Angiotensin II type 2-receptor agonist C21 reduces proteinuria and oxidative stress in kidney of high-salt fed obese Zucker rats

Abstract

Oxidative and nitrosative stress have been implicated in high sodium diet (HSD)-related hypertensive renal injury. In the present study we investigated AT₂R-mediated renoprotection in obese Zucker rats fed HSD. Obese Zucker rats were fed normal sodium-diet (NSD) or HSD 4%, for 14 days, with/without AT₂R agonist C21, delivered subcutaneously via osmotic pump, 1 mg/kg/day. Compared to NSD controls, HSD rats exhibited increase in cortical NADPH oxidase activity, urinary H₂O₂ and 8-isoprostanes, which were associated with severe glomerulosclerosis, interstitial fibrosis, decline in estimated glomerular filtration rate (eGFR), and an increase in urinary leak and activity of N-acetyl-β-D-glucosaminidase, a lysosomal enzyme and a marker of tubular damage. These changes were improved by C21 treatment. Cortical expression of endothelial nitric oxide synthase (eNOS), p-eNOS (Ser¹¹⁷⁷) and plasma nitrites were reduced after HSD intake while nitrosative stress (3-nitrotyrosine) and enzymatic defense (superoxide dismutase-to-catalase activity) remained unaltered. Albeit, C21 preserved plasma nitrites in HSD-fed OZR. C21 treatment reduced protein-to-creatinine (uPcr), albumin-to-creatinine (uAcr) as well as fractional excretion of protein (FE_pro) and albumin (FE_alb) in HSD-fed OZR, which is independent of changes in protein recycling receptors, megalin and cubilin. HSD intake also altered renal excretory and reabsorptive capacity as evident by elevated plasma urea nitrogen-to-creatinine (UN-to-cr) and fractional excretion of urea nitrogen (FE_UN), and reduced urine-to-plasma creatinine (UPcr), which were modestly, but insignificantly, improved by C21 treatment. Together results demonstrate that AT₂R activation protects against HSD-induced kidney damage in obesity plausibly by reducing NOX activity and rescuing nitrites.

Introduction

Hyperactive local angiotensin (Ang) II-AT₁R signaling in kidney can operate independently of a systemic RAS and is a key player in pathogenesis of progressive hypertensive chronic kidney diseases (CKD).^1-3 Recent analysis revealed that much of continuing increase in number of patients with CKD is seen in obese hypertensive patients.⁴ Various animal models have been employed to gain more insight into the pathogenesis of CKD associated with obesity hypertension. One of the validated models is the obese Zucker rat (OZR). These rats may or may not be hypertensive as such, but exhibit gradual increase in blood pressure⁵ and classical morphological characteristics of non-immune-spontaneous focal segmental glomerulosclerosis (FSGS) just in 14 days when fed high sodium diet (HSD, 4%).⁶ HSD intake also increased renal content of Ang-II in obese rats.^5,7 Lavaud et al⁸ and others^9,10 held non-hemodynamic factors responsible for this event; because glomerular hemodynamic function remained unchanged in obese and lean Zucker rat. Experimental and clinical reports point at essential events associated with HSD-related pathologies of hypertension and CKD: oxidative^11-13 and nitrosative stress and renal immune cell infiltration.

Progression of chronic kidney disease is predicted by (i) glomerular filtration rate, (ii) fractional excretion of protein (FE_pro) and albumin (FE_alb), and (iii) an absolute comparison of urinary albumin-to-protein ratio (uAPr). Under normal conditions, the glomerulus filters significant protein, most of which is retrieved by tubular receptors (megalin, cubilin), processed through lysosomal pathway in amino acids which are delivered back into circulation. Thus, under normal circumstances protein remains undetected in urine. FE_pro, FE_alb and uAPr have reliable predictive values in characterizing renal damage, glomerular (albuminuria>proteinuria) or tubular (proteinuria>albuminuria), as it represents glomerular filtration function with increased glomerular permeability.^14-19 In addition to these indices, determination of urinary activity of N-acetyl-β-D-glucosaminidase (NAG), one of the best characterized tubular lysosomal enzyme helps distinguish renal tubular injury. Their comprehensive assessment in OZR model of salt-induced kidney damage hasn't been reported.

