Wright State University Wright State University CORE Scholar CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2009 Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice 1 Receptor Expression in db/db Diabetic Mice Malav Navinchandra Madhu Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Repository Citation Madhu, Malav Navinchandra, "Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice" (2009). Browse all Theses and Dissertations. 310. https://corescholar.libraries.wright.edu/etd_all/310 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected].
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Wright State University Wright State University
CORE Scholar CORE Scholar
Browse all Theses and Dissertations Theses and Dissertations
2009
Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type
1 Receptor Expression in db/db Diabetic Mice 1 Receptor Expression in db/db Diabetic Mice
Malav Navinchandra Madhu Wright State University
Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all
Part of the Pharmacology, Toxicology and Environmental Health Commons
Repository Citation Repository Citation Madhu, Malav Navinchandra, "Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice" (2009). Browse all Theses and Dissertations. 310. https://corescholar.libraries.wright.edu/etd_all/310
This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected].
ANGIOTENSIN II TYPE 1 RECEPTOR EXPRESSION IN db/db
DIABETIC MICE
A thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science
By
MALAV MADHU B.Pharm., North Gujarat University, Gujarat, India 2006
2009 Wright State University
WRIGHT STATE UNIVERSITYSCHOOL OF GRADUATE STUDIES
Date: August 27, 2009
I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY MALAV MADHU ENTITLED “IMPACT OF DIABETES ON ACE/ACE2 BALANCE AND
ANGIOTENSIN II TYPE 1 RECEPTOR EXPRESSION IN db/db Diabetic MICE” BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE.
_______________________ Khalid M. Elased, R.Ph., Ph.D.
Thesis Director
________________________ Terry Oroszi
Director, Graduate Program
_______________________Mariana Morris, Ph.D. Department Chair
Committee on Final Examination __________________________ Khalid M. Elased, R.Ph., Ph.D. __________________________ James B. Lucot, Ph.D. __________________________ Mariana Morris, Ph.D. __________________________ Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies
ABSTRACT
Madhu, Malav. M.S., Department of Pharmacology and Toxicology, Wright State University, 2009. Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice.
Alterations in the renin-angiotensin system (RAS) are considered to be crucial for
the development of diabetic complications like hypertension and nephropathy. Our
previous work demonstrated role of AT1 receptors (AT1R) in the development of
hypertension in db/db diabetic mice. The aim of this study was to test the hypothesis that
there is upregulation of renal AT1R and imbalance in renal ACE/ACE2 homeostasis in
db/db mice. In addition, we hypothesize that treatment with an anti-hyperglycemic or an
AT1R blocker will correct this imbalance. Five week old control and db/db mice were
housed in metabolic cages for 24 hour collection of urine. At early age of 5 weeks, db/db
mice were obese and hyperglycemic. Urinary albumin excretion was also significantly
high in db/db mice. Changes in RAS were evaluated using enzyme activities, western
blots and immunohistochemistry. There was a significant increase in urinary ACE2
activity and ACE2 content in db/db mice at 5 weeks. There was a significant increase in
plasma ACE activity and Ang II content in db/db mice compared to controls at 8 weeks.
Western blot analysis showed significant increase in AT1R protein expression in 8, 18
and 31 week db/db mice compared to controls. There was upregulation of ACE2 and
down-regulation of ACE in kidney to compensate the effects of high plasma Ang II. To
study the effect of reduction in blood glucose and AT1R blockade, mice were treated
iii
with metformin and losartan for 12 weeks. Chronic treatment with metformin (150
mg/kg/day) and losartan (10 mg/kg/day) significantly decreased urinary albumin and
protein excretion. Metformin improved blood glucose and glucose tolerance db/db mice,
but did not affect renal expression of ACE, ACE2 and AT1R. Although chronic losartan
treatment did not alter blood glucose levels, it improved the morphology of pancreatic
islets. There was a significant increase in renal AT1R protein expression and decrease in
renal ACE2 protein expression following losartan treatment. Losartan treatment
significantly increased urinary ACE2 activity. Western blot of concentrated urine from 8
week db/db mice revealed immunoreactive bands of ACE, ACE2 and AT1R protein.
