the importance of Ang II in diabetic nephropathy development is well accepted. Ang II, an octapeptide discovered in the 1930s in the United States and characterized in Switzerland, was initially named for its first-known function: contraction of blood vessels (15). It is the most powerful biologically active peptide of the RAS, with vasoconstriction but also nonhemodynamic effects, such as electrolyte reabsorption, renal hypertrophy and tubular apoptosis in the kidneys (15).

#### **2.2.1 Receptors**

It is well established that Ang II mediates its effects mainly via binding to two G proteincoupled receptors: AT1R (which has 2 subtypes in rodents, namely AT1a and AT1b) and AT2R. AT1R, a seven-transmembrane domain receptor, is the main known mediator of Ang II actions (16); its action is summarized in **Table 1**. Ang II stimulation leads to upregulation of AT1R in the tubular compartment but downregulation of the same receptors in the glomerular compartment (17). The role of AT2R in kidneys is still not fully understood: upon stimulation by Ang II, it can counteract the effects of AT1R (18) but also activate inflammation (cf **Table 1**). In animal models of kidney damage, de novo expression of AT2R in glomeruli and vessels was induced by Ang II together with upregulation of AT2R in tubular cells (19).

Recent studies have shown the importance of 2 other receptors, the Ang1-7 or Mas receptors and the AT4 receptor (20). The latter is still under investigation and has been proven to be linked to memory. However, it is also present in vessels and kidneys (proximal and distal tubules); it increases intracellular Ca2+ levels and activates Erk and MAPK signalling (21).

#### **2.2.2 Actions**

Ang II stimulates glomerular cell proliferation and causes accumulation of extracellular matrix material by stimulating transforming growth factor 1 (TGF-1), which leads to increased protein synthesis. TGF-1 decreases protein degradation by stimulating matrix metalloproteinases, mainly MMP-2, but also plasminogen activators inhibitor-1 (PAI-1) (22-23).


Table 1. Signalling pathways stimulated by AT1, AT2 and Mas receptors

It has been demonstrated that high glucose, together with Ang II, is involved in tubular lesions seen in DN (5, 24). Indeed, high glucose and Ang II enhance angiotensinogen (Agt, the sole precursor of all angiotensins) gene expression, both in vitro in rat immortalized renal proximal tubular cells (iRPTC) (25, 26) and in vivo in streptozotocin-treated mice (a model of diabetic mice) proximal tubules (5, 24). This turns into a vicious circle, increasing tubular atrophy, as Agt is the sole substrate of the RAS and is used for synthesis of Ang II.

#### **2.3 From angiotensinogen to angiotensin II**

32 Diabetic Nephropathy

The benefits of RAS inhibition on end-organ protection in diabetic patients are well established. One of the early clinical trials on diabetic patients was performed with captopril, an angiotensin-converting enzyme inhibitor (ACE-I), and showed a reduction in the risks of death, dialysis and transplantation (10). Other trials initially used monotherapy with ACE-I, but also angiotensin receptor blockers (ARB) (11-12). Dual blockade was proposed after one of the largest clinical trials, the Candesartan And Lisinopril Microalbuminuria (CALM) Study, showed reduced albuminuria with dual therapy compared to monotherapy (13). Further clinical trials with larger sample sizes, however, have failed to confirm the superiority of dual RAS blockade compared to monotherapy;

Despite controversies over the efficacy of dual or simple RAS blockade, the importance of Ang II in diabetic nephropathy development is well accepted. Ang II, an octapeptide discovered in the 1930s in the United States and characterized in Switzerland, was initially named for its first-known function: contraction of blood vessels (15). It is the most powerful biologically active peptide of the RAS, with vasoconstriction but also nonhemodynamic effects, such as electrolyte reabsorption, renal hypertrophy and tubular apoptosis in the

