[Molecular mechanisms of nephro-protective action of enalapril in experimental chronic renal failure]

Abstract

Locally increased synthesis of angiotensin II (ANG II) in the kidney has been linked to glomerular hypertrophy, glomerulosclerosis and tubulo-interstitial fibrosis observed in chronic renal failure after subtotal nephrectomy. This action of ANG II is thought to be mediated mainly by transforming growth factor-beta (TGF-beta), which stimulates the synthesis and decreases the degradation of extracellular matrix (ECM) components, including various collagen types and fibronectin. Some recent reports indicate that reduced ANG II activity diminishes TGF-beta overexpression, and in consequence renal injury. However, no studies in SNx models concerning the influence of ANG II on gene expression regulated by TGF-beta have so far been performed. Therefore, the present study has been initiated with the following aims: 1. To develop a RT-PCR assay for evaluating gene expression concerning renin (REN), angiotensinogen (ATG) and the following ECM components: transforming growth factor-beta 1 (TGF-beta 1), fibronectin (FN), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2); 2. To assess the influence of renal mass reduction (RMR) caused by subtotal (5/6) or partial (2/6) nephrectomy on gene expression for TGF-beta 1, FN, MMP-2 and TIMP-2; 3. To evaluate the correlation between expression of these genes and activity of the circulatory or renal renin-angiotensin systems; 4. To assess the influence of treatment with enalapril (angiotensin-converting enzyme inhibitor) on renal expression of these genes, renal morphology and function in rats, relative to duration of treatment and RMR. The study consisted of two independent experiments performed in adult male Sprague-Dawley rats. Ten days prior to surgery, the animals were matched for body weight and systolic blood pressure (SBP) values and subsequently were distributed into untreated (control) and enalapril treated groups. Treatment with enalapril (EN) (50 mg/l in drinking water) was started 9 days prior to surgery. The first (short-term) experiment was performed in rats with chronic renal failure caused by subtotal nephrectomy. Remnant kidneys were taken for molecular studies at the day of SNx and 3, 7 and 21 days thereafter. Blood samples collected at the time of sacrifice served to determine plasma renin activity and plasma concentration of angiotensinogen and angiotensin II. The second (long-term) experiment was done in subtotally (5/6) and partially (2/6) nephrectomized rats. Remnant kidneys were taken for molecular and morphological studies at the day of surgery and 1 or 16 weeks thereafter. 24-hour proteinuria, hematocrit, serum creatinine and creatinine clearance values were also measured. Quantitation of renal gene expression for REN, ATG, TGF-beta 1, FN, MMP-2 and TIMP-2 was performed using RT-PCR assay and comparing amounts of respective gene mRNA with house-keeping gene mRNA encoding L19 ribosomal protein. The results obtained have led to the following conclusions: 1. The RT-PCR assay developed here ensures a reliable quantitation of gene expression for renin, angiotensinogen, transforming growth factor-beta 1, fibronectin, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2. 2. Renin gene expression in the kidney depends on renal synthesis of angiotensin II. In contrast, regulation of angiotensinogen mRNA expression seems to be independent of ANG II. 3. Long-term treatment with enalapril prevents an early increase in renal TGF-beta 1 and FN gene expression, retards the progression of chronic renal failure caused by critical renal mass reduction, and prolongs survival. 4. Intrarenal activity of the renin-angiotensin system is not a principal factor in the regulation of gene transcription for matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2.