Conclusion: 1) There is upregulation in renal AT1R protein expression in db/db mice. 2)
Chronic metformin treatment significantly reduces blood glucose and microalbuminuria
in db/db mice without affecting ACE/ACE2 balance. 3) Chronic losartan treatment had
no effect on blood glucose, but it up-regulates renal AT1R and down-regulates renal
ACE2. 4) Enzyme activity and western blot shows increased excretion of ACE2 in the
urine of db/db mice. These data show that urinary ACE and ACE2 provide good index of
intra-renal RAS status and could be used in early diagnosis and prognosis of diabetic
fold in db/db mice by the age 31 weeks (Figure 6B, p<0.05).
3.3.3 Urinary ACE2 content: Urinary ACE2 content was measured by ELISA in
50 µl urine. At 5 weeks, ACE2 content was increased significantly in db/db mice
compared to control mice (Figure 7, p<0.05).
4. Renal protein expression of ACE, ACE2 and AT1R:
To study the effect of diabetes on renal protein expression of ACE, ACE2 and AT1R
western blots were performed.
4.1 AT1R expression: Renal AT1R expression was significantly high in 8 week db/db
mice (Figure 8A, p<0.05). Higher expression of renal AT1R was also observed in 31
week db/db mice (Figure 8B, p<0.05). These renal changes may explain hypertension
associated with diabetes.
4.2 ACE expression: In young 8 week db/db mice, kidney ACE expression was
significantly less compared to their lean controls (Figure 9A, p<0.05). Old 31 week mice
60
also had lower kidney ACE expression when compared to control mice (Figure 9B,
p<0.01)
4.3 ACE2 expression: Young 8 week db/db mice exhibited significantly high renal ACE2
expression than controls (Figure 10A, p<0.05). With progression of diabetes, kidney
function declines and so does ACE2 expression. In kidneys of old 31 week mice, ACE2
expression does not differ between control and db/db mice (Figure 10B).
5. Plasma and kidney Ang II content:
5.1 Plasma Ang II: Ang II content was evaluated in 45 µl of plasma containing 1 µl
EDTA. At 8 weeks, plasma Ang II content of db/db mice was increased compared with
control mice (Figure 11A, p<0.01). Plasma Ang II levels in old 31 week db/db mice were
also significantly high compared to controls (Figure 11B, p<0.01). This finding is
supported by high plasma ACE activity in db/db mice.
5.2 Kidney Ang II: Ang II content was evaluated using 50 µl (450-500 µg) of kidney
lysate. There was no significant difference in renal Ang II content between control and
db/db mice at the age of 8 weeks and 31 weeks (Figure 12). This finding is supported by
the fact that renal ACE expression and activity are reduced in db/db mice. Upregulation
of ACE2 in kidney degrades excess Ang II, thereby keeping its deleterious effects in
control.
6. Effect of metformin:
To study the effect of reduction in glycemia on renal and urinary outcomes, 5-8 week
mice were treated with metformin (150 mg/kg/day) in drinking water for 12 weeks.
61
6.1 Effect of metformin on blood glucose: Metformin treatment in 8 week mice did not
alter blood glucose levels (data not shown). We believe that the treatment was initiated
after a significant rise in blood glucose of 8 week db/db mice. Therefore, another group
of 5 week old db/db and control mice were treated with metformin for 12 weeks.
Metformin treatment significantly reduced blood glucose of db/db mice during ad libitum
feeding (Figure 13B, p<0.05). However, the treatment did not alter blood glucose of
control mice (Fig 13A). To evaluate the effect of metformin on glucose handling, we
performed i.p. glucose tolerance test. As expected, metformin treatment significantly
improved glucose tolerance in db/db mice (Figure 16, p<0.05) but not in control mice
(Figure 15).
6.2 Effect of metformin on renal function:
6.2.1 Albumin excretion: Three weeks of metformin treatment (150 mg/kg/day)
did not alter urinary albumin excretion in db/db mice (Figure 18A). However, 9
weeks of metformin treatment significantly reduced urinary albumin excretion
(Figure 18B, p<0.001). The albumin excretion of control mice did not change
during the treatment period.
6.2.2 Total protein excretion: Treatment with metformin significantly decreased
urinary total protein excretion in db/db mice (Figure 19, p<0.0001). However,
there was no effect of the treatment on urinary total protein excretion of control
mice (Figure 19).
62
6.2.3 Creatinine excretion: Metformin did not change creatinine concentration in
urine of either db/db or control mice after 3 and 9 weeks of treatment (Figure
20A).
6.3 Effect of metformin on renal ACE and ACE2 activities: Chronic metformin treatment
has no effect on renal ACE and ACE2 activities in either db/db or control mice (Figure
21).
6.4 Effect of metformin on urinary ACE and ACE2 activities: Metformin treatment has no
effect on urinary ACE activity (Figure 22) or urinary ACE2 (Figure 23) activity in either
db/db or control mice.
6.5. Effect of metformin on renal expression of ACE, ACE2 and AT1: Metformin
treatment has no effect on the renal expression of ACE, ACE2 and AT1R in db/db and
control mice (data not shown).
6.6 Effect of metformin on morphology of pancreatic islets: Pancreas sections from 18
week mice were stained with Masson’s trichrome to study the effect of metformin
treatment on islet morphology. Untreated db/db mice show disarray of cellular
architecture and loss of their structural integrity. Chronic metformin treatment improved
islet integrity. It also reduced fibrosis by reducing collagen around islets (Figure 27).
7. Effect of losartan
To study the effect of AT1R blockade on renal and urinary outcomes 5 week mice were
treated with losartan (10 mg/kg/day) in drinking water for 12 weeks.
63
7.1 Effect of losartan on blood glucose: Chronic losartan treatment did not alter blood
glucose in control (Figure 14A) and db/db mice (Figure 14B). To evaluate the effect of
losartan on glucose handling, we performed i.p. glucose tolerance test. Losartan treatment
had no effect on glucose tolerance of control and db/db mice (Figure 15).
7.2 Effect losartan on renal function:
Urine samples were collected after 3 and 9 weeks of initiation of the treatments.
7.2.1 Albumin excretion: Three weeks of losartan treatment did not alter urinary
albumin excretion in db/db mice (Figure 18A). However, 9 weeks of losartan
treatment significantly reduced urinary albumin excretion (Figure 18B, p<0.001).
There was no effect of the treatment on urinary albumin excretion of control mice.
7.2.2 Total protein excretion: Treatment with losartan decreased urinary total
protein excretion of db/db mice (Figure 19, p<0.0001). However, there was no
effect of the treatment on urinary total protein excretion of control mice.
7.2.3 Creatinine excretion: Losartan did not change creatinine concentration in
urine of either db/db or control mice (Figure 20A).
7.3 Effect of losartan on renal ACE and ACE2 activities: Losartan treatment did not
affect renal ACE and ACE2 activity after 12 weeks of treatment (Figure 21).
7.4 Effect of losartan on urinary ACE and ACE2 activities: Losartan increased urinary
ACE2 activity in db/db mice (Figure 23, p<0.01). However, losartan had no effect on
urinary ACE2 activity of control mice. Losartan has no effect on urinary ACE activity
(Figure 22).
64
65
7.5 Effect of losartan on renal expression of ACE, ACE2 and AT1R: Chronic treatment
with losartan significantly increased AT1 receptor protein expression in both db/db and
control mice (Figure 24, p<0.05). However, the treatment decreased ACE2 protein
expression (Figure 26, p<0.05) in both db/db and control mice. Losartan treatment had no
effect on renal ACE expression (Figure 25).
7.6 Effect of losartan on morphology of pancreatic islets: Untreated db/db mice show
disarray of cellular architecture and loss of their structural integrity. Chronic losartan
treatment improved islet integrity. It also reduced fibrosis by reducing collagen around
islets (Figure 27).
