Purinergic signalling regulates renal vascular and tubular functions. P2X7 receptor (P2X7) is a non-selective cation channel activated by extra-cellular ATP which plays an important role in immune cell response and inflammation. Endothelial cells also express P2X7, including in the kidney. Studies also detected P2X7 expression in the renal tubular epithelium, notably in disease. Acute pharmacological blockade increases renal blood flow (RBF) and glomerular filtration rate (GFR), suggesting that P2X7 receptor activation promotes tonic vasoconstriction. Here, we generated a novel P2X7 global knockout (KO) rat on the F344 background, hypothesising that these rats would have enhanced RBF and would be protected from the injury caused by chronic angiotensin II (ANG II) infusion.

CRISPR/Cas9 technology introduced an early stop codon into exon 2 of P2rx7. In male and female KO and WT rats, renal P2X7 expression was measured by RTqPCR (n=4-5/group) and western blotting (n=3/group). Bone marrow-derived macrophages (BMDM) were used to assess P2X7 function by measuring interleukin (IL)-1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5-6/group). In vivo kidney function was assessed in anesthetized rats (thiopental, 50mg/kg, i.p.) at baseline and following serial arterial ligation to induce acute pressure natriuresis (n=9-10/group). Arterial blood pressure (BP) was measured directly, and urine was collected via tube cystotomy. RBF was measured using a Transonic Doppler flow probe placed around the main renal artery, and cortical and medullary flux by laser Doppler needle probes. GFR was determined by FITC-inulin clearance. Urinary sodium was measured by sodium-selective electrode analysis. Finally, male KO and WT rats (n=9-10/group) were infused with ANG II (250ng/kg/min, s.c.) for ~6 weeks. At end, one renal artery was isolated and vascular reactivity to phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP) determined by wire myography. Bladder urine was collected to measure albumin/creatinine ratio (n=4-7/group)., and kidneys were harvested for histological analyses (n=6/group). Data were analysed with unpaired t-test (two groups compared) or two-way ANOVA (two variables), with P<0.05 statistically significant.

P2rx7 mutation was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was partially decreased in kidney from male (-54%) and female (-25%) KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. In BMDM from WT rats, LPS+ATP stimulation induced IL-1β release; this response was markedly suppressed in KO BMDM (males: 2577±140 vs 413±175pg/mL, P<0.0001; females: 1889±394 vs 265±121pg/mL, P<0.0001). In vivo BP, renal haemodynamics and tubular sodium handling were not different between genotypes for either sex (P>0.05). Following chronic ANG II infusion, KO and WT rats had no renal artery dysfunction but showed similar increases in albuminuria (P=0.3587), renal perivascular fibrosis (Picrosirius Red, P=0.5393), cortical interstitium CD68-positive area (p=0.6546) and tubular casts (Periodic acid-Schiff, P=0.8803), compared with naïve controls.

Contrary to our hypothesis, global genetic deletion of P2X7 did not affect renal hemodynamics and we found no significant role for P2X7 in the modulation of tubular sodium reabsorption in health. Our data do not support a major role of P2X7 in causing renal vascular and tubular injury.