It is well established that Ang II mediates its effects mainly via binding to two G proteincoupled receptors: AT1R (which has 2 subtypes in rodents, namely AT1a and AT1b) and AT2R. AT1R, a seven-transmembrane domain receptor, is the main known mediator of Ang II actions (16); its action is summarized in **Table 1**. Ang II stimulation leads to upregulation of AT1R in the tubular compartment but downregulation of the same receptors in the glomerular compartment (17). The role of AT2R in kidneys is still not fully understood: upon stimulation by Ang II, it can counteract the effects of AT1R (18) but also activate inflammation (cf **Table 1**). In animal models of kidney damage, de novo expression of AT2R in glomeruli and vessels was induced by Ang II together with upregulation of AT2R in

Recent studies have shown the importance of 2 other receptors, the Ang1-7 or Mas receptors and the AT4 receptor (20). The latter is still under investigation and has been proven to be linked to memory. However, it is also present in vessels and kidneys (proximal and distal tubules); it increases intracellular Ca2+ levels and activates Erk and MAPK signalling (21).

Ang II stimulates glomerular cell proliferation and causes accumulation of extracellular matrix material by stimulating transforming growth factor 1 (TGF-1), which leads to increased protein synthesis. TGF-1 decreases protein degradation by stimulating matrix metalloproteinases, mainly MMP-2, but also plasminogen activators inhibitor-1 (PAI-1) (22-23).

**2.1 Clinical findings on the implications of RAS blockade in diabetic nephropathy** 

**2. The renin-angiotensin system in diabetic nephropathy** 

multicentric clinical trials are ongoing to resolve this issue (14).

**2.2 Background on the roles of angiotensin II in the kidney** 

kidneys (15).

**2.2.1 Receptors** 

tubular cells (19).

**2.2.2 Actions** 

The importance of the systemic RAS in blood pressure control and sodium homeostasis has been well accepted and Ang II has been recognized as a cardinal parameter in the development of both hypertension and kidney injury (5, 13, 15, 27). Overactivation of AT1 R by Ang II therefore represents a target for treatment, but as Ang II has many other paracrine effects (induction of inflammation, mitogenesis, cell growth, apoptosis, differentiation, migration, etc.), current therapies are not sufficient to reverse the consequences of Ang II hyperaction. Of note, local RAS have been unravelled in some organs, notably the kidneys, with luminal fluid levels of Ang II being at least 1,000-fold higher than in the plasma (28). This local RAS could also play an important role in sodium retention and blood pressure regulation and hypertension, representing both a cause and a consequence of kidney injury. Complex interactions between diabetes and hypertension due to similar etiologies of both conditions, together with the stimulating effect of hyperglycemia on Ang II production in vitro, point to an important role for local RAS in DN.

#### **2.3.1 Synthesis and degradation of angiotensin II**

The classic components of the RAS are all found in renal proximal tubules, including Agt and the enzymes (prorenin/renin, ACE, angiotensin-converting enzyme 2 (Ace2), aminopeptidases and carboxy peptidases). Upon cleavage of the prorenin into a proteolytic enzyme, renin will cleave Agt into a decapeptide: angiotensin I (Ang I). Then the dipeptidyl peptidase ACE will remove 2 amino acids from the latter and generate Ang II. Ang II is further metabolized into smaller fragments, such as Ang 1-7 and Ang III, Ang IV and Ang V, by various peptidases. Among those peptidases, Ace2 is a human homologue to ACE (42% similarity) that was discovered in 2000; it cleaves Ang I into Ang 1-9 /Ang II to Ang 1- 7, both having hemodynamic properties (29). While ACE is present in most tissues, Ace2 is specifically expressed in the kidney, and less in the testes and heart ,with neither ARB nor

Renal Angiotensinogen Gene Expression and Tubular Atrophy in Diabetic Nephropathy 35

electron donors (NADH, H+ and FADH2) and mitochondrial superoxide overproduction (33). Increased mitochondrial superoxide production activates three main pathways: the polyol/protein kinase C pathway, the hexosamine biosynthesis pathway, and increased production of advanced glycated end products (AGE) and its receptor, RAGE (33). Our transgenic (Tg) mice overexpressing rat catalase (CAT) in their RPTCs exhibit attenuated ROS generation, Agt gene expression and RPTC injury in streptozotocin (STZ)-induced diabetes in vivo (5), unequivocally demonstrating the importance of ROS in mediating Agt

**2.4 Recent findings on diabetic nephropathy using transgenic mouse models** 

In order to elucidate in vivo the importance of local intrarenal RAS, at least two systems could be used: targeted renal expression of RAS in knock out mice for any component of RAS and targeted renal overexpression of one component of the RAS in wild type mice. Our laboratory has been using the latter approach to elucidate the role of intrarenal RAS in DN.

