Volume 63, Issue 6, Pages 2050-2064 (June 2003) Effects of caspase inhibition on the progression of experimental glomerulonephritis  Bin Yang, Timothy S. Johnson, John L. Haylor, Bart Wagner, Phil F. Watson, Mohsen M.H. El Kossi, Peter N. Furness, A. Meguid El Nahas  Kidney International  Volume 63, Issue 6, Pages 2050-2064 (June 2003) DOI: 10.1046/j.1523-1755.2003.00000.x Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 1 Caspase-3 activity, proteins and mRNA in kidney tissues. (A) Caspase-3 activity assayed by the fluorometric substrate cleavage. Data represents mean ± SEM. (B) Expression of caspase-3 proteins detected by Western blot analysis. Sample immunoblots of both acute and chronic studies show 17 kD and 24 kD bands, representing the caspase-3–active subunits; a 32 kD band, representing the precursor of caspase-3; a 29 kD band, representing processing intermediate of caspase-3. Histogram data represent the result of volume density analysis as mean percentage change compared to the average of control values (mean ± SEM). (C) Tissue level of caspase-3 mRNA in rat kidneys assayed by Northern blot analysis. Histogram shows volume density analysis of autoradiographs using cyclophilin corrected for loading. Data are expressed as mean percentage change compared to the average of control values (mean ± SEM). Abbreviations are: Cyc, cyclophilin (N = 6 to 8); NS, normal serum; NTS, nephrotoxic serum; DMSO, dimethyl sulfoxide; B-D-FMK, Boc-Asp (OMe)-fluoromethyl ketone; D7, B-D-FMK administration for 7 days; and D28; B-D-FMK administration for 28 days. Symbols are: (□), NS + DMSO; (), NTS + DMSO; and (), NTS + B-D-FMK. *P < 0.05; **P < 0.01. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 2 Caspase-1 activity and proteins in kidney tissues. (A) Caspase-1 activity assayed by the fluorometric substrate cleavage. Data represents mean ± SEM. (B) Expression of caspase-1 proteins detected by Western blot analysis. Sample immunoblots of both acute and chronic studies show 20 kD band, representing the caspase-1 active subunit; a 45 kD band, representing the precursor of caspase-1. Histogram data represents the result of volume density analysis as mean percentage change compared to the average of control values (mean ± SEM) (N = 6 to 8). Abbreviations are: NS, normal serum; NTS, nephrotoxic serum; DMSO, dimethyl sulfoxide; B-D-FMK, Boc-Asp (OMe)-fluoromethyl ketone; Cyc, cyclophilin; D7, B-D-FMK administration for 7 days; and D28; B-D-FMK administration for 28 days. Symbols are: (□), NS + DMSO; (), NTS + DMSO; and (), NTS + B-D-FMK. *P < 0.05; **P < 0.01. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 3 Tissue level of interleukin-1β (IL-1β) proteins and mRNA. (A) Expression of IL-1β proteins detected by Western blot analysis. Sample immunoblots of both acute and chronic studies show 17 kD band, representing the IL-1β active subunit; a 35 kD band, representing the precursor of IL-1β. Histogram data represents the result of volume density analysis as mean percentage change compared to the average of control values (mean ± SEM). (B) Tissue level of IL-1β mRNA in rat kidneys assayed by Northern blot analysis. Histogram shows volume density analysis of autoradiographs using cyclophilin (Cyc) corrected for loading. Data is expressed as mean percentage change compared to the average of control values (mean ± SEM) (N = 6 to 8). Abbreviations are: NS, normal serum; NTS, nephrotoxic serum; DMSO, dimethyl sulfoxide; B-D-FMK, Boc-Asp (OMe)-fluoromethyl ketone; D7, B-D-FMK administration for 7 days; and D28, B-D-FMK administration for 28 days. Symbols are: (□), NS + DMSO; (), NTS + DMSO; and (), NTS + B-D-FMK. *P < 0.05; **P < 0.01. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 4 Renal cell apoptosis detected by in situ end-labeling (ISEL) the fragmented DNA. Apoptotic cells (indicated by arrows) are shown in glomeruli (a, b, and e), tubules (c, d, and f) and interstitium (g) at ×200 (a to d) and ×400 (e to g) magnification. Typical morphologic changes of apoptosis such as a shrunken cell with condensed nuclei surrounded by narrow cytoplasmic halo is shown in an enlarged picture of a glomerular apoptotic cell (a). Using electron microscopy, apoptotic cell phagocytosed by tubular cell (h, ×6858) and apoptotic cell in interstitium are shown (i, ×6858). Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 5 Renal Inflammation evaluated by ED1 staining. ED1+ cells in glomeruli (a, b, e, and f) and interstitium (c, d, g, and h) at ×100 (a to d) and ×200 (e to h) magnification. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 6 Histograms show semi-quantitative analysis of both renal apoptosis (A) and inflammation (B). Data represents mean ± SEM (N = 6 to 8). Abbreviations are: NS, normal serum; NTS, nephrotoxic serum; DMSO, dimethyl sulfoxide; B-D-FMK, Boc-Asp (OMe)-fluoromethyl ketone; Gapo, apoptotic cells per glomerulus; Tapo, apoptotic cells per 400 tubular cells; Iapo, apoptotic cells per ×400 field; Ged1, ED1+ staining cells per glomerulus; Ied1, ED1+ staining cells per ×400 field; D7, B-D-FMK administration for 7 days; and D28; B-D-FMK administration for 28 days. Symbols are: (□), NS + DMSO; (), NTS + DMSO; and (), NTS + B-D-FMK. *P < 0.05; **P < 0.01. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 7 Renal fibrosis analyzed on Masson's Trichrome–stained sections and by collagen I, III, and IV mRNAs. Glomerular injury and sclerosis and tubulointerstitial fibrosis in Masson's Trichrome–stained sections at ×200 (A to D) and ×400 (E to H) magnification. Histogram (I) shows semiquantitative analysis of renal fibrosis. Data represents mean ± SEM. Tissue levels of collagen I (J), III (K), and IV (L) mRNAs in rat kidneys assayed by Northern blot analysis. Histogram shows volume density analysis of autoradiographs using cyclophilin (Cyc) corrected for loading. Data is expressed as mean percentage change compared to the average control value (mean ± SEM) (N = 6 to 8). Abbreviations are: NS, normal serum; NTS, nephrotoxic serum; DMSO, dimethyl sulfoxide; B-D-FMK, Boc-Asp (OMe)-fluoromethyl ketone; D7, B-D-FMK administration for 7 days; and D28; B-D-FMK administration for 28 days. Symbols are: (□), NS + DMSO; (), NTS + DMSO; and (), NTS + B-D-FMK. *P < 0.05; **P < 0.01. Kidney International 2003 63, 2050-2064DOI: (10.1046/j.1523-1755.2003.00000.x) Copyright © 2003 International Society of Nephrology Terms and Conditions