1. Inhibition of glomerular cell apoptosis by heparin
Yoshihisa Ishikawa, Masanori Kitamura 
Kidney International 
Volume 56, Issue 3, Pages (September 1999)
DOI: /j x
Copyright © 1999 International Society of Nephrology Terms and Conditions

2. Figure 1
Effect of heparin on oxidant-triggered apoptosis of mesangial cells. Confluent rat mesangial cells were pretreated with (+) or without (-) heparin (5 to 100 U/ml) for 1.5 hours in the presence of 1% fetal calf serum (FCS) and were exposed to hydrogen peroxide (H2O2; 100 to 150 μ M) for up to 24 hours. Heparin 100 U/ml was generally used for experiments. (A) Phase-contrast microscopy: 150 μ M H2O2 for 16 hours. (B) Trypan blue analysis. After induction of apoptosis (100 μ M H2O2, 24 hr), mesangial cells were gently trypsinized and mixed with the same volume of trypan blue solution. Percentages of viable cells were evaluated by light microscopy. Assays were performed in quadruplicate. Data were expressed as means ± SE. Asterisks indicate statistically significant differences (P < 0.05). (C) Trypan blue analysis: dose-dependent effect. Cells were pretreated with 0 to 100 U/ml heparin, exposed to 150 μ M H2O2 for 24 hours, and subjected to trypan blue analysis. (D) Hoechst staining. After induction of apoptosis (150 μ M H2O2, 24 hr), cells were fixed and stained by Hoechst for one hour and subjected to fluorescence microscopy. Apoptotic cells were identified by nuclear condensation and/or fragmentation, and percentages of apoptotic cells were calculated. Assays were performed in quadruplicate. An asterisk indicates a statistically significant difference against H2O2-treated, heparin-untreated cells (P < 0.05). (E) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After induction of apoptosis (150 μ M H2O2, 6 hr), cells were subjected to TUNEL, as described in the Methods section (fluorescence microscopy). (F) Ladder detection assay. Cells were pretreated with 0 to 100 U/ml heparin, exposed to 150 μ M H2O2 for 16 hours, and subjected to agarose gel electrophoresis. (G) Effect of heparan sulfate proteoglycan (HSPG). Mesangial cells were pretreated with (+) or without (-) HSPG (500 μg/ml) for 1.5 hours in the presence of 1% FCS and exposed to H2O2 (150 μ M) for 24 hours. Percentages of apoptotic cells were evaluated by Hoechst staining. An asterisk indicates a statistically significant difference (P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

3. Figure 1
Effect of heparin on oxidant-triggered apoptosis of mesangial cells. Confluent rat mesangial cells were pretreated with (+) or without (-) heparin (5 to 100 U/ml) for 1.5 hours in the presence of 1% fetal calf serum (FCS) and were exposed to hydrogen peroxide (H2O2; 100 to 150 μ M) for up to 24 hours. Heparin 100 U/ml was generally used for experiments. (A) Phase-contrast microscopy: 150 μ M H2O2 for 16 hours. (B) Trypan blue analysis. After induction of apoptosis (100 μ M H2O2, 24 hr), mesangial cells were gently trypsinized and mixed with the same volume of trypan blue solution. Percentages of viable cells were evaluated by light microscopy. Assays were performed in quadruplicate. Data were expressed as means ± SE. Asterisks indicate statistically significant differences (P < 0.05). (C) Trypan blue analysis: dose-dependent effect. Cells were pretreated with 0 to 100 U/ml heparin, exposed to 150 μ M H2O2 for 24 hours, and subjected to trypan blue analysis. (D) Hoechst staining. After induction of apoptosis (150 μ M H2O2, 24 hr), cells were fixed and stained by Hoechst for one hour and subjected to fluorescence microscopy. Apoptotic cells were identified by nuclear condensation and/or fragmentation, and percentages of apoptotic cells were calculated. Assays were performed in quadruplicate. An asterisk indicates a statistically significant difference against H2O2-treated, heparin-untreated cells (P < 0.05). (E) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After induction of apoptosis (150 μ M H2O2, 6 hr), cells were subjected to TUNEL, as described in the Methods section (fluorescence microscopy). (F) Ladder detection assay. Cells were pretreated with 0 to 100 U/ml heparin, exposed to 150 μ M H2O2 for 16 hours, and subjected to agarose gel electrophoresis. (G) Effect of heparan sulfate proteoglycan (HSPG). Mesangial cells were pretreated with (+) or without (-) HSPG (500 μg/ml) for 1.5 hours in the presence of 1% FCS and exposed to H2O2 (150 μ M) for 24 hours. Percentages of apoptotic cells were evaluated by Hoechst staining. An asterisk indicates a statistically significant difference (P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

4. Figure 2
Effect of heparin on the H2O2-triggered activation of activator protein 1 (AP-1). (A) Northern blot analysis. Mesangial cells were exposed to H2O2 (100 μ M) for 0.5 to 2 hours in the presence (+) or absence (-) of heparin (100 U/ml) and were subjected to Northern blot analysis of c-fos and c-jun. As a loading control, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown on the bottom. (B) Reporter assay. Mesangial cells were transfected with a reporter plasmid pTRE-LacZ that introduces a β-galactosidase gene (lacZ) under the control of 12-O-tetradecanoate 13-acetate response element (TRE). After incubation for 48 hours in the presence of 1% fetal calf serum (FCS), the cells were pretreated with (+) or without (-) heparin for 1.5 hours and stimulated by H2O2 (100 μ M) for 24 hours. The cells were fixed and subjected to 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) assay. Activity of AP-1 was evaluated by counting X-gal-positive cells in each well. Data are shown as a fold increase against the value of untreated control. Assays were performed in quadruplicate. Asterisks indicate statistically significant differences (P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

5. Figure 3
Effects of heparin on apoptosis of mesangial cells triggered by different stimuli. Confluent mesangial cells were pretreated with (+) or without (-) heparin (100 U/ml) for 1.5 hours in the presence of 1% FCS and were exposed to (A) staurosporine (1.5 μ M), (B) pyrrolidine dithiocarbamate (PDTC, 50 μ M), or (C) ultraviolet light (UV, 200 mJ) for 20 to 24 hours. Cells were fixed, stained by Hoechst 33258, and subjected to fluorescence microscopy. Assays were performed in quadruplicate. Asterisks indicate statistically significant differences compared with the cells exposed to apoptotic stimuli without heparin (P < 0.05).
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

6. Figure 4
Effects of heparin on H2O2-initiated apoptosis in other cell types. NRK49F fibroblasts and MDCK epithelial cells were pretreated with (+) or without (-) heparin (100 U/ml) for 1.5 hours in the presence of 1% FCS and were exposed to H2O2 (150 μ M for NRK49F and 350 μ M for MDCK) for 16 to 20 hours. Cells were then stained by Hoechst or subjected to ladder detection assay. Assays were performed in quadruplicate. Asterisks indicate statistically significant differences compared with H2O2-exposed, heparin-untreated cells (P < 0.05). (A) Hoechst staining of NRK49F cells (fluorescence microscopy). (B) Hoechst staining of detached MDCK cells. In contrast to mesangial cells and NRK49F cells, the majority of apoptotic MDCK cells detached from the culture plate. To evaluate apoptosis of MDCK cells quantitatively, detached cells and attached cells were stained by Hoechst separately, and total percentages of apoptotic MDCK cells were calculated. (C) Ladder-detection assay.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions

7. Figure 5
Effect of heparin on spontaneous apoptosis in explanted glomeruli. (A) Ladder detection assay. Isolated, normal rat glomeruli were incubated at 37°C for two hours in culture medium containing 1% FCS and 0 to 100 U/ml of heparin and subjected to agarose gel electrophoresis. (B) TUNEL assay. Isolated glomeruli were incubated at 37°C for 20 minutes in the presence (+) or absence (-) of heparin (100 U/ml) and were subjected to TUNEL assay, as described in the Methods section. Glomeruli were examined by fluorescence microscopy.
Kidney International  , DOI: ( /j x)
Copyright © 1999 International Society of Nephrology Terms and Conditions