Research On The Mechanisms Of Aldosterone-Induced Renal Injury

ObjectivePatients with chronic kidney disease ( CKD) have a high burden of mortality and cardiovascular morbidity. It is critical to explore the mechanisms underlying renal scarring leading to end stage renal disease (ESRD) and develop new strategies to prevent and/or slow this progression. Recently, attention has focused on the role of aldosterone in the pathophysiology of hypertension and organ fibrosis. But the mechanisms responsible for renal fibrosis remain to be elucidated.The present studies aimed to investigate the mechanisms underlying aldosterone induced glomeruli sclerosis and renal interstitial fibrosis. We will prepare chronic aldosterone/salt infusion - induced hypertensive SD rats model to determine the role of reactive oxygen species ( ROS) and mitogen - activated protein kinase ( MAPK) in the pathogenesis of aldosterone/salt - induced renal injury.Materials and Methods1 Animal preparationMale Sprague - Dawley rats ( Clea, Japan) , weighing 220 to 258 g at the beginning of the experiments, were randomly treated with one of the following combinations for 6 weeks; Group - 1; tap water + vehicle (0. 5% ethanol, SC, n = 6) , Group - 2; 1 % NaCl in the drinking solution + vehicle ( n = 8) , Group-3; 1%NaCl + aldosterone (0.75μg/H, SC, n=8) , Group-4; 1% NaCl + aldosterone + eplerenone (0.125% in chow, n=8) , and Group-5; 1 % NaCl + aldosterone + tempol (3 mmol/L in drinking solution, n = 8). Atthe beginning of the experiments, rats were anesthetized with sodium pentobarbi-tal (50 mg/kg, IP) , and an osmotic minipump (model 2002, Alza Co. Palo Alto, CA) was implanted subcutaneously at the dorsum of the neck to infuse vehicle or aldosterone. Systolic blood pressure (SBP) was measured in conscious rats by tail - cuff plethysmography (BP -98A, Softron Co. , Tokyo, Japan) and 24 hour urine samples were collected at the 1, 3 and 6th weeks. Blood and kidney samples were harvested at the end of the 6th week. After decapitation, the kidney was removed, snap -frozen in liquid nitrogen and stored at -80^. Renal cortical mRNA expression of NAD( P) H oxidase components p22phox, Nox-1 and gp91phox were quantitatively analyzed by real -time PCR. The activities of MAPKs, including extracellular signal - regulated kinases (ERK1/2) , c- Jun -NH2 -terminal kinases (JNK) , p38 MAPK and ERK5 in renal cortical tissues were measured by Western blot analysis. Urinary protein excretion was determined using a protein assay kit ( microTP -test, Wako Co. , Tokyo, Japan ). We determined the degree of lipid peroxidation using biochemical assays of TBARS in renal cortical tissues and urine. Renal cortical tissue collagen content was determined on the basis of the hydroxyproline concentration.2 Cell cultureRat mesangial cells ( RMCs) were obtained from intact glomeruli of 4 - to 6 - week - old Sprague - Dawley rat and characterized according to published methods. RMCs were used between passages 5 and passages 10. Protein levels of MR in RMCs were analyzed by Western blot. The cells were divided into the follow groups; Group - 1; control, Group -2; IOjjlM PD98059, Group -3; 1 jxM eplerenone, Group - 4; lOOnM aldosterone, Group - 5; aldosterone + PD98059, Group - 6; aldosterone + eplerenone. Cell proliferation in RMCs was evaluated by 3H - thymidine incorporation. ERK1/2 activity was measured by Western Blot.3 Statistical analysisThe values are presented as means ± SE. Statistical comparisons of the differences were performed using Statview 5. 0 software. P <0. 05 was considered statistically significant.Results1 Compared with 1% NaCl infused rats, aldosterone/salt infused rats showed higher blood pressure (165 ±5 vs 118 ±3mmHg) , left kidney weight to body weight ratio (5. 39 ±0. 28 vs 3.57 ±0. 23% ) ,renal cortical collagen content (16.0 ±0.7 vs 13. 1 ± 1. 1 |xg/g) and UproteinV (101 ±24 vs 9 ±3mg/ d)( P <0.05).2 Compared with 1% NaCl infused rats, aldosterone/salt infused rats increased renal cortical TBARS level (0. 23 ±0. 02 vs 0. 09 ±0. 01 nmol/mg protein, P < 0. 05 ) and NAD ( P) H oxidase components p22phoxNNox - 4 and gp91phox mRNA expression (2. 3 ±0. 2, 4. 3 ±0. 8 and 3. 0 ±0. 3 fold,respectively, P <0.05).3 Compared with 1% NaCl infused rats, aldosterone/salt infused rats showed increased renal cortical ERK1/2 ,JNK and ERK5 activities (3.3 ±0. 3 , 2. 3 ±0. 3 and 3.0 ± 0. 2 fold, respectively, P < 0.05).4 eplerenone, a selective mineralocorticoid receptor blockade, prevented aldosterone/salt induced hypertension (165 ±5 vs 127 ±2 mmHg) , reduced left kidney weight to body weight ratio (5. 39 ±0. 28 vs 3. 57 ±0. 23% ) ,decreased renal cortical collagen content (16.0±0.7 vs 13.1 ±1. lfxg/g) and UproteinV (101 ±24 vs 9 ±3mg/d, P <0. 05) , normalized renal cortical TBARS level, NAD( P) H wcidase components p22phoxNNox -4 and gp91phox mRNA expression and ERK1/2JNK and ERK5 activity.5 Similarly, tempol, a SOD mimetic, also prevented aldosterone/salt induced hypertension (125 ±5mmHg) , significantly decreased left kidney weight to body weight ratio (4.05±0.21%), reduced renal cortical collagen content (12. 0 ± 0. 6jxg/g) and UproteinV (9 ± 2mg/d) , normalized renal cortical TBARS level and ERK1/2JNK and ERK5 activity. Tempol did not alter renal cortical NAD( P) H oxidase component Nox -4 and gp91phox mRNA level in aldosterone/salt infused rats.6 There are mineralocorticoid receptor protein expression in RMCs. Aldo-sterone increased ERK1/2 activity(3. 6 ±0.4 fold, P <0. 05) and 3H - thymi-...