8. Immunohistochemistry of kidney:
To confirm the results obtained from western blot analysis, immunohistochemistry was
performed. As expected, AT1R expression was increased significantly in kidney tubules
of db/db mice compared to controls (Figure 28).
9. Western blots of concentrated urine:
Urinary excretion of ACE, ACE2 and AT1R protein was studied using western blots. A
total of 80 µg of concentrated urinary protein was added in each lane. Immunoblot of
ACE revealed two immunoreactive bands at 190 kDa and ~70 kDa. The smaller band
may be a degradation fragment of intact 190 kDa ACE. Immunoblot of ACE2 revealed
only one band at ~70 kDa which represents degradation fragment of integral ACE2.
Single immunoreactive bands of AT1R and β-actin were also detected at 43 kDa and 42
kDa respectively (Figure 29). The bands of β-actin were not consistent for each sample
so as to be used as a control protein.
DISCUSSION
This study tested the hypothesis that there is upregulation of AT1 receptors and
imbalance in ACE/ACE2 homeostasis in the kidneys of db/db mice. In addition we
studied the effects of chronic metformin and losartan treatment on renal RAS. Metformin
is widely prescribed as a blood glucose lowering drug for type 2 diabetics. It is an insulin
sensitizer which is thought to act by reducing hepatic glucose output and enhancing
peripheral glucose uptake (Stumvoll et al., 1995;Cusi et al., 1996). Initially a group of 8
week old db/db and control mice were treated with 150 mg/kg/day metformin in drinking
water. The treatment did not affect blood glucose of either db/db or control mice. Eight
week db/db mice had very high average blood glucose (more than 500 mg/dL) at the start
of the treatment. Therefore, we think that metformin was ineffective in reducing blood
glucose. To achieve glycemic control, young 5 week db/db and control mice were treated
with metformin (150 mg/kg/day) for 12 weeks. On this occasion metformin treatment
improved both glycemia and glucose tolerance in db/db mice. The time of initiation of
metformin treatment is critical for lowering blood glucose.
In present study, losartan treatment had no effect on blood glucose and glucose tolerance
of db/db and control mice. Some studies indicate that AT1 receptor blockers improve β-
cell function and glucose tolerance and delay the onset of type 2 diabetes in humans
(Lindholm et al., 2002) and in mouse (Chu et al., 2006). Some epidemiological data
indicates that RAS blockade delays the onset of type 2 diabetes in patients with
66
hypertension (Yusuf et al., 2005). ARBs and ACEIs are thought to affect glucose
metabolism by improving insulin sensitivity (Moan et al., 1996;Fogari et al., 1998).
However, our previous study shows that chronic losartan treatment reduces blood
pressure in db/db mice without affecting glucose tolerance (Senador et al., 2009). One
reason for improved glucose tolerance by AT1R blockade could be timing of initiation of
treatment. Our finding agrees with previous studies on db/db mice who initiated the
treatment after glucose had started to rise (Mathew et al., 2005;Shao et al., 2006;Sugaru
et al., 2007). Moreover, studies on streptozotocin induced diabetes and ob/ob mice
reported failure of chronic losartan treatment to improve blood glucose (Raimondi et al.,
2004;Erbe et al., 2006).
Although losartan treatment does not improve glucose tolerance, it improves morphology
of pancreatic islets in db/db mice. RAS components like ACE, angiotensinogen and
AT1R are reported be present in pancreatic islets (Lau et al., 2004). Activation of AT1R
is believed to inhibit insulin release in response to glucose loading (Carlsson et al., 1998).
Ang II also activates NAD(P)H oxidase and thus causes oxidative stress induced β-cell
dysfunction and apoptosis (Nakayama et al., 2005). Treatment of db/db mice with
candesartan improves granulation and reduces fibrosis and loss of endothelial cells in
islets (Shao et al., 2006). In present study, losartan treatment improves islet morphology
and integrity of β-cells. It should be noted that losartan treatment may increase insulin
release but may not improve insulin resistance. Therefore, it may not alter glucose levels
significantly.
