Expression of inter-α-trypsin inhibitor and tumor necrosis factor-stimulated gene 6 in renal proximal tubular epithelial cells  Ulf Janssen, Gareth Thomas, Tibor Glant, Dr Aled Phillips  Kidney International  Volume 60, Issue 1, Pages 126-136 (July 2001) DOI: 10.1046/j.1523-1755.2001.00779.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Current view of the structure of the IαI family of protease inhibitors. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Constitutive expression of bikunin and H3 mRNA. Growth-arrested HK2 cells were stimulated with 5mmol/L d-glucose, 25mmol/L d-glucose, or interleukin (IL)-1β (1 ng/mL) in the absence of serum. Total cellular RNA was isolated and mRNAs for bikunin (A) and heavy chain 3 (H3) mRNA (B) examined by RT-PCR (36 cycles of amplification). Ethidium bromide stained PCR products were separated on a 3% agarose gel. Scanning densitometry confirmed the lack of induction of either bikunin or H3 mRNA expression in the presence of either IL-1β or 25mmol/L d-glucose. One representative gel of four individual experiments is shown. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Protein expression of the inter-alpha-trypsin inhibitor (IαI) family in membrane isolates of HK2 cells. Growth-arrested HK2 cells were stimulated with either 5mmol/L d-glucose (5 Gl) or IL-1β (1 ng/mL) for up to 24 hours in the absence of serum. Membrane preparations were isolated, and equal amounts of HK2-derived membrane protein (19 μg/lane) were loaded onto 3 to 12% SDS-PAGE gradient gels. Western blot analysis was carried out as detailed in the Methods section. In parallel experiments, growth-arrested cells were stimulated with either 5mmol/L d-glucose (5 Gl), 25mmol/L d-glucose (25 Gl), or 25mmol/L L-glucose (25 L-Gl), for up to 96 hours. The antiserum used recognizes intact IαI family members [including pre-αI (120 to 130 kD)], individual heavy chains (75 to 80 kD), and bikunin (35 kD), as well as IαI associated with TSG-6 (TSG-6, molecular weight 42 kD). The multiple bands following Western analysis therefore represent intact IαI family members, individual chains of IαI, and complexes bound to TSG-618. Scanning densitometry confirmed that there were no differences in IαI expression under any of the experimental conditions. One representative experiment of four independent experiments is shown. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Induction of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA by interleukin-1β (IL-1β) or 25mmol/L D-glucose. Growth-arrested HK2 cells (A) or HPTCs (B) were stimulated with 5mmol/L d-glucose, 25mmol/L d-glucose, or IL-1β (1 ng/mL) in the absence of serum. Total cellular RNA was isolated at various time points up to 96 hours, and TSG-6 mRNA was examined by RT-PCR. Ethidium bromide-stained PCR products were separated on a 3% agarose gel. One representative gel of four individual experiments is shown. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 Induction of TSG-6 protein following IL-1β stimulation. Growth-arrested HK2 cells were stimulated with 5mmol/L d-glucose (5 Gl) or IL-1β (1 ng/mL) in the absence of serum. Membrane preparations were isolated at 24-hours poststimulation (A). Equal amounts of PTC-derived membrane protein (18 μg/lane) was loaded onto 3 to 12% SDS-PAGE gradient gels. In parallel experiments, supernatants were collected, concentrated, and loaded onto 3 to 12% SDS polyacrylamide gradient gels (20 μL of ×8, concentrated supernatant/lane; B). Western blot analysis was carried out as detailed in the Methods section. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Induction of TSG-6 protein following 25mmol/L D-glucose stimulation. Growth-arrested HK2 cells were stimulated with 5mmol/L d-glucose (5 Gl) or 25mmol/L d-glucose (25 Gl) or 25mmol/L L-glucose (25 L-Gl). Membrane preparations (A) or supernatant samples (B) were collected at 72-hours and 96-hours poststimulation, respectively. Equal amounts of PTC-derived membrane protein (19 μg/lane) were loaded onto 3 to 12% SDS-PAGE gradient gels. Supernatant were concentrated using centricon microconcentrators and loaded onto 3 to 12% SDS-PAGE gradient gels (20 μL/lane). Western blot analysis was carried out as detailed in the Methods section. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Effect of cycloheximide upon the IL-1β and 25mmol/L D-glucose–induced TSG-6 mRNA. Growth-arrested HK-2 cells were stimulated with IL-1β (1 ng/mL) in the presence or absence of cycloheximide (5 μg/mL) for up to 24 hours. Similarly, growth-arrested cells were stimulated with 25mmol/L d-glucose in the presence or absence of cycloheximide (5 μg/mL) for up to 72 hours prior to isolation of total cellular RNA. In addition, RNA was isolated from unstimulated control cells at time zero, and cells were incubated with cycloheximide alone at time points up to 72 hours. TSG-6 mRNA was examined by RT-PCR. Subsequently, PCR products were separated by electrophoresis on a 3% agarose gel (A). The results are expressed as the normalized densitometric ratios of TSG-6 mRNA to α-actin following stimulation with either 25mmol/L d-glucose or IL-1β (1 ng/mL) in the absence of cycloheximide (□) to the ratio following stimulation in the presence of cycloheximide (▪) (B). One representative gel of four individual experiments is shown. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 8 Inhibition of plasmin activity in supernatants after stimulation with IL-1β or 25mmol/L D-glucose. Growth-arrested HK-2 cells (A) or HPTCs (B) were stimulated with 5mmol/L d-glucose (▴), 25mmol/L d-glucose (•), or IL-1β (▪; 1 ng/mL) in the absence of serum up to 96 hours. Plasmin activity of supernatants was determined as detailed in the Methods section. Results were normalized to the time zero medium control for each experimental condition and the data represents mean ± SD of three independent experiments. *P < 0.05 IL-1β vs. 5mmol/L d-glucose; #P < 0.05 25mmol/L vs. 5mmol/L d-glucose. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 9 Immunoprecipitation of TSG-6 abrogates plasmin inhibitory activity in IL-1β and 25mmol/L D-glucose stimulated cell culture supernatants. Supernatant samples were collected from HK-2 (A) cells or HPTCs (B) stimulated with either IL-1β (1 ng/mL) for 24 hours or 25mmol/L d-glucose for 96 hours. TSG-6 was immunoprecipitated and plasmin activity measured. Data were normalized to the time zero medium control for each experimental condition. Results show the plasmin activity prior (□) to and following immunoprecipitation with anti-TSG-6 antibody (▪) or rabbit IgG control (). Values represent the mean ± SD of four independent experiments. *P < 0.05 for TSG-6 immunoprecipitated samples vs. nonimmunoprecipitated and rabbit IgG control. Kidney International 2001 60, 126-136DOI: (10.1046/j.1523-1755.2001.00779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions