Bailey, Matthew

Baker, Andrew

Nespoux, Josselin Georges

2023-06-12T09:57:13Z

2023-06-12T09:57:13Z

2023-06-12

Purinergic signaling regulates numerous intrarenal mechanisms contributing to the long-term control of blood pressure. The P2X7 receptor is an ionotropic receptor activated by extra-cellular ATP that participates in inflammation and activation of immune cells. P2X7 is also expressed in the vascular endothelium, including in the kidney where its function remains poorly understood. Previous research identified the gene P2rx7, which encodes the P2X7 receptor, as a candidate gene for susceptibility to hypertensive renal vascular injury in Fischer (F344) rats. Higher expression of P2X7 was reported in several rat models of hypertensive kidney injury and both pharmacological blockade and P2X7 deletion were found renoprotective. However, studies raised concerns regarding the specificity of the broadly used P2X7 antagonists which have generated conflicting results. Here, I hypothesized that global genetic deletion of P2X7 in F344 rats will prevent the development of renal inflammation and injury in a model of chronic angiotensin II (Ang II)-induced hypertension. In this study, I sought to characterize the contribution of P2X7 to basal renal vascular and tubular functions in male and female rats. I also aimed to determine whether absence of P2X7 exerts renoprotective effects during chronic Ang II infusion, and to provide cellular and molecular mechanistic evidence for such protection using a combination of in vivo, ex vivo and in vitro studies. To this end, I used F344 rats to generate a novel CRISPR-Cas9-designed P2X7 knockout (KO) and showed that it is a true global KO with no functional P2X7 protein expressed. Using wire myography, I found that P2X7 KO impaired endothelial-dependent vasodilation in the renal artery of male, but not female, rats that may reflect diminished nitric oxide (NO) production in response to acetylcholine. NO is a key paracrine factor regulating the renal pressure natriuresis mechanism and blood pressure (BP). Thus, I assessed kidney function in anesthetized male and female P2X7 KO and wild-type (WT) rats at baseline and following acute stepwise increases in renal perfusion pressure. Overall, results showed no significant genotype effect on renal haemodynamics, BP and NO production. Moreover, my data do not support a major role of P2X7 in the modulation of tubular sodium reabsorption. I next attempted to generate an experimental model of chronic Ang II infusion-induced hypertensive kidney and vascular injury in F344 WT and P2X7 KO rats. After 5-6 weeks of infusion, rats developed modest renal damage consisting of perivascular fibrosis and tubular injury. P2X7 did not contribute to the development of renal perivascular fibrosis and tubular injury during chronic Ang II infusion. Finally, I investigated the implication of P2X7 to endothelial-to-mesenchymal transition (EndMT) using the potent P2X7 antagonist A438079 in vitro. I found that P2X7 blockade did not affect EndMT. In summary, I propose that P2X7 receptors modestly promote endothelial NO production in healthy rat renal arteries and inhibit tubular sodium reabsorption in a sex-specific manner. The role of P2X7 in renal vascular functions may become more relevant in a pathological context and requires further investigations with better disease models.

https://hdl.handle.net/1842/40652

http://dx.doi.org/10.7488/era/3413

en

The University of Edinburgh

P2X7

Renal haemodynamics

Kidney function

CRISPR/Cas9

Hypertension

Kidney injury

Purinergic

Pressure natriuresis

Rat

EndMT

endothelial-to-mesenchymal transition

ATP

Role of the purinergic P2X7 receptor in renal haemodynamic physiology and hypertensive renovascular injury

The role of the purinergic P2X7 receptor in renal haemodynamic physiology and hypertensive renovascular injury

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy