Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis

Abstract

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.

Fig 1. tPA activity in the presence of antimicrobial agents used for the treatment of PD peritonitis.

The activity of tPA in the presence of different antimicrobial agents used for the treatment of PD peritonitis was assessed with reference to controls. Results are expressed as the mean and SD of percentage of activity in control wells from a representative experiment. Control = dialysate only, V+G = Vancomycin and Gentamicin, A = Amoxicillin, C = Cefazolin and F = Fluconazole.

Fig 2. DNase activity after 6 hours incubation with tPA and antimicrobials or tPA and bacteria.

The activity of DNase in the presence of different antimicrobial agents (Panels A & B) or bacteria (Panels C & D) was measured through digestion of 1 μg of pcDNA3 DNA. V+G = Vancomycin and Gentamicin, A = Amoxicillin, C = Cefazolin and F = Fluconazole. The effects of bacteria on DNase activity was assessed at baseline (T = 0) and following 6 hours incubation (T = 6) at 37°C. Samples were incubated with 5 μg/mL tPA and/or 2.5 μg/mL DNase, as applicable.

Fig 3. DNase activity in the presence of vancomycin and gentamicin.

The effect of increasing concentrations of gentamicin on DNase activity was measured in the presence of a constant concentration of Vancomycin. Inhibition of DNase activity, visible as smearing (incomplete digestion) of DNA (Lanes 4–8), was evident at Gentamicin concentrations >35 μg/mL. Samples contained 5 μg/mL tPA and 2.5 μg/mL DNase, as appropriate.

Fig 4. tPA and DNase do not directly affect the viability of E. coli and S. aureus.

Fig 5. tPA and DNase do not influence antimicrobial activity.

The effect of tPA and DNase on antimicrobial activity was assessed by measuring the survival of bacteria (with known sensitivity) to the antimicrobial agents over time in the presence or absence of tPA and DNase. All assessments were performed in triplicate and bacterial counts were calculated through spot counting of serial dilutions of bacteria. Data show is the mean number of cfu/mL at 6 and 24 hours as a % of baseline (error bars are the standard deviation). A = Amoxicillin, C = Cefazolin, V&G = Vancomycin and Gentamicin.

Fig 6. H&E sections of kidney, liver, peritoneum and spleen.

Microscopic analysis of abdominal organs and peritoneum was performed following intraperitoneal administration of dialysate (A-D) or dialysate with tPA and DNase (E-H) in the presence or absence of LPS (data with LPS not shown). Samples contained 5 μg/mL tPA and 2.5 μg/mL DNase, as appropriate.