Anticubilin Antisense RNA Ameliorates Adriamycin-Induced Tubulointerstitial Injury in Experimental Rats Jun Liu, MD, Kailong Li, MD, Yani He, MD, Jianguo.

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Anticubilin Antisense RNA Ameliorates Adriamycin-Induced Tubulointerstitial Injury in Experimental Rats Jun Liu, MD, Kailong Li, MD, Yani He, MD, Jianguo Zhang, MS, Huiming Wang, MD, Jurong Yang, MD, Jun Zhan, BS, Haijun Liang, MS The American Journal of the Medical Sciences Volume 342, Issue 6, Pages 494-502 (December 2011) DOI: 10.1097/MAJ.0b013e31821952a2 Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 1 Expression of green fluorescent protein (GFP) in tubular epithelia in rats on day 28 after transfection (on day 28). (A) Normal control group. (B) Vector control group. (C) pAdEasy-CUB group. (D) pAdEasy-ACUB group. Only weak fluorescent signals could be detected in group A; meanwhile, there were high levels of GFP fluorescent signals in tubules in groups B, C and D (n=6). No detectable signal in the normal control group by immune blotting; in contrast, significant signals were seen from the other groups, but there was no difference among the groups after semi-quantification. (E) Representative blot of GFP expression. (F) Semi-quantificational value of left blot. The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 2 Expression of cubilin in tubules on day 28. Major signals of positive cubilin localized on the brush membranes of tubules (indicated in red). (A) Normal control group. (B) Vector control group. (C) pAdEasy-CUB group. (D) pAdEasy-ACUB group (n=18 in each group). Magnification: 200×. (E) Representative blot of cubilin expression; actin serves as a normalized control (left panel). (F, G) Semi-quantificational value of left blot (right panel). The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 3 Deposition of albumin in tubules at days 14 and 28. Positive staining of albumin was indicated as red spots. Both E and J showed fewer depositions of albumin compared with B and G, respectively. (A, F) Normal control group. (B, G) Adriamycin model group. (C, H) Vector control group. (D, I) pAdEasy-CUB group. (E, J) pAdEasy-ACUB group. (A–E) Day 14. (F–J) Day 28. Magnification: 200× (n=18). The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 4 Expression of MCP-1 in tubulointerstitial in adriamycin-induced rats. Positive staining of MCP-1 was indicated in brown. Major signals paralleled with dilated tubules. Groups G, H and J showed stronger expression of MCP-1 compared with B, C and D, respectively, whereas E and J showed reduced signals compared with B and G, respectively. (A, F) Normal control group. (B,G) Adriamycin model group. (C, H) Vector control group. (D, I) pAdEasy-CUB group. (E, J) pAdEasy-ACUB group. (A–E). Day 14. (F–J) Day 28. Magnification: 200× (n=18). The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 5 Expression of cubilin, MCP-1, TGF-β and RNATES in kidney. (A) Representative blots. (B) Semi-quantitative results of cubilin. (C) Semi-quantitative results of MCP-1. (D) Semi-quantitative results of TGF-β. (E) Semi-quantitative results of regulated on activation in normal T-cell expressed and secreted The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 6 Number of ED-1–positive cells in tubulointerstitial area in adriamycin-induced rats on day 28 after transfection. #P < 0.01 compared with normal control group; ΔP < 0.01 compared with disease control group. (A, F) Normal control group. (B, G) Adriamycin model group. (C, H) Vector control group. (D, I) pAdEasy-CUB group. (E, J) pAdEasy-ACUB group. (A–E) Day 14. (F–J) Day 28. (K) Semi-quantitative results of ED-1. The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions

Figure 7 Antisense cubilin ameliorates peritubular capillary injury in nephrotic rats on days 14 and 28. Positive staining of JG-12 was indicated as red spots. (A, F) Normal control group. (B, G) Adriamycin model group. (C, H) Vector control group. (D, I) pAdEasy-CUB group. (E, J) pAdEasy-ACUB group. (A–E) Day 14. (F–J) Day 28. The American Journal of the Medical Sciences 2011 342, 494-502DOI: (10.1097/MAJ.0b013e31821952a2) Copyright © 2011 Southern Society for Clinical Investigation Terms and Conditions