J Korean Med Sci.  2012 Feb;27(2):160-169. 10.3346/jkms.2012.27.2.160.

Comparison of Early and Late Conversion of Sirolimus in Experimental Model of Chronic Cyclosporine Nephropathy

• Kim JY
• Ghee JY
• Lim SW
• Piao SG
• Chung BH
• Yoon HE
• Hwang HS
• Choi BS
• Kim J
• Yang CW

• KMID: 1120153
• DOI: http://doi.org/10.3346/jkms.2012.27.2.160

Abstract

Sirolimus (SRL) is a promising drug for replacing calcineurin inhibitors. We performed this study to determine the optimal time of conversion from cyclosporine (CsA) to SRL in an experimental model of chronic CsA nephropathy. Three separate studies were performed. In the first study, SRL was given to rats with or without CsA for 4 weeks. In the second study, rats were treated initially with CsA for 1 week, and then switched to SRL (early conversion). In the third study, CsA was given for 4 weeks and then replaced by SRL for 4 weeks treatment of CsA (late conversion). The influence of SRL on CsA-induced renal injury was evaluated by assessing renal function, histopathology (interstitial inflammation and fibrosis), and apoptotic cell death. Combined CsA and SRL treatment significantly impaired renal function, increased apoptosis, and interstitial fibrosis and inflammation compared with CsA or SRL treatment alone. Early conversion to SRL did not change renal function, histopathology, or apoptosis compared with early CsA withdrawal. By contrast, late conversion to SRL significantly aggravated these parameters compared with late CsA withdrawal. In conclusion, early conversion from CsA to SRL is effective in preventing CsA-induced renal injury in a setting of CsA-induced renal injury.

Keyword

Cyclosporine; Conversion; Nephrotoxicity; Sirolimus

MeSH Terms

Animals
Apoptosis/drug effects
Chronic Disease
Cyclosporine/*toxicity
Immunosuppressive Agents/*pharmacology
Intestines/drug effects/pathology
Kidney Diseases/chemically induced/*pathology
Male
Models, Animal
Rats
Rats, Sprague-Dawley
Sirolimus/*pharmacology

Figure

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  Fig. 1 Experimental design. Three separate studies were performed. (A) Combined sirolimus and cyclosporine, (B) early conversion, and (C) late conversion. S.C., subcutaneous injection; VH, vehicle.

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  Fig. 2 Influence of combined treatment of SRL and CsA on interstitial fibrosis in chronic CsA nephropathy. (A) Trichrome staining. The CsA group shows typical striped interstitial fibrosis in the cortex whereas the VH4 and VH4 + SRL4 groups does not show any change. The CsA4 + SRL4 group shows further interstitial fibrosis compared with the CsA4 and VH4 + SRL4 groups (original magnification, × 200). (B) Quantitative analysis of TIF. Note the markedly greater interstitial fibrosis in the CsA4 + SRL4 group compared with the CsA4 and VH4 + SRL4 groups. *P < 0.01 vs VH4 or VH4 + SRL4 groups; †P < 0.01 vs CsA4 group.

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  Fig. 3 Influence of combined SRL and CsA treatment on interstitial inflammation in chronic CsA nephropathy. (A) ED-1 immunohistochemistry. (B) Quantitative analysis of ED-1 positive cells. Note the significantly higher number of ED-1-positive cells in the CsA4 + SRL4 group than the CsA4 group. *P < 0.01 vs VH4 or VH4 + SRL4 groups; †P < 0.05 vs CsA4 group. (C) Immunohistochemistry of OPN protein. (D) Northern blot analysis for osteopontin (OPN) mRNA. Note the greater OPN mRNA expression in the CsA4 + SRL4 group compared with the CsA4 and VH4 + SRL4 groups. The data are presented as relative optical density with the VH4 group designated as 100% reference and are normalized to 18S. *P < 0.01 vs VH4 or VH4 + SRL4 groups; †P < 0.05 vs CsA4 group.

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  Fig. 4 Influence of combined of SRL and CsA treatment on apoptotic cell death in chronic CsA nephropathy. (A) Representative photomicrographs of the TUNEL assay (original magnification, × 200). Arrows indicate the TUNEL-positive cells. (B) Quantitative analysis of TUNEL-positive cells. Note that the number of TUNEL-positive cells was higher in the CsA4 + SRL4 group compared with the CsA4 and VH4 + SRL4 groups. (C) Immunoblots for active caspase-3. Active caspase-3 protein levels are referenced against β-actin and the relative optical density is presented with the VH4 group designated as 100% reference and normalized with β-actin. * P < 0.01 vs C VH4 or VH4 + SRL4 group; †P < 0.05 vs CsA4 group.

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  Fig. 5 Effect of early conversion from CsA to SRL on interstitial inflammation and fibrosis. (A) Quantitative analysis of ED-1-positive cells. There was no difference for the number of ED-1-positive cells between the CsA early withdrawal and early conversion groups. (B) Northern blotting for OPN mRNA. OPN mRNA did not differ between the early conversion and the CsA-early withdrawal groups. The data are presented as relative optical densities with the VH1 + W3 group designated as 100% reference and are normalized to 18S. (C) Quantitative analysis of TIF. The CsA early withdrawal group shows minimal striped interstitial fibrosis in the cortex. The amount of interstitial fibrosis does not differ between the CsA early withdrawal and early conversion groups.

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  Fig. 6 Effect of early conversion from CsA to SRL on apoptotic cell death. (A) Quantitative analysis of TUNEL-positive cells. The number of TUNEL-positive cells is slightly higher in the CsA early withdrawal group than in the VH group (*P < 0.01 vs VH1 + W3 or VH1 + SRL3 group). The number of TUNEL-positive cell does not differ significantly between the CsA early withdrawal and early conversion groups. (B) Immunoblot analysis for active caspase-3. Active caspase-3 protein levels does not differ between the CsA early withdrawal and early conversion groups. Active caspase-3 protein levels were referenced against β-actin and the relative optical density is presented with the VH1 + W3 group designated as 100% reference and normalized with β-actin.

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  Fig. 7 Effect of late conversion from CsA to SRL on interstitial inflammation and fibrosis. (A) Quantitative analysis of ED-1-positive cells. The number of ED-1-positive cells is high in the CsA4 + W4 group compared with the VH4 + W4 group or the VH4 + SRL4 group. Late conversion from CsA to SRL at 4 weeks further increases the number of ED-1-positive cells compared with the CsA late withdrawal group. (B) Northern blotting for OPN mRNA. Late conversion from CsA to SRL significantly increases OPN mRNA expression in the CsA late withdrawal rat kidney. The data are presented as relative optical density with the VH4 + W4 group designated as 100% reference and are normalized to 18S. *P < 0.01 vs VH4 + W4 or VH4 + SRL4 group; †P < 0.05 vs CsA4 + W4 group. (C) Quantitative analysis of TIF. Late conversion from CsA to SRL causes greater interstitial fibrosis than that observed in the CsA late withdrawal group. *P < 0.01 vs VH4 + W4 or VH4 + SRL4 groups; †P < 0.05 vs CsA4 + W4 group.

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  Fig. 8 Effect of late conversion from CsA to SRL apoptotic cell death. (A) Quantitative analysis of TUNEL-positive cells. The number of TUNEL-positive cells is much higher in the later conversion group than in the CsA-late withdrawal group. (B) Immunoblot analysis of active caspase-3. Note the further increase in active caspase-3 protein level in the late conversion group. Active caspase-3 protein levels were referenced against β-actin and the relative optical density was presented with the VH4 + 4W group designated as 100% reference and normalized with β-actin. *P < 0.01 vs VH4 + W4 or VH4 + SRL4 groups; †P < 0.05 vs CsA4 + W4 group.

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