Role of Brain Angiotensin-II in Development of Experimental Diabetic Nephropathy in Wistar Rats

Abstract The renin-angiotensin-aldosterone system (RAAS) plays a key role in diabetic nephropathy (DN). Angiotensin-II secreted during the RAAS pathway increases nephropathy. It stimulates oxidative stress which can quench nitric oxide. Reduced nitric oxide level aggravates Ang-II-induced vasoconstriction. Ang-II has also emerged as a central mediator of the glomerular hemodynamic changes that are associated with renal injury. Deletion of ACE2 is also noted due to increased Ang-II level which leads to the development of DN. We hypothesize that nephropathy caused by Ang-II in the periphery may be controlled by brain RAAS. ACE inhibitors and ARBs may show the renoprotective effect when administered through ICV without crossing the blood-brain barrier. DN was observed after 8 weeks of diabetes induction through alloxan. Administration of captopril and valsartan once and in combined therapy for 2 weeks, significantly reduced urine output, blood urea nitrogen, total protein in the urine, serum cholesterol, serum creatinine, serum triglycerides, and kidney/body weight ratio as compared to diabetic control rats. Further, combination therapy significantly increased the body weight and serum nitrate level as compared to diabetic control animals. However, increased ACE2 levels in the brain may reduce the sympathetic outflow and might have decreased the peripheral activity of Ang-II which shows beneficial effects in DN.


INTRODUCTION
Recent studies say that globally around 382 million people are suffering from diabetes which will rise to 592 million by 2035 and will become the most challenging health-related problem of the 21st century (Bertoncello et al., 2015).India is also known as the diabetic capital of the world as it projected 109 million diabetic individuals by 2035 (Kaveeshwar, Cornwall, 2014).Currently, diabetic mellitus (DM) affects more than 62 million people in India which is more than 7.1% of the total population.The estimated survey shows that around 1 million people die in India due to diabetic complications every year (Kaveeshwar, Cornwall, 2014;Unnikrishnan et al., 2007).Diabetic nephropathy (DN) remains the most common cause of end-stage renal disease (ESRD) worldwide (Zhang et al., 2015).Patients suffering from DN have symptoms like proteinuria & microalbuminuria and show a progressive decline in glomerular function.DN is a serious chronic complication in type 1 diabetes mellitus.Along with kidney complications nephropathy caused by diabetes also causes a rise in arterial blood pressure which gets develops in 30 to 40 % of all types of diabetic patients (Lewis et al., 1993;Trevisan, Dodesini, Lepore, 2009).
The exact mechanism of DN is not very much clear but it has been well reported that the renin-angiotensinaldosterone system (RAAS) plays a major role in the regulation of DN and that is why the first line treatment therapy includes blockade of the RAAS pathway to treat Page 2/12 Braz.J. Pharm.Sci.2023;59: e20200 Anubhav Kumar Singh, Niraj Kumar Singh, Ahsas Goyal, Bhupesh Chander Semwal, Harlokesh Narayan Yadav DN (Trojacanec et al., 2013).Angiotensin-II (Ang-II) one of the main components of RAAS shows an important role in both renal physiology and the pathogenesis of chronic kidney disease (Kobori et al., 2013).The Ang-II of the RAAS gets increased in DN.Ang-II affects the kidney in many ways such as it causes cellular differentiation, cell proliferation, apoptosis, renal hypertrophy, tubular sodium restoration, and most importantly vasoconstriction (Leehey et al., 2000).
The RAAS pathway is present in various organs, including the heart, adipose tissues, kidney, adrenal gland, and brain.It has been well documented that brain RAAS plays a critical role in the development of kidney failure and heart failure (Mckinley et al., 2001;Chawla, Sharma, Singh, 2010).Angiotensinogen is common the precursor molecule for the synthesis of angiotensin I, II, and III in the brain as well as in the liver (Campbell et al., 2003).The RAAS pathway starts from the release of renin from the kidney after which this renin acts on plasma angiotensinogen to convert it into angiotensin-I (Ang-I).Angiotensin-converting enzyme (ACE) converts Ang-I into Ang-II which shows a deleterious effect on the kidney to cause renal damage (Doughue et al., 2000;Crackower et al., 2002).After that formation of angiotensin-converting enzyme-2 (ACE-2) takes place from ACE, this is a monocarboxypeptidase compound.This ACE-2 is one of the new elements of the RAAS that cleaves decapeptide Ang-I and octapeptide Ang-II to form Angiotensin (1-7) & Angiotensin (1-9) respectively (Tikellis et al., 2008).When ACE2 removes amino acid from the carboxy-terminal of Ang-II, Ang (1-7) gets synthesized.Ang (1-7) which is a heptapeptide compound shows beneficial effect in DN as it has opposite actions than Ang-II (Battle, Soler, Ye, 2010).
Renin and Ang-II secreted during the RAAS pathway increase nephropathy as it shows rapid effects like increased vasoconstriction and increased aldosterone secretion (Santos et al., 2013;Sharma, Sharma, 2013).Ang-II stimulates oxidative stress which can quench nitric oxide, an endothelium-dependent vascular relaxant, and thereby aggravate Ang-II-induced vasoconstriction (Ruster, Wolf, 2006).Ang-II also emerged as a central mediator of the glomerular hemodynamic changes associated with progressive renal injury (Alberti, Zimmet, Shaw, 2006).Ang (1-7) and Ang (1-9) counter-regulate the negative consequences of Ang-II by the activation of Mas receptors (Giani, Munoz, Pons, 2011).Moreover, the deletion of ACE2 is noted due to increasing Ang-II levels which lead to the development of DN (Zini et al., 1996).
The ACE2 enzyme found in the brain plays an important role in the regulation of peripheral activity of RAAS (Baltatu, Campos, Bader, 2011).It has been believed that over-expression of ACE2 in the brain reduces the sympathetic outflow and decreases the peripheral activity of Ang-II (Feng et al., 2010).
However, in this study, we want to investigate how brain RAAS may control the periphery RAAS pathway without crossing the blood-brain barrier itself.Hence, in this research work, we have hypothesized that nephropathy caused by angiotensin-II in the periphery may be controlled by brain RAAS.Drugs like ACE inhibitors and ARBs may show a renoprotective effect when administered through ICV-route.

Animals
A total 36 Wistar rats of either sex with the body weight of 200g ± 10% were used in this study.Animals were randomly divided into six groups (Normal Control, Diabetic Control, Vehicle treated, Captopril treated, Valsartan Treated, and Captopril + Valsartan Treated) having six rats in each group.Rats were fed with standard food and water ad libitum.The animals were housed in cages with a 12 h light/dark cycle and temperaturecontrolled environment (20±2ᴼC).The experimental protocol was approved by Institutional Animal Ethical Committee (GLAIPR/CPCSEA/IAEC/2015/P.Col/R1) for the use of laboratory animals.

Drugs and Chemicals
Alloxan monohydrate, Valsartan, and Captopril (Sigma-Aldrich, Hyderabad, India) were freshly prepared before the administration.All the other regents used in the present study were analytical grade and freshly prepared before use.

Surgery and administration of drugs
Thiopental sodium (Thiosol Sodium, Neon laboratories ltd, Mumbai; 30 mg/Kg, i.p) was used to anesthetize the rats (Kushwaha et al., 2011).A minor cut (approx 1-2cm) was applied on the head on either side of the midline (Haley, McCormick, 1957).Extra tissues were removed to expose the skull.After that rats were placed on stereotaxic apparatus to determine the ICV position.The location concerning the lambda point was 0.8mm Anteroposterior (AP), 1.8mm Mediolateral (ML), and 3.6mm Dorso-ventral (DV) deep to the skull surface.After the ICV point was determined, the drilling was done manually by using a driller in both hemispheres, and polypropylene cannula were placed in each hole for smooth administration of drugs, regularly for 2 weeks.The drug was administered by using a Hamilton syringe at 1μL/min rate and 5μL drug or vehicle was introduced in each hemisphere.

Inductions and assessment of experimental diabetic nephropathy
Randomly selected rats were allocated to receive alloxan monohydrate (120 mg/Kg, i.p.; Once) for induction of experimental type 1 diabetes mellitus (Sharma et al., 2010).Animals having serum glucose levels more than 200 mg/dL were considered as diabetic.Blood samples were collected from the retro-orbital sinus.The presence of diabetes mellitus was confirmed by measurement of serum glucose level by GOD-POD method (Trinder, 1969) using a commercially available enzymatic kit (Span diagnostics ltd., Surat, India).

Assessment of diabetic nephropathy
After 8 weeks of diabetes induction, DN was confirmed.It was evaluated biochemically by accessing the blood urea nitrogen (BUN), serum creatinine, total protein in the urine, and serum nitrite level weight (Vaishya, Singh, Lal, 2008) using diagnostic kits (Span diagnostics ltd., Surat, India).

Estimation of serum nitrates
Unlike NO, nitrite can be measured easily and nitrite concentration can be used to infer the level of NO production.Nitrite release in coronary effluent was measured.Greiss reagent 0.5 ml (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in water) was added to 0.5 ml of serum.The optical density at 550 nm was measured using a spectrophotometer.Nitrite concentration was calculated by comparison with the spectrophotometer reading of standard sodium nitrite.The result was expressed as micromoles per liter (μM/L).

Estimation of serum cholesterol and triglyceride
The serum cholesterol and triglyceride were estimated by blood samples collected by retro-orbital sinus by using commercially available kits (Span Diagnostics Ltd., Surat, India).

Estimation of kidney weight/body weight ratio
Kidney weight/Body weight ratio was estimated by removal of both kidneys, renal fascias were removed and kidneys were weighed individually.The kidney weight/ body weight (%) was calculated by following the formula (Sinuani et al., 2006).

Statistical analysis
All values were expressed as mean ± standard deviation (S.D.).Statistical analysis was performed by using GraphPad and Sigmastat software.Data obtained from various groups were significantly analyzed using one-way ANOVA, p<0.05 was considered to be statistically significant.

RESULTS
At the start of the experiment, an assessment of different parameters was done.After a single administration of alloxan (120mg/Kg, i.p.), parameters were accessed each week till the 10 th week.DN was confirmed at the 8 th week and treatments were continued further up to the 10 th week after alloxan administration.All parameters were accessed up to the 10 th week and findings were compared between the treatment group vs. normal and diabetic control group.

Effect of various pharmacological interventions on body weight
The effect of treatments on body weight was illustrated in Figure 1 (A).Significant (p<0.05)change was found in the body weight of diabetic rats after the administration of alloxan monohydrate as compared to normal rats.Vehicle-treated group rats do not show any significant decrease in body weight when compared to diabetic control rats.Drug-treated groups i.e. captopril ICV treated group (2μg/day) & valsartan ICV treated group (100 nmol/day) alone and in combination shows significant (p<0.05)increase in body weight when they are compared to the diabetic control group after treatment.

Effect of various pharmacological interventions on kidney weight/body weight
Figure 1 (B) depicted the effect of treatment on the kidney weight/body weight ratio.A significant increase in kidney weight /body weight was noted in diabetic rats as compared to normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduced the elevated kidney weight/body weight ratio as compared to diabetic rats.

Effect of various pharmacological interventions on blood urea nitrogen
The effect of treatments on BUN was illustrated in Figure 2 (A).The concentration of BUN significantly (p<0.05)increases in diabetic rats as compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the elevated level of BUN as compared to diabetic rats.

Effect of various pharmacological interventions on serum triglyceride level
Figure 3 (B) depicted the effect of treatment on serum triglyceride levels.Serum triglyceride levels also get elevated significantly (p<0.05) in diabetic rats when compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/ day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the high serum triglyceride level as compared to diabetic rats.

Effect of various pharmacological interventions on urine output/24-hour
The effect of treatments on serum urine output/24hour was illustrated in Figure 4 (A).Administration of

Effect of various pharmacological interventions on serum total cholesterol
The effect of treatments on serum cholesterol levels was illustrated in Figure 3 (A).A significant (p<0.05)increase in serum cholesterol was noted in diabetic rats when compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the elevated level of serum cholesterol as compared to diabetic rats.

Effect of various pharmacological interventions on total protein in urine
Figure 4 (B) depicted the effect of treatment on total protein in the urine.The concentration of total protein in urine significantly (p<0.05)increases in diabetic rats as compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the elevated level of total protein in the urine as compared to diabetic rats.

Effect of various pharmacological interventions on serum glucose level
The effect of treatments on serum glucose levels was illustrated in Figure 5 (A).A single administration of alloxan (120 mg/Kg; i.p.) significantly (p<0.05)increases the serum glucose concentration as compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration does not affect the serum glucose level in diabetic rats.alloxan (120 mg/Kg; i.p.) significantly (p<0.05)increases the urine output as compared to normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/ day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the elevated urine output in diabetic rats.

Effect of various pharmacological interventions on serum creatinine
Figure 5 (B) depicted the effect of treatment on serum creatinine.Serum creatinine concentration was significantly ( p<0.05) increased in diabetic rats as compared with normal rats.Treatment with captopril (2μg/day; ICV) and valsartan (100 nmol/day; ICV) alone or in combination for 2 weeks after 8 weeks of alloxan administration significantly (p<0.05)reduces the elevated serum creatinine level as compared to diabetic rats.

DISCUSSION
In this study, it has been demonstrated for the first time that brain RAAS may control the DN.Diabetes mellitus has been induced by a single administration of alloxan at 120 mg/Kg i.p. (Sharma et al., 2010) which develops nephropathy in 8 weeks.Assessment of parameters was continued up to the 10 th week.In the present study, ACE Inhibitor captopril at the dose of 2 μg/day; ICV (Baum, Becker, Suberts, 1983) and ARB valsartan at the dose of 100nmol/day; ICV (Nakamura et al., 2014) were administered to rats.Captopril and valsartan administered through the ICV route may control the peripheral activity of Ang-II by reducing the sympathetic outflow without crossing the blood-brain barrier (Feng et al., 2010).Rats of the vehicle-treated group receive ACF through the ICV route which does not show any significant change as compared to the diabetic control group.While captopril and valsartan either alone or in combined therapy show subsequent renoprotection in severe nephropathic rats.
Alloxan is a kind of β-cytotoxin that damages β-cells of the pancreatic islet which leads to insufficient insulin release that causes type-1 diabetes.Alloxan generates ROS which preferentially accumulates in β-cells through the GLUT2 glucose transporter to destroy it.Treatment with captopril and valsartan alone or in combined form does not show any significant change in increased glucose level which has been supported by Onozato et al., 2002.In type 1 diabetes mild reduction in body weight is common due to flushing of glucose & calories through urine (Rosenfalck et al., 2002).Treatment with captopril, valsartan, and their combination shows significant alteration in body weight as compared to the diabetic control group.
DN causes many characteristic changes to the structure of the glomerulus mainly known as glomerulosclerosis (Cockcroft, Gault, 1976).Due to nephropathy, extracellular deposits get increased inside the renal corpuscle, with decreased surface area for filtration.It also makes the glomerular basement membrane thicker and leakier, due to which polyuria occurs (Makaya et al., 2013).The diabetic control group significantly increases the urine output as compared to the normal group.While captopril & valsartan alone or in combined therapy significantly reduces the elevated urine output.Restoration in glomerular basement membrane permeability may be the reason for it.
The serum creatinine, BUN, and urinary total protein level are commonly used parameters to check the kidney function in DN which gets abnormally elevated in the case of DN.Serum creatinine is the most important indicator that shows renal health because it is a byproduct of muscle metabolism that is excreted by kidneys (Bakris, Weir, 2000;Maschio et al., 1996).A damaged kidney was unable to filter creatinine which causes increased serum creatinine levels (Zatz et al., 1987).Urea is produced by the liver in the urea cycle as a waste product of digested protein (Viberti et al., 1994).In nephropathic conditions, the kidney fails to remove excess urea from the body which causes elevation of BUN.Macroalbuminuria is the most common cause of DN.Alteration in kidney function causes excess albumin secretion in urine which is commonly known as proteinuria (Kelly et al., 2007;Mogenson, 1987).It has been seen that persistent albuminuria causes dysfunction of several cells like glomerular endothelial cells, podocytes, and proximal tubule epithelial cells due to which proteinuria occurs in DN (Fioretto, Stechower, Mauer, 1998).BUN, serum creatinine &urinary protein level were significantly reduced by alone or combined treatment with captopril & valsartan through the ICV route.This may be due to improved glomerular filtration rate & reduction in glomerular permeability due to which protein loss gets reduced (Chawla, Sharma, Singh, 2010).
Assessment of lipid profile has been done to determine the effect of nephropathy on serum cholesterol level and serum triglyceride level.It was observed that diabetic rat shows a marked increase in both cholesterol and triglyceride levels as compared to normal rats.Dyslipidemia due to diabetes tends to cause hypercholesterolemia in which LDL, VLDL, and triglyceride levels get increased in serum (Goldberg, 2001).The mechanism involved behind this is the loss of urinary protein that stimulates an increased LDL synthesis from the liver which causes upregulation of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA).While impaired chylomicrons and VLDL clearance is a dominant factor that increases serum triglyceride concentration (Vaziri, 2003;Vaziri, Sato, Liang, 2003).Alone or combined treatment with captopril & valsartan through the ICV route reduces the elevated level of serum cholesterol and serum triglyceride significantly.This may be due to the ability of ACE inhibitors and ARB to restore proteinuria which helps in maintaining high lipid complications in DN (Trevisan, Dodesini, Lepore, 2006).
Due to vascular endothelial dysfunction (VED) in DN reduced serum nitrite concentration was observed (Jindal, Singh, Balakumar, 2007).Serum nitrite level and kidney weight/body weight were considered as the most important marker that indicates renal insufficiency (Remmuzi, Bertani, 1990).Again alone or combined treatment with captopril and valsartan through the ICV route significantly elevates the reduced serum nitrite level.The most significant elevation in serum nitrite level is seen in combined drug therapy (captopril & valsartan) which may be due to VED restoration.However, the ratio of kidney weight/body weight in diabetic rats gets increased significantly due to the presence of renal hypertrophy which increases the weight of the kidney in both diabetic and vehicle groups (Zhang et al., 2015).Treatment with captopril & valsartan alone or combined therapy reduces the elevated kidney weight/body weight significantly.This may be due to a reduction of Ang-II level which causes cell proliferation & renal hypertrophy (Remuzzi et al., 1991).

