Sixma, M.L. (Marije) (2013) Sepsis-induced acute kidney injury. “The role of the P2X7 receptor (P2X7R) in renal injury and sepsis through the local production of cytokines and chemokines”. thesis, Medicine.
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Abstract
Introduction: Sepsis is an exaggerated inflammatory response to infection that may lead to multiple organ failure and death. The kidney is commonly affected in the septic process leading to dysfunction or complete failure. Of note, survivors of sepsis-induced acute kidney injury (AKI) have an increased risk of developing end-stage chronic kidney disease. Release of pro-inflammatory cytokines such as IL-1β and IL-18 are associated with the pro-inflammatory cascade and immunological factors are likely to contribute to the development of sepsis-induced AKI. However, initial release of these mediators is, in itself, insufficient. A considerable role for the post-transcriptional processing and release of mature cytokines is via the formation of a large multiprotein complex, the NLRP3 inflammasome. The cell surface P2X7 receptor (P2X7R) facilitates assembly of NLRP3. As well as mediating inflammatory responses, the P2X7R is also involved in apoptotic cell death. Upregulation of this receptor is associated with renal injury. Moreover, A-438079, a selective P2X7 antagonist, attenuated this response in a rat model of glomerulonephritis. These findings suggest that the P2X7R could function as a potential therapeutic target. P2X7 expression is best known in immune cells such as macrophages which play a critical role in the initiation, maintenance, and resolution of inflammation. Macrophages both release and are influenced by chemokines such as MCP-1 that play a prominent role in the acute inflammatory response in several models of kidney disease. Moreover, IL-1 β (after maturation and release by the P2X7/inflammasome) induces expression of MCP-1 by epithelial cells. Aims: With a working hypothesis that P2X7R upregulation causes renal injury through local production of cytokines and chemokines, I sought to determine whether renal P2X7R expression increases in a fluid-resuscitated three-day rat model of faecal peritonitis, and specifically to define its relationship and co-localization to histopathological changes and macrophage infiltration. In a separate longer term (2-week) rat model of zymosan-induced peritonitis, I sought to describe renal P2X7R expression and pathological changes from the early stages into the recovery phase. Methods: For the acute model, sepsis was induced in instrumented, awake male Wistar rats by intraperitoneal injection of faecal slurry followed by fluid resuscitation at 10 ml/kg/hr commencing 2 hours post-induction of sepsis. Sham animals received a similar fluid regimen but no i.p. injection. Septic and sham-operated animals were sacrificed post-sepsis at either 6 hours (n = 6 per group) or 24 hours (n= 8 per group). Kidneys were harvested and blood analyzed for serum creatinine, histological evidence of renal injury, P2X7 expression, macrophage infiltration, and cytokine and chemokine expression. Comparison was made against naïve, non-instrumented animals (n=6). In the long-term model, non-instrumented male Wistar rats were given i.p. zymosan (a yeast cell wall product) as a more prolonged septic insult. Kidneys were harvested at day 2 (4 sham, 4 sepsis animals) and day 14 (4 sham, 8 sepsis), and analyzed for renal injury, macrophage infiltration and chemokine expression. Continuous variables are presented as mean ± SD. Parametric and non-parametric data were compared by unpaired t-test and Mann Whitney U tests, respectively. One-way ANOVA assessed differences between more than two groups of continuous variables. Post-hoc Tukey’s test ascertained between-group differences with P values <0.05 being taken as statistically significant. Results: In the acute septic AKI model, serum creatinine was significantly higher at 24 hours (p<0.05). However, no significant renal histological damage was seen at either 6 or 24 hour timepoints. In septic kidneys, P2X7R expression increased at 24 hours (p< 0.05) though a trend was apparent even at 6 hours. Severely septic animals (with a 3-fold increase in serum creatinine) showed evidence of intraluminal debris that stained intensely for P2X7. Increased expression of P2X7 correlated with elevated serum and renal tissue IL-1β levels. Serum IL-1β were elevated in septic animals, though levels were falling by 24 hours (p<0.05). By contrast, renal tissue IL-1β levels were elevated at 6 hours after sepsis (p=0.05) but further increased at 24 hours (p<0.01). Consistent with these findings, septic AKI was associated with increased renal expression of caspase-1 and pro-IL-18. No macrophage infiltration was seen at either timepoint and there was only minimal elevation in renal MCP-1 levels. In the long-term model of sepsis, there was also minimal evidence of acute renal injury at Days 2 or 14. P2X7 staining has yet to be analyzed; however, macrophage infiltration was evident in both glomeruli and interstitium in all Day 14 septic animals but in none of the sham controls. Renal MCP-1 levels were also significantly elevated in septic animals (p<0.01) at this timepoint. By contrast, macrophage infiltration was only seen in one of the four septic animals on Day 2 and renal MCP-1 levels were similar to sham animals. Conclusions: A significant increase in renal P2X7 expression was seen in septic animals after 24 hours, correlating with elevated levels of IL-1β. In severely septic animals more renal injury was apparent with a distinct pattern in staining, suggestive of a role in cell death. This finding, together with an absence of early renal macrophage infiltration, suggests renal P2X7 is likely to be expressed by intrinsic kidney cells. While significant histological damage was not seen, reflecting findings in septic patients, there was biochemical evidence of renal dysfunction. Further research is needed to evaluate whether P2X7 antagonism offers protection. By contrast, the longer term model (in which P2X7 staining has not yet been analyzed) did show significant renal macrophage infiltration within the glomeruli and interstitium with a corresponding increase in renal MCP-1 at day 14. Whether this contributes to long-term renal damage requires further study.
| Item Type: | Thesis (Thesis) |
|---|---|
| Supervisor name: | Kallenberg, Professor C.G.M. |
| Supervisor name: | Singer, Professor Mervyn and University College London and Research Department of Clinical Physiology |
| Faculty: | Medical Sciences |
| Date Deposited: | 25 Jun 2020 10:51 |
| Last Modified: | 25 Jun 2020 10:51 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/1199 |
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