| PrefaceChronic renal disease is one of the main causes that endanger human lives. It is critical to explore the mechanism underlying renal scarring leading to end stage renal disease( ESRD) and develop new strategies to prevent or slow the progression.It has been demonstrated that aldosterone was substantially involved in hypertension and organ fibrosis. Recently increasing evidence supported potential roles of aldosterone in the pathogenesis of renal injury. However the precise mechanism that is responsible for aldosterone-induced renal injury remains unclear.Rho-kinase, an effector of the small G protein Rho, is activated by several stimuli and controls actin-cytoskeleton assembly and formation of stress fiber and focal adhesion, and thereby contributes to various cellular functions including smooth muscle contraction, cell adhesion, migration, proliferation, cytokinesis and transformation, all of which might be involved in the pathogenesis of renal injury . Indeed, several studies have demonstrated that treatment with Rho-kinase inhibitors attenuated renal injury, independent of blood pressure changes . Recently, Kobayashi et al. showed that in Dahl salt-sensitive hypertensive rats, severe renal injury was associated with increases in renal tissue Rho-kinase mRNA levels and myosin light chain phosphorylation, and treatment with a mineralocorticoid receptor (MR) antagonist prevented myosin light chain phosphorylation and improved renal injury. These data suggest that the aldosterone/Rho-kinase pathway plays a role in mediating renal injury in Dahl salt-sensitive hypertensive rats. However, to date, there is no convincing evidence indicating the potential contribution of Rho-kinase to aldosterone-induced renal injury.Angiotensin II (Angll) has been demonstrated to be a profibrosis factor. Furthermore, it has been shown to activate the Rho/Rho-kinase pathway via its type I receptor. Recently it was reported that increased corticol Angll level was induced in aldosterone/salt-induced renal injury. But to date there is no study reporting the relationship between Rho-kinase and AngII in aldosterone/salt-induced renal injury.ObjectiveThe present study aims to investigate whether renal injury induced by chronic treatment with aldosterone is associated with activation of Rho-kinase and the possible mechanism . Further studies were also performed to examine the effects of a specific Rho-kinase inhibitor, fasudil, on renal injury induced by chronic treatment with aldosterone.MethodsFive-week-old male Sprague-Dawley rats weighing 182-206 g at the beginning of the experiment underwent right uninephrectomy under anesthesia with sodium pentobarbital (50 mg/kg, IP). After 2 weeks recovery from surgery, rats were treated for 5 weeks with 1%NaCl in a drinking solution and one of the following: 1) vehicle (2% ethanol, SC, n=9); 2) aldosterone (0.75μg.H-1, SC, n=9; ); 3) aldosterone (0.75μg.H-1, SC) + fasudil (10 mg.kg-1.day-1, SC, n=8; ). An osmotic minipump was implanted subcutaneously to infuse vehicle and aldosterone and replaced every 2 weeks under sodium pentobarbital anesthesia. Body weight was measured every week; Systolic blood pressure (SBP) was measured in conscious rats by tail-cuff plethysmography, and 24-hr urine samples for protein and creatinine assay were collected at 0, 1,3 and 5 weeks. Blood and kidney samples were collected at the end of week 5 under sodium pentobarbital anesthesia. PAS and masson staining were done to the renal tissue specimens to evaluate the severity of glomerular proliferative and sclerotic lesion and tubulo-interstitial fibrosis. To investigate whether Angll was involved in the pathogenesis of aldosterone-induced renal injury, we also assayed Angll content in renal cortex. Since Rho-kinase inhibits myosin phosphatase by phosphorylating its myosin binding subunit, myosin phosphate target subunit-1 (MYPT1), we measured phosphorylated levels of MYPT1 in renal tissues as a marker of Rho-kinase activity by western blotting, as well as phospho-Smad2/3 and ACE protein expression. Furthermore, cortical mRNA expressions of TGF-β1, CTGF, type I and type III collagen were all measured by real-time PCR.Results1. Aldosterone-infused rats showed a reduced body weight (BW) compared with vehicle-infused animals, but treatment with fasudil did not alter the BW of aldosterone-infused rats. On the other hand, kidney weight (KW) and the KW/BW ratio were much higher in aldosterone-infused rats than vehicle-infused rats. Treatment with fasudil significantly decreased the KW and KW/BW ratio of aldosterone-infused rats. In addition, aldosterone-infused rats showed increased plasma creatinine levels and decreased creatinine clearance compared with vehicle-infused rats. Treatment with fasudil prevented aldosterone-induced changes in creatinine clearance and plasma creatinine levels.2. SBP was almost identical in the 3 groups at the beginning of the protocol (2 weeks after uninephrectomy). Aldosterone-infused rats progressively developed hypertension (193±3 mmHg at week 5). Treatment with fasudil did not significantly alter SBP in aldosterone-infused rats (191±9 mmHg at week 5 ). At the same time, aldosterone-infusion resulted in severe proteinuria (362±93 mg.day-1 at week 5), and treatment with fasudil markedly reduced UproteinV in aldosterone-infused rats (96±22 mg.day-1 at week 5 ).3. Compared with vehicle-infused rats, aldosterone infusion significantly increased cortical AngII levels, which were not altered by fasudil treatment.4. Vehicle-infused rats showed almost normal glomeruli and tubulointerstitium; However, aldosterone-infused rats exhibited injured glomeruli characterized by cell proliferation and sclerosis . In aldosterone-infused rats, severe tubulointerstitial fibrosis was also observed. Treatment with fasudil significantly attenuated aldosterone-induced renal injuries mentioned above.5. Western blot analysis revealed that the expressions of MYPT1 and Smad2/3 phosphorylation and ACE protein in renal cortical tissues were markedly increased in aldosterone-infused rats (5.46±0.70-fold increase, 4.88±0.20-fold increase and 3.01±0.75-fold increase versus vehicle-infused rats respectively); The aldosterone-induced increases in renal cortical MYPT1 and Smad2/3 phosphorylation were prevented by treatment with fasudil, but cortical ACE expression was unaffected.6. Compared with vehicle-infused rats, aldosterone-infused rats showed increased renal cortical mRNA levels of types I and III collagen, TGF-β1 and CTGF by 3.86±0.20, 2.63±0.14, 2.64±0.10 and 3.23±0.20-fold , respectively. Treatment with fasudil significantly decreased these mRNA levels in aldosterone-infused rats. Conclusions1. These results provided evidence, for the first time, that Rho-kinase was substantially involved in aldosterone-induced renal injury through activation of TGF-β-dependent pathway;2. The renoprotection of fasudil was independent of blood pressure lowering;.3. Aldosterone infusion induced augmented renal cortical angll levels and increased ACE expression which were not altered by Rho-kinase inhibition, suggesting that the activated Rho-kinase mediated the effect of augmented Angll levels in the aldosterone/salt-induced renal injury. |
PDF Full Text Request