P2X7 receptor protects against inflammation induced renal fibrosis during pyelonephritis with a‐hemolysin producing Escherichia coli
Authors: Therkildsen Jacob R , Christensen Mette G , Skals Marianne , Praetorius Helle A. 
Published: November 2023

Urinary tract infections are regularly caused by sub‐strains of E. coli that produce the virulence factor α‐hemolysin (HlyA). The more severe the infection is, the likelier it is to isolate HlyA‐producing E. coli. Severe or repeated ascending urinary tract infections can cause renal scarring and failure. There is substantial evidence that local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigates the effect of the P2X7 receptor in a murine pyelonephritis model.MethodThe experiments were performed by installing 108 HlyA‐producing E. coli (ARD6), 108 none‐HlyA producing E. coli strain (D2103) or saline in the urinary bladder of P2X7+/+ or P2X7−/−‐mice. After monitoring the mice for 1, 5 or 14 days, urine and plasma samples were collected and the mice were perfusion fixed for evaluation of the renal histology. The infection status of the kidney was assessed by ligation of the renal artery and removal of the left kidney before fixation and following culturing of kidney homogenate on blood agar plates.ResultBy this method, we were able to induce pyelonephritis already after 24 hours, confirmed by growth of HlyA‐producing E. coli from kidney homogenate in the mice subjected to these bacteria only. After 24 hours, the plasma levels of interleukin‐6 (IL‐6) was 13.8 ± 5.2 pg/ml in the control mice and 57.15 ± 21.6 pg/ml in mice inoculated with ARD6. Similarly, we found that the IL‐8 analogue keratinocyte chemoattractant (KC) was 26.3 ± 8.2 pg/ml in control and 127.7 ± 60.0 pg/ml in ARD6 subjected mice. The plasma concentration of IL‐1b and TNFa were similar in the two groups. The reference E. coli strain (D2103) caused cystitis but not pyelonephritis. The mice still showed pyelonephritis after 5 days, with positive growth on kidney homogenates in the animals subjected to ARD6, although the cytokine‐level decreased and no longer showed statistical significance. This may suggest, that the increased cytokine production caused by the infection already decreases after a few days. Sirius staining of the kidney sections did not reveal any renal fibrosis on day 5.Spot urine samples showed that the urinary tract infection was slowly cleared during a 5–14 days period. After 14 days, we could, however, clearly show an increased density of collagen in the renal medulla and a massive infiltration by immune cells particularly in the renal medulla of the ARD6‐infected mice compared to controls. Moreover, diffuse areas in the cortex showed signs of scarring (by Sirius stain). The susceptibility to pyelonephritis is similar in P2X7−/− and P2X7+/+ mice with the same number of spontaneous death. Moreover, the same bacterial load caused similar degree of pyelonephritis in P2X7−/− and P2X7+/+ mice. However, the P2X7−/− mice showed reduced renal fibrosis in response to infection, reduced infiltration by immune cells and only limited diffuse scarring of the cortex after 14 days compared to P2X7.+/+ConclusionThese findings suggest that the P2X7 receptor is involved in the renal scarring following pyelonephritis induced by HlyA‐producing E. coli. Thus, we are now testing whether P2X7R antagonist may be used as a therapeutic intervention in urinary tract infections – even after an infection is established.Support or Funding InformationThe study is supported by:The Danish Council for Independent Research, Lunbeck Foundation and the Recerch Centre MEMBRANES