The first sign of nephropathy in diabetics is presence of persistent albuminuria. In db/db
mice, significant microalbuminuria develops as early as 8 weeks (Sharma et al., 2003).
67
In this study, microalbuminuria was evident at the age of 5 weeks in db/db mice. At the
same time total protein excretion was not different between control and db/db mice. At
this age db/db mice were hyperglycemic. Moreover, blood pressure in db/db mice starts
to rise after the age of 11 weeks (Senador et al., 2009). Therefore in initial stages of
kidney damage is triggered by high blood glucose levels. Hyperglycemia and RAS
contribute in the development of nephropathy (Larkins & Dunlop, 1992;Andersen et al.,
2000). As mice become old, kidney function declines, measured by glomerular filtration
rate (Sharma et al., 2003). At the age of 31 weeks, albumin excretion of db/db mice
increases 10 fold compared to 5 week mice. Hyperinsulinemia has been found to increase
transcapillary escape of albumin in non-diabetic subjects, providing a link between
albuminuria and insulin resistance (Niskanen & Laakso, 1993). There are conflicting
reports in the literature on the effect of metformin on albuminuria. One study reports
reduction in urinary albumin excretion after metformin treatment in patients with type 2
diabetes (Amador-Licona et al., 2000) while others did not find any difference between
the treated and untreated groups (Imano et al., 1998;UKPDS,1998). The fact that
metformin reduces both fasting (Fujita et al., 2005) and non-fasting (Fruehwald-Schultes
et al., 2002) serum insulin levels, explains why it is effective in reducing albuminuria. On
the contrary, the effectiveness of losartan in reducing albuminuria in normotensive
(Zandbergen et al., 2003) and hypertensive (Brenner et al., 2001;Lozano et al.,
2001;Andersen et al., 2002) diabetic patients is well-known. Losartan blocks AT1R and
attenuates many of the deleterious actions of Ang II in kidneys such as contraction of
mesangial cells and glomerular arterioles (Manley, 2000), increase in membrane pore
radius (Remuzzi et al., 1993), glomerular sclerosis and modulation of extra-cellular
68
matrix (Leehey et al., 2000). In present study, metformin and losartan significantly
reduced urinary total protein excretion after 3 weeks of treatment. However, reduction in
urinary albumin excretion was only noticeable after 9 weeks. The duration of treatments
and time-points for urine collections were selected to compare our results with the
literature (Hu et al., 2009;Chu et al., 2006).
Western blot analysis of kidney shows that there is increase in ACE2 expression and
decrease in ACE expression in young db/db mice. This combination attenuates Ang II
accumulation and produces more Ang (1-7) in kidneys. Ang II over-activity is believed to
play an important role in the pathogenesis of DN (Parving et al., 2001). Ang (1-7),
produced by ACE2, is a vasodilator and anti-proliferative peptide that opposes the action
of Ang II (Koitka et al., 2008). Interestingly, ACE2 expression decreases and ACE
expression increases in the glomerulus of db/db mice, leading to altered glomerular
permeability and albuminuria (Ye et al., 2006). Ang II can increase intraglomerular
pressure by constricting both afferent and efferent arteriole, thereby stimulating urinary
albumin excretion (Remuzzi & Bertani, 1998). It has also been revealed that in animals
with experimental diabetes, intragmolerular pressure is increased even before systemic
blood pressure rises (Hostetter et al., 1982). Therefore, kidney upregulates ACE2 to
counteract pro-hypertensive processes and maintain its function. As they become old,
ACE2 expression goes down and kidney function deteriorates.
One of the key findings in the present study is upregulation of AT1R in kidneys of db/db
mice. AT1Rs are up regulated by conditions that increase Ang II like dehydration in rats
(Barth & Gerstberger, 1999;Sanvitto et al., 1997) and sodium deficiency in mouse (Chen
et al., 2003). These findings indicate that expression of AT1 receptors is affected by its
69
agonist Ang II. In a recent study, researchers found that systemic Ang II infusion
increases AT1 receptor mRNA expression in brain tissue of rats (Wei et al., 2009). An
increase in oxidative stress also leads to an increase in renal AT1R protein and mRNA
expression causing stimulation of sodium transporters and hypertension (Banday &
Lokhandwala, 2008). High blood glucose levels can increase reactive oxygen species and
eventually oxidative stress to upregulate AT1R. AT1R mRNA and protein levels are also
increased in vascular smooth muscles of type 2 diabetic patients (Hodroj et al., 2007).
Another study reported upregulation of AT1 receptor together with over expression of
Ang II in cavernous tissue of type 1 diabetic rats suggesting its possible role in erectile
dysfunction (Yang et al., 2009). High levels of circulating insulin, as seen type 2
diabetics, can also upregulate AT1 receptors in vascular smooth muscles (Hodroj et al.,
2007;Nickenig et al., 1998). Additionally, AT1R mRNA expression also increases in
pancreatic islets of db/db mice (Chu et al., 2006). This study shows that db/db mice have
high levels of circulating Ang II. Therefore, it is plausible that high expression of AT1
receptors in response to high circulating Ang II could lead to diabetes related
hypertension in db/db diabetic mice.
Treatment with losartan increases Ang II levels in the plasma in Lewis rats (Ferrario et
al., 2005). As a result, losartan also increases cardiac ACE2 activity promoting
production of Ang (1-7) form Ang II. High plasma Ang II may also cause stimulation of
AT2 receptors to exert its beneficial effects. There are conflicting reports on effect of
ARBs on AT1R expression. Some report an increase in AT1R with ARB treatment
(Wang et al., 1997;Hu et al., 2009) while others report down regulation (Wei et al.,
2009). Our data show that renal AT1R protein expression increases following chronic
70
losartan treatment in both db/db and control mice. As mentioned earlier db/db mice
already have high levels of circulating Ang II and an increase in Ang II results in an
increase in AT1R. Therefore, losartan treatment is likely to up regulate AT1R in kidney,
where Ang II is sequestered from plasma and is also a primary site of its action. Many
reports in the literature suggest that blockade of AT1R increases ACE2 (Soler et al.,
2009;Ferrario et al., 2005;Igase et al., 2005;Whaley-Connell et al., 2006). In contrast,
study by Xia and others did not find any significant change in ACE2 expression after
losartan treatment (Xia et al., 2009). In this study, losartan treatment significantly
reduced renal ACE2 protein expression in db/db and control. A recent study shows down
regulation of ACE2 mRNA in rat astrocytes after Ang II treatment (Gallagher et al.,
2006). Therefore, an increase in plasma Ang II levels by losartan may cause
downregulation of ACE2. Although AT1Rs are blocked by losartan, they can be
constitutively active and may act as autoreceptors (Zou et al., 2004). In this case, AT1R
no longer need Ang II to initiate signaling and may affect expression of ACE2. Another
possible mechanism for ACE2 downregulation may be action of Ang II or its derivatives
on angiotensin receptors. There was no change in expression of renal ACE protein after
losartan treatment. This finding agrees with study by Gallagher and others (Gallagher et
al., 2006). However, chronic metformin treatment did not affect RAS parameters in either
db/db or control mice.
To confirm the data obtained from western blot, renal ACE and ACE2 activities were
determined. As expected, ACE2 activity was increased and ACE activity was decreased
in kidneys of 8 week db/db mice. On the contrary, plasma ACE activity and Ang II
content were higher in 8 week db/db mice compared to controls and at this age mice are
71
not hypertensive (Senador et al., 2009). This means that kidney keeps deleterious effects
of Ang II in check by upregulating ACE2. As mice become old, kidneys cannot keep up
and systemic hypertension develops (Figure 5 vs. Figure 21). A ratio of high ACE2 to
low ACE favors less Ang II accumulation in tissues like kidney, helping to maintain their
function. Studies in the literature suggest that losartan treatment increases ACE2 activity
(Ferrario et al., 2005;Xia et al., 2009;Jessup et al., 2006). In this study, losartan treatment
does not change ACE activity in either db/db or control mice. Chronic losartan treatment
reduces expression of ACE2 protein in the kidney, but it doesn’t change ACE2 activity.
One reason for this could be sequestration of plasma Ang II by the kidney. Reduction in
ACE2 protein caused by losartan in the kidney is compensated by high ACE2 activity to
cleave the harmful Ang II.
It is reasonable to assume that up regulation of ACE2 in kidneys may be reflected in
urine samples. A study by Wakahara et. al. has shown that ACE2 mRNA expression in
human renal tissue marginally correlated with the degree of proteinuria (Wakahara et al.,
2007). A strong correlation between proteinuria and urinary expression of ACE and
ACE2 has also been observed in humans (Wang et al., 2008). In this study, we measured
urinary ACE2 activity and ACE2 content in young 5 week mice. At this age, db/db mice
excreted more ACE2 in urine compared to controls. Additionally, ACE2 activity in the
urine of db/db mice was also higher compared to controls. Kidney upregulates ACE2
sensing the change glycemia which is detected by urine analysis. At 5 weeks, urinary
albumin excretion was also high in db/db mice but urinary total protein excretion was not
different between the two groups. Furthermore, our study shows presence of ACE, ACE2
and AT1 proteins in the urine of 8 week old db/db mice. Urinary ACE and ACE2 enzyme
72
73
activities were also high in 8 week db/db mice. ACE and ACE2 are both type 1 integral
membrane protein comprising a large extracellular catalytic domain. ACE has been
shown to be proteolytically released from cell surface (Parkin et al., 2004). Catalytically
active forms of ACE have also been found in urine (Hooper, 1991) and plasma (Senador
et al., 2009). On the contrary, ACE2 activity is not detectable in plasma (Elased et al.,
2006). Therefore, it likely that source of urinary ACE2 is kidney and that of urinary ACE
is plasma. This study shows presence of ACE2 in urine of young db/db mice, using both
sensitive catalytic assay and immunoblotting. Urinary ACE2 levels reflect intra-renal
RAS status and have potential for diagnosis and prognosis of diabetic renal disease.
CONCLUSION
Cardiovascular and renal diseases are long term complications of diabetes and are leading
cause of morbidity and mortality. There is evidence of activation of RAS in diabetic
animals and humans. Our previous study showed effectiveness of AT1R blockade in
reduction of blood pressure in db/db mice. Present study shows for the first time, that
there is upregulation of AT1R protein expression in kidneys of db/db mice that may
contribute in the development of diabetes related hypertension. We also show that,
chronic losartan (10 mg/kg/day) treatment has no effect on blood glucose levels although
it improves morphology of pancreatic islets. In addition, losartan treatment increased
AT1R protein expression but decreased ACE2 protein expression in both db/db and
control mice. Losartan treatment also reduces urinary albumin and protein excretion in
db/db mice.
To investigate the effect of blood glucose reduction, mice were treated with metformin
(150 mg/kg/day). As expected, metformin treatment improved blood glucose and glucose
tolerance in db/db mice. Our study shows effectiveness of metformin in reducing urinary
albumin and protein excretion. However, chronic metformin treatment did not change
renal expression or activity of ACE and ACE2.
One of the aims of this study was to examine if ACE2 could be used as a predictor for
early phase of diabetic nephropathy. We show that there is an increase in urinary ACE2
activity and ACE2 content in young 5 week, db/db mice. Present study also shows
74
presence of ACE, ACE2 and AT1R proteins in concentrated urine samples of 8 week
mice. Our previous studies show that there is no detectable ACE2 activity in plasma.
Therefore, kidney is the primary source of urinary ACE2. These findings suggest that
urinary ACE2 could be a potential non-invasive biomarker of diabetic nephropathy.