To obtain specific overexpression of the rat Agt gene (rAgt) in RPTC, our laboratory used the Kidney-specific Androgen regulated Promoter 2 (KAP2) (34, 35). The cDNA encoding full-length rAgt fused with HA-tag at the carboxyl terminal and NotI restriction enzyme site attached at both 5'- and 3'-termini was thus inserted into the KAP2 promoter and thereafter

Fig. 2. Schematic map of the kidney androgen-regulated promoter (KAP2)-rat Agt construct

Studies using this rAgt-transgenic (Tg) mice model have demonstrated that overexpression of renal rAgt alone induces hypertension and albuminuria and that RAS blockade reverses these abnormalities (34). Thereafter the same model was used to assess a possible synergic deleterious action of local RAS overactivity and high glucose on RPTCs, which could contribute to the pathophysiology of DN and help unravel new

STZ was used to induce diabetes in non-transgenic (non-Tg) and Tg mice. As far as systemic hypertension is concerned, neither STZ-induced diabetes nor insulin treatment changed the blood pressure levels of Tg mice or non-Tg mice. STZ administration led, four weeks later, to diabetes, increased kidney/body weight and albuminuria, and were normalized by insulin treatment. RAS blockers did not affect glucose levels but reversed

gene expression and in the development of DN.

**2.4.1 The angiotensinogen transgenic mouse model** 

protective mechanisms.

microinjected into one-cell fertilized mouse embryos as shown below:

**2.4.2 Tubular apoptosis in diabetic angiotensinogen transgenic mice** 

ACE-I, which can inhibit its activities (29). Ace2 levels in glomerules and proximal tubules are decreased in patients with chronic kidney disease and DN (30).

The following diagram illustrates the pathway for the synthesis and degradation of Ang II.

Fig. 1. Synthesis and degradation of Ang II

When Ace2 null mice were bred with the Akita model of type 1 diabetes, the obtained Ace2(-/y) Ins2(WT/C96Y) mice exhibited increased mesangial matrix scores, urinary albumin excretion rates and glomerular basement membrane thicknesses compared to Ace2(+/y)Ins2(WT/C96Y) with the same blood glucose levels (31). This highlights once more the role of RAS in the development of kidney injury in cases of chronic hyperglycemia.

#### **2.3.2 Importance of angiotensinogen in diabetic nephropathy**

Our laboratory has previously demonstrated that both ARB and ACE-I block Agt gene expression and induction of hypertrophy stimulated by high glucose levels in immortalized rat RPTCs and that renal Ang II acts in an autocrine manner to stimulate TGF-ß1 expression and, subsequently, TGF-ß1 enhances cellular hypertrophy and collagen α1 (type IV) expression in RPTCs (32). Our experiments on RPTCs have shown that high glucose stimulates Agt gene expression via at least 4 pathways:


The latter have been extensively studied within the frame of elucidating the molecular mechanisms of hyperglycemia action in DN. It is now accepted that elevated glucose levels enhance PKC activation, augment membrane lipid peroxidation in glomeruli and induce Agt gene expression in rat RPTCs via ROS generation (25). Excessive intracellular accumulation of glucose (seen in chronic hyperglycemia) leads to disturbances at the level of the TriCarboxylic Acid (TCA) pathway, followed by the formation of high quantities of electron donors (NADH, H+ and FADH2) and mitochondrial superoxide overproduction (33). Increased mitochondrial superoxide production activates three main pathways: the polyol/protein kinase C pathway, the hexosamine biosynthesis pathway, and increased production of advanced glycated end products (AGE) and its receptor, RAGE (33). Our transgenic (Tg) mice overexpressing rat catalase (CAT) in their RPTCs exhibit attenuated ROS generation, Agt gene expression and RPTC injury in streptozotocin (STZ)-induced diabetes in vivo (5), unequivocally demonstrating the importance of ROS in mediating Agt gene expression and in the development of DN.

#### **2.4 Recent findings on diabetic nephropathy using transgenic mouse models**

In order to elucidate in vivo the importance of local intrarenal RAS, at least two systems could be used: targeted renal expression of RAS in knock out mice for any component of RAS and targeted renal overexpression of one component of the RAS in wild type mice. Our laboratory has been using the latter approach to elucidate the role of intrarenal RAS in DN.

#### **2.4.1 The angiotensinogen transgenic mouse model**

34 Diabetic Nephropathy

ACE-I, which can inhibit its activities (29). Ace2 levels in glomerules and proximal tubules

The following diagram illustrates the pathway for the synthesis and degradation of Ang II.

When Ace2 null mice were bred with the Akita model of type 1 diabetes, the obtained Ace2(-/y) Ins2(WT/C96Y) mice exhibited increased mesangial matrix scores, urinary albumin excretion rates and glomerular basement membrane thicknesses compared to Ace2(+/y)Ins2(WT/C96Y) with the same blood glucose levels (31). This highlights once more the role of RAS in the development of kidney injury in cases of chronic hyperglycemia.

Our laboratory has previously demonstrated that both ARB and ACE-I block Agt gene expression and induction of hypertrophy stimulated by high glucose levels in immortalized rat RPTCs and that renal Ang II acts in an autocrine manner to stimulate TGF-ß1 expression and, subsequently, TGF-ß1 enhances cellular hypertrophy and collagen α1 (type IV) expression in RPTCs (32). Our experiments on RPTCs have shown that high glucose

The latter have been extensively studied within the frame of elucidating the molecular mechanisms of hyperglycemia action in DN. It is now accepted that elevated glucose levels enhance PKC activation, augment membrane lipid peroxidation in glomeruli and induce Agt gene expression in rat RPTCs via ROS generation (25). Excessive intracellular accumulation of glucose (seen in chronic hyperglycemia) leads to disturbances at the level of the TriCarboxylic Acid (TCA) pathway, followed by the formation of high quantities of

are decreased in patients with chronic kidney disease and DN (30).

**2.3.2 Importance of angiotensinogen in diabetic nephropathy** 

stimulates Agt gene expression via at least 4 pathways:

p38 MAP Kinase;

ROS.

Hexosamine biosynthesis;

Protein Kinase C via de novo synthesis of diacylglycerol;

Fig. 1. Synthesis and degradation of Ang II

To obtain specific overexpression of the rat Agt gene (rAgt) in RPTC, our laboratory used the Kidney-specific Androgen regulated Promoter 2 (KAP2) (34, 35). The cDNA encoding full-length rAgt fused with HA-tag at the carboxyl terminal and NotI restriction enzyme site attached at both 5'- and 3'-termini was thus inserted into the KAP2 promoter and thereafter microinjected into one-cell fertilized mouse embryos as shown below:

Fig. 2. Schematic map of the kidney androgen-regulated promoter (KAP2)-rat Agt construct

Studies using this rAgt-transgenic (Tg) mice model have demonstrated that overexpression of renal rAgt alone induces hypertension and albuminuria and that RAS blockade reverses these abnormalities (34). Thereafter the same model was used to assess a possible synergic deleterious action of local RAS overactivity and high glucose on RPTCs, which could contribute to the pathophysiology of DN and help unravel new protective mechanisms.

#### **2.4.2 Tubular apoptosis in diabetic angiotensinogen transgenic mice**

STZ was used to induce diabetes in non-transgenic (non-Tg) and Tg mice. As far as systemic hypertension is concerned, neither STZ-induced diabetes nor insulin treatment changed the blood pressure levels of Tg mice or non-Tg mice. STZ administration led, four weeks later, to diabetes, increased kidney/body weight and albuminuria, and were normalized by insulin treatment. RAS blockers did not affect glucose levels but reversed

Renal Angiotensinogen Gene Expression and Tubular Atrophy in Diabetic Nephropathy 37

**(a) db/m+ (b) db/m+ Cat-Tg** 

**PT PT PT**

**(c) db/db (d) db/db Cat-Tg**

**G**

**PT**

**PT**

**G G**

Fig. 3. Apoptosis in male non-Tg and Tg mouse kidneys at week 20, analyzed by TUNEL

Using DNA chip microarrays technology, our laboratory recently identified 2 proapoptotic genes, Bcl-2 modifying factor (*Bmf*) and *Caspase-12,* which are differentially upregulated in renal proximal tubules of db/db mice but normalized in db/db CAT-Tg

> **change (db/db vs db/m+)**

1454880\_s\_at Bcl2-modifying factor 3.07 0.0099 3.07 0.0098 1449297\_at caspase 12 1.82 0.0069 1.81 0.0070 1431875\_a\_at E2F transcription factor 1 1.19 0.0065 1.19 0.0064

Fig. 4. List of genes up-regulated in microarray chips of db/db vs db/m+ and db/db vs

**p-value (db/db vs db/m+)** 

**Foldchange (db/db vs db/db-CAT Tg)** 

1.28 0.0039 1.29 0.0038

1.99 0.0073 1.97 0.0074

**p-value (db/db vs db/db-CAT Tg)** 

staining. Arrows indicate apoptotic cells. G, glomerulus; PT, proximal tubule.

**G**

**PT**

**PT**

**PT PT**

**PT**

**Probe Set ID Gene Title Fold-**

Tnf receptor-associated

1450231\_a\_at baculoviral IAP repeatcontaining 4

factor 1

Magnification x600.

mice (39) as shown below:

1423602\_at 1445452\_at

db/db CAT-Tg mice

the deleterious effects of rAgt-overexpression in diabetic mice. Renal injury found in Tg mice was more severe in STZ-treated Tg mice, with loss of brush borders in RPTC and marked tubular luminal dilatation. In addition, glomerular and RPTC hypertrophy and increased tubular luminal area were markedly attenuated by insulin and RAS blockers in Tg and non-Tg STZ-treated mice, while a combination of both treatments completely reversed these abnormalities. Apoptotic assays (TUNEL) and immunohistochemistry using caspase-3 antibody showed increased levels of apoptosis in RPTC of Tg mice compared to non-Tg, the latter having higher levels than non-STZ treated mice. Investigations of the molecular pathways involved reveal an increased level of Bax and concomitant downregulation of Bcl-xL. One hypothesis could therefore be that hyperglycemia enhanced tubular apoptosis by increasing the Bax/Bcl-xL ratio, thus having a pro-poptotic effect. STZ-induced diabetes leads to apoptosis in RPTCs and to a lesser degree in distal tubules, but not in the glomeruli, confirming previous findings of a pro-apoptotic effect of diabetes on RPTCs (36). Treatment with insulin and/or RAS blockers leads to an almost complete absence of apoptosis in kidneys of non Tg and Tg mice. Another salient finding in Agt-Tg mice is the persistent kidney injury despite hydralazine treatment. In fact, hydralazine treatment markedly reduced systemic blood pressure but did not affect albuminuria and tubular apoptosis. Further investigations into the underlying mechanism of high glucose and Ang II action were performed on Tg mice overexpressing catalase (CAT-Tg) in their RPTCs. STZ-induced diabetic CAT-Tg mice exhibited attenuated ROS generation and tubular apoptosis (5). Furthermore, in double Tg mice having Agt and CAT specifically expressed in their RPTCs, ROS generation, NADPH activity and levels of hemoxygenase 1 (HO-1) were significantly lowered by CAT overactivity compared to Agt-Tg mice. Levels of collagen type IV, monocyte chemotactic protein-1 (MCP-1), TGF-1 and plasminogen activator inhibitor-1 were also lowered by CAT overexpression in double Tg mice compared to Agt Tg mice (37). Thus, CAT overexpression alleviates oxidative stress in RPTC and reduces the toxicity of Ang II and chronic hyperglycemia on the kidneys.