CONCLUSION
Based on the above discussion, it may be concluded that co-administration of valsartan and captopril attenuates diabetic nephropathy.These observed renoprotective effects may be due to the decreased central sympathetic outflow and peripheral activity of Ang II.It is the most responsible component in the RAAS pathway that causes nephropathy as it has effects like increased oxidative stress and most importantly vasoconstriction.The RAAS present in the body either in the brain or periphery controls the physiological activity of the renal function.Deletion of ACE2 is noted due to increasing Ang-II levels which lead to the development of DN.The experiments done, the analysis performed & the result obtained promotes us to rather strongly claim that nephropathy developed in the rat is due to increased secretion of Ang-II.Treatment was given with single and combined drug therapy of captopril and valsartan through the ICV route in the brain which show beneficial results in DN.The mechanism which may involve in this would be overexpression of the ACE2 level in the brain.Increased ACE2 levels in the brain may reduce the sympathetic outflow and might have decreased the peripheral activity of Ang-II which helps in the treatment of DN.

FIGURE 1 -
FIGURE 1 -Effect of vehicle and drug treatment on body weight (A) and kidney weight/body weight % (B) on 10 th week.Values are expressed as mean ± S.D., a=p<0.05 vs. normal control; b=p<0.05 vs. diabetic control.

FIGURE 2 -
FIGURE 2 -Effect of vehicle and drug treatment on blood urea nitrogen (BUN; A) and serum nitrite (B) level on 10 th Week.Values are expressed as mean ± S.D., a=p<0.05 vs. normal control; b=p<0.05 vs. diabetic control.

FIGURE 3 -
FIGURE 3 -Effect of vehicle and drug treatment on serum cholesterol (A) and triglyceride (B) level on 10 th Week.Values are expressed as mean ± S.D., a=p<0.05 vs. normal control; b=p<0.05 vs. diabetic control.

FIGURE 4 -
FIGURE 4 -Effect of vehicle and drug treatment on urine output/24-hr (A) and total protein in the urine (B) on 10 th Week.Values are expressed as mean ± S.D., a=p<0.05 vs. normal control; b=p<0.05 vs. diabetic control.

FIGURE 5 -
FIGURE 5 -Effect of vehicle and drug treatment on serum glucose (A) and creatinine (B) level on 10 th Week.Values are expressed as mean ± S.D., a=p<0.05 vs. normal control.