75
APPENDIX A
Urinary, but Not Plasma Angiotensin-Converting Enzyme 2 (ACE2), Is Associated with Diabetic Nephropathy
Malav N Madhu, Rendong Quan, Nathan M Weir, Wenfeng Zhang, Mariana Morris, Khalid M Elased
Boonshoft School of Medicine, Wright State University, Dayton, OH
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide. Alteration in the renin angiotensin system (RAS) is widely believed to contribute to kidney injury in diabetes. Currently, detection of urinary albumin is the only non-invasive technique used for diagnosis of DN. However, microalbuminuria is a poor predictor for DN, while proteinuria is only detectable in late stage nephropathy. Recent studies suggested that ACE2 is renoprotective in 8 week old murine model of type II diabetes (db/db mice). The aim of this study was to test the hypothesis that urinary ACE2 is an early marker for intra-renal RAS status and nephropathy in db/db diabetic mice. Individual urine samples were collected from 5-8 week old male db/db diabetic and control mice over 24 hours. Renal and urinary ACE2 activities were determined using the conventional fluorogenic substrate, Mca-APK (Dnp) and also using SELDI-TOF mass spectrometry (MS). Urinary ACE2 content was evaluated by ELISA. Urinary albumin, creatinine and protein were measured as an index of kidney damage. Renal ACE and ACE2 expression were determined by western blot. At an early age (5-6 weeks) db/db mice developed moderate hyperglycemia, hyperinsulinemia and mild albuminuria (p<0.01). Five week old db/db mice showed a significant increase in urinary ACE2 content (p<0.05) and activity (p<0.01) compared to controls. This finding was confirmed by SELDI-TOF MS. There was increase in renal ACE2 expression and activity of 8 week db/db mice (p<0.05) while renal ACE expression was decreased in db/db mice (p<0.01) compared to controls. Interestingly, plasma ACE activity was significantly higher in db/db mice compared to controls (p<0.01). There was no detectable ACE2 activity in plasma suggesting urinary ACE2 was derived from the kidney. In conclusion, urinary ACE2 warrants further investigation as a potential non-invasive marker for early diabetic nephropathy.
Poster accepted in the 63rd High Blood Pressure Research Conference, Chicago 2009
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APPENDIX B
Upregulation of Angiotensin AT1 Receptors in Hypertensive db/db Diabetic Mice
Malav N Madhu, Nathan M Weir, Rendong Quan, Denielle Senador, Wenfeng Zhang, Mariana Morris, Khalid M Elased
Boonshoft School of Medicine, Wright State University, Dayton, OH
Hypertension is a major cause of cardiovascular and renal disease in diabetics. In our previous studies we showed that high blood pressure in obese db/db diabetic mice can be reduced by angiotensin AT1 receptor (AT1R) blockade. The aim of this study was to assess the changes in renal and aortic AT1R expression and pressor response to chronic angiotensin II (Ang II) infusion in db/db mice. Western blot analysis was used to measure renal and aortic protein expression in 8-18 week old male db/db mice and their lean controls. AT1R expression was significantly increased in kidneys and aorta of db/db mice compared to controls (p<0.01). Immunohistochemistry showed increased immunostaining for AT1R in cortical kidney tubules and glomeruli of db/db mice compared to controls. There was also a significant increase in plasma ACE activity (p<0.01) and plasma Ang II (p<0.05) in db/db mice compared to controls. Another group of 8 week old db/db mice were implanted with carotid telemetric probes and 24 hr mean arterial pressure (MAP), heart rate (HR) and activity were monitored weekly. MAP began to increase in db/db mice after the age of 11 weeks during both light and dark periods when compared to controls (p<0.01). At 15 weeks, Ang II was infused at 1000 ng/kg/h for 4 weeks to evaluate its effect on pressor response. The infusion elicited a spike in MAPs of both control and db/db mice compared to the previous week (p<0.01). After the initial increase, MAP of control mice regressed towards baseline. However, MAP of db/db mice remained elevated throughout the infusion period (p<0.05). This is the first report to demonstrate increases in AT1R expression in db/db mice. The prolonged pressor response to Ang II by db/db mice could, in part, be attributed to the upregulation of AT1R and preexisting elevation in plasma Ang II.
Poster accepted in the 63rd High Blood Pressure Research Conference, Chicago 2009
77
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