#### **3. Conclusion and perspectives**

Agt and chronic hyperglycemia act together at the level of the RPTC, leading to tubular atrophy due to pro-apoptotic activities and interstitial fibrosis. This unravels the importance of the local RAS in the development of DN. Both *in vitro* and *in vivo* experiments of overexpression of Agt indicate that the latter stimulates RPTC hypertrophy and apoptosis, but significant effects on the glomeruli remain to be determined. However, because tubular atrophy seems to be a better predictor of disease progression than glomeruli lesions, this finding may be considered of significant clinical importance, as therapeutics reproducing the effects of CAT may be specifically developed to impede or even stop the progression of DN.

Further directions include studying the effect of the local RAS on glomeruli and deciphering the molecular pathways by which Agt and chronic hyperglycemia induce RPTC apoptosis. One important clue is the role of ROS, which is induced by both intrarenal RAS overactivity and chronic hyperglycemia. Indeed, we have reported an increase of apoptotic cells in RPTCs of db/db mice (type II diabetic mouse model) and normalization by overexpression of catalase (CAT) in their RPTCs (db/db CAT-Tg mice) (38) as shown below:

the deleterious effects of rAgt-overexpression in diabetic mice. Renal injury found in Tg mice was more severe in STZ-treated Tg mice, with loss of brush borders in RPTC and marked tubular luminal dilatation. In addition, glomerular and RPTC hypertrophy and increased tubular luminal area were markedly attenuated by insulin and RAS blockers in Tg and non-Tg STZ-treated mice, while a combination of both treatments completely reversed these abnormalities. Apoptotic assays (TUNEL) and immunohistochemistry using caspase-3 antibody showed increased levels of apoptosis in RPTC of Tg mice compared to non-Tg, the latter having higher levels than non-STZ treated mice. Investigations of the molecular pathways involved reveal an increased level of Bax and concomitant downregulation of Bcl-xL. One hypothesis could therefore be that hyperglycemia enhanced tubular apoptosis by increasing the Bax/Bcl-xL ratio, thus having a pro-poptotic effect. STZ-induced diabetes leads to apoptosis in RPTCs and to a lesser degree in distal tubules, but not in the glomeruli, confirming previous findings of a pro-apoptotic effect of diabetes on RPTCs (36). Treatment with insulin and/or RAS blockers leads to an almost complete absence of apoptosis in kidneys of non Tg and Tg mice. Another salient finding in Agt-Tg mice is the persistent kidney injury despite hydralazine treatment. In fact, hydralazine treatment markedly reduced systemic blood pressure but did not affect albuminuria and tubular apoptosis. Further investigations into the underlying mechanism of high glucose and Ang II action were performed on Tg mice overexpressing catalase (CAT-Tg) in their RPTCs. STZ-induced diabetic CAT-Tg mice exhibited attenuated ROS generation and tubular apoptosis (5). Furthermore, in double Tg mice having Agt and CAT specifically expressed in their RPTCs, ROS generation, NADPH activity and levels of hemoxygenase 1 (HO-1) were significantly lowered by CAT overactivity compared to Agt-Tg mice. Levels of collagen type IV, monocyte chemotactic protein-1 (MCP-1), TGF-1 and plasminogen activator inhibitor-1 were also lowered by CAT overexpression in double Tg mice compared to Agt Tg mice (37). Thus, CAT overexpression alleviates oxidative stress in RPTC and reduces the toxicity of Ang II and

Agt and chronic hyperglycemia act together at the level of the RPTC, leading to tubular atrophy due to pro-apoptotic activities and interstitial fibrosis. This unravels the importance of the local RAS in the development of DN. Both *in vitro* and *in vivo* experiments of overexpression of Agt indicate that the latter stimulates RPTC hypertrophy and apoptosis, but significant effects on the glomeruli remain to be determined. However, because tubular atrophy seems to be a better predictor of disease progression than glomeruli lesions, this finding may be considered of significant clinical importance, as therapeutics reproducing the effects of CAT may be specifically developed

Further directions include studying the effect of the local RAS on glomeruli and deciphering the molecular pathways by which Agt and chronic hyperglycemia induce RPTC apoptosis. One important clue is the role of ROS, which is induced by both intrarenal RAS overactivity and chronic hyperglycemia. Indeed, we have reported an increase of apoptotic cells in RPTCs of db/db mice (type II diabetic mouse model) and normalization by overexpression

of catalase (CAT) in their RPTCs (db/db CAT-Tg mice) (38) as shown below:

chronic hyperglycemia on the kidneys.

**3. Conclusion and perspectives** 

to impede or even stop the progression of DN.

Fig. 3. Apoptosis in male non-Tg and Tg mouse kidneys at week 20, analyzed by TUNEL staining. Arrows indicate apoptotic cells. G, glomerulus; PT, proximal tubule. Magnification x600.

Using DNA chip microarrays technology, our laboratory recently identified 2 proapoptotic genes, Bcl-2 modifying factor (*Bmf*) and *Caspase-12,* which are differentially upregulated in renal proximal tubules of db/db mice but normalized in db/db CAT-Tg mice (39) as shown below:


Fig. 4. List of genes up-regulated in microarray chips of db/db vs db/m+ and db/db vs db/db CAT-Tg mice

Renal Angiotensinogen Gene Expression and Tubular Atrophy in Diabetic Nephropathy 39

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#### **4. References**


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**4. References** 


**3** 

*Dayton, OH* 

*USA* 

**Diabetic Nephropathy:** 

Jayson Yap and Mohammad G. Saklayen

**Role of Aldosterone and Benefits of** 

**Therapy with Aldosterone Receptor Blocker** 

*VA Medical Center and Wright State University Boonshoft School of Medicine,* 

ACE-inhibitor and angiotensin receptor blocker (ARB) have been shown to reduce proteinuria and progression of renal dysfunction in both type 1 and type 2 diabetics (1-11). However, even with optimal use of ACE-Inhibitor or ARB, the progression of renal dysfunction is not completely stopped. Even in studies where ACEI or ARB therapy showed improved outcome, a very high percentage of patients still progress. This scenario is now highlighted in a recent study published in JAMA, which shows the incidence of diabetic nephropathy increasing in USA in excess of what can be accounted for by the increased incidence of type 2 DM in the population (12). The present treatment strategy is therefore not adequate and other additional

Since the publication of the landmark RALES study (13) showing a significant survival benefit in patients with systolic heart failure when treated with aldosterone inhibition in addition to ACEI and beta-blockers, the interest in the vasculotoxic effect of aldosterone and the beneficial therapeutic effects of aldosterone receptor blocker drugs like spironolactone

In addition to its classical action in the distal nephron, aldosterone is now known to exert many other effects on other areas of kidneys as well as in cardiovascular tissues. (14 )

Aldosterone is now known to have a significant role in renal hemodynamics, independent of Angiotensin II. In a remnant kidney model in the rat Greene et al showed that there was >10-fold rise in aldosterone in the remnant kidney rats (REM) compared to sham operated ones (15). As expected, proteinuria, hypertension and glomerulosclerosis in the REM rats were attenuated with treatment using ACE-inhibitor or angiotensin receptor blockers. However, when these treated rats (REM AIIA) were given an aldosterone infusion the extent of proteinuria, hypertension and glomerulosclerosis were similar to untreated (REM) rats, suggesting deleterious renal hemodynamic effects of aldosterone independent of angiotensin II. Use of spironolactone in these rats transiently reduced proteinuria and

**2. Aldosterone biology and lessons learnt from animal models** 

**1. Introduction** 

effective treatment strategies are urgently needed.

has been steadily increasing (14).

lowered arterial pressure.

converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000; 87:E1-9.