Renal AT₂R, while sparsely expressed in healthy tissues, is upregulated in obesity,²⁰ diabetes²¹ and tissue damage²² and considered endogenous defense by virtue of its own actions and by antagonizing AT₁R-mediated deleterious actions. Recently, AT₂R activation by preferential agonists C21 and CGP42122A has showed natriuretic and blood pressure reducing effects, as well as GFR improvement in HSD-fed obese rats.⁵ AT₂R also have anti-inflammatory,²³ anti-proliferative and anti-fibrotic effects,²⁴ which are important in preventing disease progression. Also, there is indication that AT₂Rs may be involved in anti-oxidative stress activities.²⁵ Pharmacological inhibition of AT₂R by PD123319 has shown to increase oxidative stress in vitro^26,27 and in vivo.²⁸ AT₂R stimulation by CGP42112 caused a decrease in (rotenone+Ang-II)-induced NADPH oxidase (NOX) activity and mRNA expression of NOX units in in vitro CATH.a model of Parkinson's disease.²⁹ Recently, C21, which is a non-peptide orally active AT₂R agonist, has been shown to reduce superoxide formation in cryo-sections of aorta of stroke-prone spontaneously hypertensive rats²⁴ and frozen sections of ischemic cerebral hemisphere.³⁰ However, in study of Rehman et al²⁴ C21 did not reduce NOX activity. It is unknown whether direct AT₂R stimulation by a preferential agonist reduces oxidative stress and protects against salt-induced kidney damage in obesity. Therefore, the present study investigated the role of AT₂R selective activation by C21 in oxidative stress and reno-protection in terms of kidney structure and function in high salt fed obese Zucker rats.

Methods

Experimental protocol

Male obese Zucker rats (12-14 week) were purchased from Harlan, Indianapolis, IN. After arrival, the animals were housed in the University of Houston animal care facility. Animal experimental protocols used in this study were approved by the IACUC at the University of Houston and adhere to the NIH Guide for the Care and Use of Laboratory Animals. Animals were treated with AT₂R agonist C21 (1 mg/kg/day) subcutaneously via Alzet^® osmotic pump (Model 2ML2, Durect Corporation, Cupertino, CA) while maintained on either normal (NSD, 0.4%) or high sodium diet (HSD, 4%) for 14 days. In the present study, the subcutaneous delivery rate ∼0.42 μg/min (or ∼605 μg/day) was chosen to maintain the daily dose of C21 (1 mg/kg/day) as we reported earlier.⁵ This dose of C21 is considered AT₂R selective as another study from our laboratory⁷, i.v.)-induced natriuresis was blocked by AT₂R antagonist PD123319. Control obese rats were implanted with osmotic pumps filled with normal sterile saline while maintained on either NSD or HSD for 14 days. Each of the groups had n=8 animals. Rats were placed in metabolic cages during terminal four days for urine collection over the last 3 days. Twenty four-hour food and water intake was recorded. After treatment, blood was collected via cardiac puncture, processed for plasma preparation and stored at -80°C. Kidney cortices were snap-frozen and stored at -80°C. The kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1 and protease inhibitors.

For detailed various assay methods, please refer to the online-only Data Supplement.

Statistical analysis

Data are presented as mean±SEM. The data were analyzed using GraphPad Prism 5 and subjected to one-way ANOVA with Bonferroni's Multiple Comparison post hoc Test, *p<0.05, n=8 per group.

Results

General parameters

In comparison to obese control rats, HSD-fed rats drank more water (HSD: 32.96±3.69 vs. CT: 23.54±2.1 mL/day) and exhibited marked diuresis (HSD: 16.33±1.78 vs. CT: 8.96±0.88 mL/day). C21 treatment did not affect water consumption and diuresis in rats fed with either NSD or HSD. Food consumption, body weight and changes in total kidney weight of all animal groups remained unchanged over 2-week study period (Table 1). No animals died during the study.

Table 1.

General and renal parameters of control and C21-treated obese rats fed with either normal (NSD) or high sodium diet (HSD).

