Sex Difference in Renal Artery Contractility in a Novel CRISPR/Cas9‐Generated P2X7 Knockout Rat

Abstract

Purinergic signaling regulates numerous physiological processes, including renal hemodynamics. The P2X7 receptor (P2X7) is a non‐selective cation channel activated by extra‐cellular adenosine triphosphate (ATP) which plays an important role in immune cell response and inflammation. P2X7 is also expressed in endothelial cells. In the kidney, acute pharmacological blockade of P2X7 increases renal perfusion in healthy rats (Kidney Int. 88(5): 1079–1087, 2015). Here, we generated a novel P2X7 knockout (KO) rat and examined ex vivo contractility of the renal artery in male and female rats.A P2rx7 global KO rat was generated on a F344 IcoCrl background using CRISPR/Cas9 technology. KO was achieved via a two‐adenine insertion predicted to introduce a premature stop codon in exon 2. RNA and protein were extracted from KO and wild‐type (WT) kidneys to detect P2X7 by RTqPCR (n=4‐5/group) and western blotting (n=2/group). Bone marrow‐derived macrophages (BMDM) from KO and WT rats 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). RNA from BMDM was extracted after LPS to detect P2rx7, Il1b, Tnf and Il6 mRNA by RTqPCR (n=3/group), and Griess reaction was used to assess BMDM nitric oxide (NO) production (n=5‐6/group). One main renal artery from 2.5‐to‐4.5‐month‐old male and female KO and WT rats (n=8‐11/group) was mounted on a wire myograph to assess the response to 125 mM potassium physiological solution (KPSS) and to cumulative doses (1 nM‐30 μM) of phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP).The P2rx7 mutation in exon 2 was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was decreased by ~60% in kidney and ~53% in BMDM from KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. P2X7 KO did not alter BMDM polarization after LPS stimulation. The induction of NO production was not different between genotypes (2.14±0.26 vs 2.31±0.43 μM, NS) and mRNA expression of M1 markers Il1b, Tnf and Il6 was similarly increased. In BMDM from male WT rats, ATP stimulation induced IL‐1β release; this response was markedly suppressed in KO BMDM (2577±70 vs 413±19 pg/mL, p<0.0001). In the renal artery, the constriction induced by KPSS depolarization or by PE was not different between genotypes for either sex. In male rats, the maximum vasodilation induced by ACh was significantly decreased in KO compared to WT (IC50=0.25 vs 0.20 μM, NS; %Imax= 24±3 vs 10±2%, p=0.0015); this was not observed in females (IC50=0.11 vs 8.28 μM, NS; %Imax= 19±4 vs 12±4%, NS). The dilation induced by the direct NO donor, SNP was not affected by either genotype or sex.Global genetic deletion of P2X7 impaired endothelial‐dependent vasodilation in the renal artery of male rats that may reflect diminished NO production in response to ACh. Thus, the increased renal perfusion induced by P2X7 inhibitor may arise from off‐target effects. The potential sexual dimorphism in vascular expression and/or function of P2X7 requires further investigation.