Effects of sequential application of immunosuppressive agents according to the cell cycle in adriamycin-induced nephropathy rats.

Abstract

The aim of this study was to observe the therapeutic effects of three sequential drug-based treatments according to the cell cycle in rats with adriamycin-induced nephropathy. A rat model of adriamycin-induced nephropathy was prepared by two injections, and three experimental groups were set up: control group (n=8); adriamycin-induced nephropathy rat group (n=8); and Meprednisone (MP), Ciclosporin (CsA), and mycophenolate (MMF) treatment group (n=8). Twenty-four-hour urine protein was quantified and serum total protein (TP), albumin (ALB), cholesterol (Chol), triglyceride (TG), urea nitrogen (BUN), and serum creatinine (Scr) were measured by an automatic biochemical analyzer. Pathological changes in renal tissues were observed by light microscopy. Serum matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-β1 (TGF-β1) were evaluated by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Connective tissue growth factor (CTGF) expression was measured by Western blotting. Expression of nephrin and podocin in podocytes was compared by immunohistochemistry and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Compared with the control group, 24-h urine protein in nephropathy group was significantly reduced at 2, 4, 8, and 12 weeks (p<0.01). Twenty-four-hour urine protein in the three treatment groups was significantly decreased compared with nephropathy group at 8 and 12 weeks (p<0.05). There were no significant differences among treatment groups (p>0.05), but their levels were higher than those in control group. TP and ALB levels in nephropathy group were decreased compared with control group (p<0.01) and increased compared with treatment groups (p<0.05), while TG and Chol levels in nephropathy group were increased compared with control group (p<0.01) and decreased compared with treatment group (p<0.05). There were no significant differences in biochemical parameters among the treatment groups. TGF-β1 levels were decreased, MMP-2 and MMP-9 levels were increased, and CTGF expression was reduced in the three therapeutic groups. Among the treatment groups, the combination of MP, CsA, and Rapa significantly inhibited fibrosis. The protein and mRNA levels of nephrin and podocin were significantly decreased in nephropathy group and their expression and distribution were partially restored in treatment groups. The present findings suggest that the sequential therapeutic treatments based on the cell cycle significantly improved the pathological changes in adriamycin-induced nephropathy rats. The sequential treatments significantly reduced urine protein levels, increased TP, ALB, MMP-2, and MMP-9 levels, decreased TG, Chol, and TGF-β1 levels, restored expression of nephrin and podocin in renal tissues, and significantly improved renal fibrosis.