General parameters	CT	C21	HSD	HSD+C21
Food consumed, g/day	29.96±0.68	28.83±0.71	28.77±2.12	31.99±1.69
Water consumed, mL/day	23.54±2.10	26.54±2.16	32.96±3.69	29.58±3.44
Total kidney weight, g	3.24±0.13	3.16±0.06	3.14±0.11	3.00±0.07
Plasma protein, mg/mL	2.70±0.07	2.51±0.08	2.42±0.09	2.30±0.04
Total body weight, g	619.4±14.4	541.6±17.2	630.0±19.8	613.1±6.5
Urine formation, mL/day	8.96±0.88	11.58±1.55	16.33±1.78*	16.75±1.87
Urinary urea nitrogen (UN), mg/dL	32.58±0.66	32.05±0.96	27.63±0.21*	27.30±0.43
Urinary creatinine (Cr), mg/dL	20.47±2.41	15.81±1.08	7.83±0.34*	9.12±0.62
Plasma urea nitrogen (UN), mg/dL	26.51 ±3.08	30.16±2.74	36.03±2.14*	33.66±2.03
Plasma creatinine (Cr), mg/dL	3.69±0.26	3.44±0.59	5.98±0.44*	4.83±0.80
Plasma total nitrite, μmol/L	23.67±1.75	21.00±2.16	15.35±0.49*	22.08±2.16†

^*significantly different from control obese rats;

^†significantly different from HSD-fed rats (HSD); Values are represented as mean±SEM; One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; N=8 in each group. CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD.

Indices of oxidative stress in vivo

HSD feeding significantly increased the activity of NADPH oxidase (NOX), a major superoxide radical anion (O₂^−•) producing enzyme (HSD: 0.37±0.07 vs. CT: 0.13±0.04 ΔRLU/μg protein/min), which was reduced by C21 in HSD-fed rats (HSD+C21: 0.11±0.04 ΔRLU/μg protein/min), but not in NSD-fed control rats (Fig. 1A). Renal activities of superoxide dismutase (SOD), superoxide dismutating enzyme (Fig. 1B), and catalase (CAT), H₂O₂ metabolizing enzyme (Fig. 1C), remained unchanged. Mean plasma content of H₂O₂ (HSD: 323.80±25.85 vs. CT: 173.20±38.58 μM/mL) (Fig. 1D) and urinary excretion of H₂O₂ (HSD: 41.70±4.72 vs. CT: 23.01±2.13 μM/mg Cr (creatinine)) (Fig. 1E) were increased in HSD-fed rats. Co-treatment with C21 reduced urinary (HSD+C21: 30.51±3.00 μM/mg Cr), but not plasma content of H₂O₂ (HSD+C21: 328.80±22.31 μM/mL) in HSD-fed rats. Urinary excretion of 8-isoprostane was increased as well in HSD-fed rats (HSD: 7.90±1.65 pg/μg Cr), which was reduced by C21 treatment (HSD+C21: 4.12±1.01 pg/μg Cr) (Fig. 1F). Renal carbonyl content was also reduced by C21 treatment in HSD fed rats (HSD+C21: 5.47±1.42 vs. HSD: 10.51±0.92 μM/mg protein) (Fig. 1G). C21 did not affect carbonyl content in NSD fed obese rats.

Figure 1.

Oxidative stress indices such as activities of NADPH oxidase (A), SOD (B) and CAT (C), plasma H₂O₂ (D), urinary excretion of H₂O₂ (E) and 8-isoprostanes (F), and protein carbonyls (G) of control and C21-treated obese rats fed with either normal of high sodium diet for 14 days. Effect of C21-treatment on hyperosmolar salt (0.25 M NaCl)-induced superoxide formation in HK-2 cells (H). Values are represented as mean±SEM; One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD.

Superoxide formation in vitro

To selectively isolate the effect of AT₂R activation on NOX-mediated superoxide formation in absence of in vivo hemodynamic or renal changes, we have stimulated HK-2 cells with high salt (0.25 M NaCl, final) to generate superoxide formation (control treatment had 113 mM NaCl). Salt induced significant superoxide formation (∼12 fold compared to control) which was reduced by AT₂R agonist C21 (1μM) (∼3 fold compared to salt). Pre-treatment with AT₂R antagonist PD123319 (1μM) abolished C21-inhibition of salt-induced superoxide formation. In fact under normal salt condition, treatment with PD123319 increased superoxide formation (∼9 fold) (Fig. 1H).

Expression of endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser¹¹⁷⁷) and 3-nitrotyrosine

In comparison to obese control rats, HSD fed rats show a decreased expression of eNOS (∼130 kDa) by ∼50%, which was not affected by C21 treatment (Fig. 2A). Phospho-eNOS remained statistically unchanged among treatment groups (Fig. 2B). Changes in renal expression of 3-nitrotyrosine were remained non-significant. Although, there was a trend of decrease in the expression of 3-nitrotyrosine due to HSD feeding (by ∼20%), which was further decreased by C21 in HSD-fed rats (by ∼20%) (Fig. 2C).

Figure 2.

Effect of C21-treatment on expression of (A) endothelial nitric oxide synthase (eNOS), (B) p-eNOS (Ser¹¹⁷⁷), and (C) 3-nitrotyrosine (a marker of nitrosative stress) in kidney of obese rats fed with either normal or high sodium diet for 14 days. Phospho-eNOS was quantitated by PathScan^® sandwich ELISA. Values are represented as mean±SEM; One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD.

Plasma nitrites

HSD feeding of obese rats significantly reduced plasma nitrites (HSD: 15.35±0.49 vs. CT: 23.67±1.75 μmol/L). This decrease in plasma nitrites was prevented by concurrent treatment with C21 (HSD+C21: 22.08±2.16 μmol/L) (Table 1).

Morphological assessment of kidney

Periodic acid-Schiff staining was performed to study renal structural changes (Fig. 3, Table 2). In NSD fed OZR: glomeruli were characterized by mild to moderate focal segmental hypercellularity, mesangial expansion, matrix deposition and thickening of basement membrane and narrowing of Bowman's space; tubules were characterized by mild increase in cellularity, moderate vacuolization, lipid deposition, narrowing of lumen, and sloughing of brush border and basement membranes; interstitium showed mild infiltration of immune cells. HSD feeding of obese rats accelerated such structural changes; in glomeruli, which extended to crescent formation, visceral epithelial cell hypertrophy in urinary space, focal segmental collapse of tuft, breaking of capillaries and loss of nephron elements; in tubules, it extended to dilatation, cast formation and atrophy; in interstitium, it extended to significant infiltration and fibrosis. Our findings are in agreement with other reports.³¹ C21 treatment significantly prevented these structural changes, albeit reduction of glomerular indices was higher than that of tubular (Table 2). Other improvements in tubular structural changes by C21 such as cast formation, lipid deposition and narrowing of tubular lumen, vacuolization, sloughing of brush border and basement membranes, visceral epithelial cell hypertrophy may have been insufficient to be detected by our semi-quantitative assessment.

Figure 3.

morphological assessments of periodic acid-Schiff (PAS)-stained kidney sections of obese rats treated with C21 and fed with either normal or high sodium diet for 14 days (A). Glomerular cellularity score (B), decrease in Bowman's space (C), Corpuscle occlusion score (D), Glomerulosclerosis score (E), interstitial infiltration score (F), Interstitial fibrosis score (G). B-G: values are represented as mean±SEM; One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD. Original magnification, X40.

Table 2.

Qualitative assessment of glomerular, tubular and interstitial injury in C21-obese rats fed with either normal (NSD) or high sodium diet (HSD).

Morphological changes	CT	C21	HSD	HSD+C21
Glomerular changes
Focal segmental cellularity and mesangial cell expansion	++	++	+++	++
Thickening of basement membrane	++	++	+++	++
Narrowing of Bowman's space	++	+	+++	++
Crescent formation	+	+	++	+
Focal segmental collapse of glomerular tuft	+	-	++	+
Obliterated capillaries	+	-	++	+
Loss of nephron elements	+	-	++	+
Tubular changes
Cellularity	+	+	+++	++
Dilatation	-	-	+++	++
Atrophy	+	+	+++	+++
Lipid deposits	+	+	++	+
Cast formation and narrowing of lumen	+	+	++	++
Vacuolization	++	++	+++	+++
Sloughing of brush border and basement membrane	++	++	+++	+++
Visceral epithelial cell hypertrophy in urinary space	+	-	++	++
Interstitial changes
Infiltration	+	+	+++	++
Fibrosis	+	+	++	+

Note: Several morphological indices were highlighted as bar graph in Fig. 4. Intensity was graded as mild (+), moderate (++) and severe (+++).

Indices of renal function

HSD feeding caused a significant decrease in urinary excretion of Cr (HSD: 7.83±0.34 vs. CT: 20.47±2.41 mg/dL) and urea nitrogen (UN) (HSD: 27.63±0.21 vs. CT: 32.58±0.66) (Table 1), which were not affected by C21 co-treatment. These HSD-related urinary changes were associated with elevated plasma levels of Cr (HSD: 5.98±0.44 vs. CT: 3.69±0.26 mg/dL) and UN (HSD: 36.03±2.14 vs. CT: 26.51±3.08 mg/dL) in HSD-fed rats (Table 1), which were also not reduced with C21 treatment. Based on plasma and urinary values of Cr and UN, glomerular filtration rate (GFR) was estimated (Fig. 4A). HSD feeding significantly reduced GFR (HSD: 15.15±2.3 vs. CT: 43.50±9.90 μL/min), which was improved with C21 treatment (HSD+C21: 27.43±4.91 μL/min), albeit at non-significant extent compared either with HSD or CT. HSD feeding significantly decreased urine-to-plasma Cr (UPcr) (HSD: 1.37±0.14 vs. CT: 5.80±0.89) (Fig. 4B), and increased FE_UN values (HSD: 60.45±5.83 vs. CT: 26.08±3.68) (Fig. 4C). C21-treatment increased UPcr (HSD+C21: 2.32±0.40) and decreased FE_UN (HSD+C21: 46.05±9.30), albeit at non-significant extent. Surprisingly, HSD intake did not alter plasma UN-to-Cr in obese rats (HSD: 6.17±0.40 vs. CT: 7.31±0.92). C21 treatment increased plasma UN-to-Cr values in obese rats fed either NSD (C21: 11.16±2.19) or HSD (HSD+C21: 8.66±1.74) (Fig. 4D). FE_pro was increased by HSD intake (HSD: 0.18±0.02 vs. CT: 0.12±0.02 μg/min) (Fig. 5A), while FE_alb remained unchanged (Fig. 5B). C21 was able to reduce significantly, both, FE_pro (HSD+C21: 0.11±0.02 μg/min) and FE_alb (HSD+C21: 0.08±0.01 μg/min) in HSD fed rats. HSD feeding of obese rats showed a significant increase in uPcr (HSD: 36±1.63 vs. CT: 17±1.14) (Fig. 5C), but did not affect uAcr (Fig. 5D). C21 treatment of HSD-fed rats decreased both, uPcr (HSD+C21: 29±1.56) and uAcr (HSD+C21: 22±2.47). Similarly, albumin-to-total protein ratio (uAPr) was also decreased in urine of HSD fed rats (HSD: 89±15 vs. CT: 268±55%), which was not affected by C21 (Fig. 5E). Intake of HSD resulted in tubular damage and leakage of NAG in urine. NAG activity was significantly increased in urine of HSD-fed rats compared to NSD controls (HSD: 3.32±0.31 vs. CT: 2.22±0.24 RFU/30 min/μg Cr). Treatment of HSD-fed rats with AT₂R agonist C21 reduced tubular damage and urinary NAG activity (HSD+C21: 2.46±0.13 RFU/30 min/μg Cr) (Fig. 5F). Dot blot analysis of kidney homogenate of HSD fed rats showed significantly reduced expression of protein recycling receptors, cubilin (HSD: 0.77±0.16 vs. CT: 1.27±0.14) (Fig. 5G) and megalin (HSD: 0.25±0.02 vs. CT: 0.47±0.07) (Fig. 5H); C21 had no further effects on the expression of these proteins.

Figure 4.

Effect of C21-treatment on indices of renal function of obese rats fed with either normal or high sodium diet for 14 days: estimated GFR (A), urine-to-plasma creatinine ratio (UPcr) (B), fractional excretion of urea nitrogen (FE_UN) (C), plasma urea nitrogen-to-creatinine ratio (UN-to-Cr) (D). (A) Values are represented as mean±SEM; (B-D) individual values are represented as scattered plot along with their respective mean. One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD. ^ΦStatistically significant at n=12, 95% CI, 10% beta error.

Figure 5.

Effect of C21-treatment on urinary fractional excretion of protein (FE_pro) (A), fractional excretion of albumin (FE_alb) (B), protein-to-creatinine (uPcr) (C), albumin-to-creatinine (uAcr) (D), albumin-to-total protein (uAPr, percent) (E), activity of NAG, Nacetyl-β-D-glucosaminidase, a lysosomal enzyme and a marker of tubular damage (F) and expression of protein recycling receptors cubilin (G) and megalin (H) in kidney of obese rats fed with either normal or high sodium diet for 14 days. G-H: Immune-reactivity was normalized against amido black. Values are represented as mean±SEM; One-way ANOVA followed by Bonferroni's Multiple Comparison Test, p<0.05; CT-obese control, C21-obese treated with C21, HSD-obese treated with HSD, HSD+C21-obese treated with C21 and HSD. ^ΦStatistically significant at n=12, 99% CI, 10% beta error.

Qualitative assessment of all biochemical indices is summarized in Table 3.

Table 3.

Summary of effects of HSD or C21 during HSD feeding in OZR.

Indices	CT vs. HSD	HSD vs. HSD+C21
Food consumption	↔	↔
Water consumption	↔	↔
Total kidney weight	↔	↔
Total body weight	↔	↔
Urinary indices
Urine formation	↑	↔
Urea nitrogen	↓	↔
Creatinine	↓	↔
Fractional excretion of protein	↑	↓
Fractional excretion of albumin	↔	↓
H2O2 excretion	↑	↓
8-isoprostane excretion	↑	↓
N-acetyl-β-D-glucosaminidase activity	↑	↓
Protein-to-creatinine	↑	↓
Albumin-to-creatinine	↔	↓
Albumin-to-total protein, percent	↓	↔
Plasma indices
Protein	↔	↔
Urea nitrogen	↑	↔
Creatinine	↑	↔
Nitrite	↓	↑
H2O2	↑	↔
Renal cortical indices
eNOS expression	↓	↔
Phosphor-eNOS (Ser1177)	↓	↔
3-nitrotyrosine expression	↔	↔
NADPH oxidase activity	↑	↓
Superoxide dismutase activity	↔	↔
Catalase activity	↔	↔
Protein carbonyls	↔	↓
Cubilin expression	↓	↔
Megalin expression	↓	↔
Estimated glomerular filtration rate	↓	↔
Urine-to-plasma creatinine	↓	↔
Fractional excretion of urea nitrogen	↑	↔
Plasma urea nitrogen-to-creatinine	↔	↔

Discussion

A novel and significant contribution of our study is to demonstrate that AT₂R activation exerts anti-oxidative effects and preserve kidney structure and function under HSD feeding in obese rats. This finding is supported by reduced renal NOX activity, urinary excretion of H₂O₂, 8-isoprostanes and renal protein carbonyls in AT₂R selective agonist C21 treated HSD fed rats. Interestingly, C21 was unable to lower plasma content of H₂O₂ which indicates renal specific effects of AT₂R agonist treatment. Unlike our previous report,⁵ we did not observe C21-mediated diuretic and reduced body weight gain effect in HSD fed OZR. This may have been due to different route of C21 administration i.e., oral bolus earlier vs. osmotic pump continuous delivery in the present study.

One of the mechanisms by which chronic salt intake in obesity causes formation of superoxide is activation of the renal RAS component, Ang-II-AT₁R leading to NOX activation and oxidative stress. Our study show that the HSD-induced increase in superoxide and oxidative stress are due to increased NOX activity, a major superoxide generating enzyme and not due to superoxide catabolizing enzymes, SOD and CAT, which were not altered by HSD intake in obese rat kidneys. These findings are consistent with earlier studies.³² Superoxide can directly influence tubular reabsorption of sodium^33,34 in setting of hypertension⁵ and renal injury in obese rats as seen in the present study and in Dahl S (salt-sensitive) rats.³⁵ The AT₂R agonist C21 treatment has shown to reduce renal Ang-II,⁵ expression of p47phox – NOX subunit,³⁶ superoxide formation³⁷ and salt-sensitive rise in blood pressure.⁵ Since Ang-II via AT₁R^38,39 and high blood pressure⁴⁰ are potent stimuli to cause oxidative stress, it is likely that a reduction in renal Ang-II levels and blood pressure in response to C21,⁵ contributed to the antioxidative properties of the AT₂R in these animals. On the other hand, our in vitro study performed in HK-2 cells and another in vivo study²⁸ provide evidence suggesting that AT₂R activation may have a direct role in reducing oxidative stress. However, present in vivo study does not allow us to conclude whether C21 actually reduced oxidative stress independent of the changes in blood pressure in obese animals. A separate study is warranted to delineate AT₂R-linked anti-oxidative stress activity from hypertension and Ang-II in salt-induced renal injury in obese rats.

Our study does not support the role of nitrosative stress in HSD-related renal pathologies in obesity. OZR have reduced expression and activity of NOS and bioavailability of nitric oxide.⁴¹ HSD intake further reduced eNOS expression and induced superoxide formation.⁴² If increase in expression/activity of NOS is involved per se, we would expect a change in renal 3-NT during HSD intake because HSD intake increases superoxide formation. However, 3-NT remained unchanged; therefore in such conditions, we do not expect peroxynitrite and nitrosative stress to rise. This notion is supported by our results of reduced expression of renal eNOS and plasma nitrites and unchanged renal 3-nitrotyrosine in HSD fed obese rats. C21 did not prevent reductions of eNOS or p-eNOS, but preserved plasma nitrites in obese rats fed HSD, and this effect may be owed to reduction of NOX activity, superoxide formation and oxidative stress indices.

Obese Zucker rats exhibit modest monocyte-macrophage infiltration, glomerular hypertrophy, mesangial matrix expansion and basement membrane thickening as early as 7-8 week of age, which is resolvable by AT₂R agonist C21.⁴³ At this age, animals do not show membranoproliferative FSGS, tubular fibrosis or proteinuria. Older OZRs (12-14 week of age) exhibit mild FSGS and fibrosis and decline in GFR at 30 week of age.⁴³ OZR in our study at 12-14 week of age showed morphological changes which remained statistically unchanged by C21 treatment. This discrepancy observed with C21 treatment in our earlier⁴³ and present study may be due to age of OZR (7-8 week vs. 12-14 week). Present study data suggest that HSD intake caused an interstitial infiltration and hastened tubulointerstitial fibrosis secondary to FSGS and reduced GFR. Similar changes also have been observed in HSD fed Dahl salt-sensitive⁴⁴ and obese SHR rats.⁴⁵ Thus, immune cell infiltration and fibrosis may represent a nonspecific response to HSD intake, which can plausibly contribute to kidney damage, for example by augmenting oxidative stress.^42,46 Our recent studies, both in vivo and in vitro, indicated that AT₂R activation with C21 exert anti-inflammatory response via altering cytokine profiles of renal epithelial cells and macrophages.^23,43 Consistent with those findings,^23,43 in the present study, AT₂R activation by C21 significantly reduced interstitial infiltration of immune cells in HSD fed OZR and in turn may have been a reason for reduced tubulointerstitial fibrosis. Anti-inflammatory properties of AT₂R activation are dependent on activation of phosphatases⁴⁷ and decreased NF- B responses as well.⁴⁸ It is likely that C21-mediated reduction of fibrotic injury may involve reduced expression of inflammatory and fibrotic mediators^49-52 or decrease in tissue inhibitors of metalloproteinases (MMP), resulting in increased MMP activity, as seen in diabetic ApoE-/- mouse model.³⁶

Kidney damage was also assessed by functional changes. NSD fed OZR exhibited primarily glomerular proteinuria, uAcr>uPcr, which might have originated from glomerular leakage, defective retrieval of tubular protein or both. HSD intake caused additional tubular proteinuria without an increase in albuminuria as determined by decreased uAPr in HSD fed OZR in current study and Dahl S rats.⁴⁶ HSD-induced tubular proteinuria may have been due to (a) altered retrieval of excess filtered albumin by endocytosis as demonstrated by loss of megalin-cubilin and/or (b) abnormal protein degradation by lysosomes and urine proteases,⁵³ which return non-immunoreactive peptide fragments of albumin to tubular lumen for excretion that remained undetected by antibodies specific to intact albumin.^54,55 Other tubular protein retrieval mechanisms exist,⁵⁶ but how exactly HSD intake in obesity affects these mechanisms is not known. AT₂R agonist C21 reduced proteinuric indices independent of protein recycling receptors (megalin-cubilin), which suggest that C21 improved either glomerular protein permeability or lysosomal degradation of protein, and thus reduced secretion of nephritic proteins into the tubular lumen. This finding is in accordance with that of Gelosa et al⁵⁰ who have shown that C21 (10 mg/kg/day, for 7 week) treatment significantly delayed the time of proteinuria development in stroke-prone SHR. This conclusion is further supported by reduced urinary marker of tubular lysosomal dysfunction (NAG activity) in C21-treated HSD-fed rats.

Despite resolved microscopic changes in AT₂R agonist C21 treated HSD fed OZR, improvements in uAcr and eGFR did not achieve statistical significance. This may have been due to methodology. Light microscopy is a semi-quantitative method while PRM method of urinary protein estimation and eGFR are quantitative. Proteinuria itself is a variable index of renal damage and is neither always correlated with renal structural changes^38,57 nor it predicts renal pathology and progression. The difference in uAcr between HSD and HSD+C21 has 91.1% statistical power. Similarly, it should be noted that eGFR of C21-treated rats was neither statistically different from control nor HSD fed rats. We calculated statistical power of 77.3% to assess any significant difference in eGFR between HSD and HSD+C21 group. C21 would show improvement in eGFR and reduction in uAcr, perhaps with higher sample size (for eGFR, n=12, beta error level 10%, 95% CI; for uAcr, n=9, beta error level 10%, 99% CI) or longer treatments. As established earlier^58-60 and seen clinically,^61-63 HSD-induced decline in GFR occurred in parallel with decreasing urinary concentrating ability as demonstrated by significantly reduced UPcr and elevated FE_UN in HSD fed OZR, which were appeared to be not significantly improved by C21 treatment (Fig. 4). This may have been due to wide range of values that are possible for abnormal concentration of UN and Cr in plasma and urine.⁶¹ Overall, the present study provides evidence suggesting that AT₂R activation with C21 exerts reno-protection during high salt intake in obesity.

Perspectives

Progression of salt-induced proteinuric renal injury occurs via oxidative stress and inflammation, as major pathways that subsequently form a vicious cycle for CKD leading to a faster decline of GFR in obesity. Clinically angiotensin converting enzyme-1 (ACE) inhibitor (ACEi)^64,65 or AT₁R blockers^66,67 help control such pathologies, but they are inconsistent. Our series of experiment highlights AT₂R stimulation as a therapeutic avenue in managing salt-induced oxidative pathologies, especially in obesity where AT₂R are upregulated.²⁰ More importantly, we show direct evidence that AT₂R can preserve indices of kidney injury, via protecting plasma nitrites by reducing oxidative stress and limit exposure of nephritic proteins in the tubular lumen by preserving glomerular integrity, tubular protein recycling or lysosomal processing. AT₂R agonist can be considered as potentially promising therapy for protecting kidney structure and function in obesity. However, further studies are needed to arrive on more definite conclusion regarding the use of the AT₂R agonists as a therapeutic target.

Novelty and Significance.

What is New?

• Selective activation of AT₂R by C21 is renoprotective during high sodium intake in obesity.

• AT₂R agonist C21 reduces exposure of nephrotic proteins to tubular lumen and preserves renal structure and function in HSD fed obese rats.

• Superoxide is known to scavenge nitrites with formation of toxic peroxynitrite. AT₂R agonist C21 reduces NOX activity and thereby reduces superoxide formation and thus preserves plasma nitrites.

What is Relevant?

• Obesity, excessive sodium intake and renal dysfunction are highly implicated in the pathogenesis of hypertension.

• AT₂R activation by C21 during HSD feeding is renoprotective plausibly by reducing oxidative stress which in turn will have an impact on blood pressure control in obesity.

Summary

Our study demonstrates that AT₂R agonist treatment protects against HSD-induced glomerular and tubular damage and preserves kidney function in obesity; these changes in the kidney structure and function are associated with reduced oxidative stress. Moreover, in vitro and vivo studies suggest that AT₂R activation reduces NOX activity and thereby reduces superoxide formation and preserves plasma nitrites, which all act in concert to reduce salt-induced kidney injury in obese rats.