Characterization of P2rX7 knockout in PCK (polycystic kidney) rats
Arkhipov SN, Potter D, Geurts AM, and Pavlov TS. Characterization of P2rX7 knockout in PCK (polycystic kidney) rats. FASEB Journal 2018; 32(1 Suppl):624.5.
Accumulating evidence suggests that the autocrine and paracrine effects of Adenosine-3-phosphate (ATP) could be detrimental for the progression of polycystic kidney diseases (PKD). P2X7 targeting in cell cultures and zebrafish was shown to decrease cyst progression. In this project, CRISPR/Cas9 approach was employed to knockout P2X7 receptor in the PCK rat strain, a model of autosomal recessive PKD (ARPKD), to study involvement of P2X7 in PKD progression in mammals. Mendelian distribution was observed in the litters showing no embryonic lethality. Adult knockout animals exhibited moderate hypertension (mean arterial blood pressure 123±6 and 132±5 mmHg at 9AM/9PM as registered with continuous DSI telemetry). General morphological evaluation of the kidneys revealed that total P2X7 knockout caused exacerbated cystogenesis compared to wild-type littermates. Impaired renal function in the knockout group was also confirmed with metabolic studies and inulin clearance studies in conscious animals. Thirteen weeks old P2rx7-/+ and P2rx7-/-animals had higher body mass than P2rx7+/+ littermates (421±9g and 430±10.5g vs 392±12g; n=5-14). Plasma sodium and chloride (not potassium) were slightly but significantly lower in the knockout group. We observe significant proteinuria of 35-40 mg/day/100g body weight in both groups (at 13 weeks) but with aging (at 24 weeks), it reached 89±13 and 157±19 mg/day/100g in wild-type and knockout animals, respectively. Glomerular filtration rate was also lower in the P2rx7-/-group (0.88±0.06 vs 1.22±0.11 ml/min/100g body weight) in young rats, however, with aging GFR significantly decreased in both groups and did not differ (0.42 and 0.49, respectively). Purinergic signaling is known as a powerful down-regulator of ENaC-mediated sodium reabsorption. Thus, patch clamp analysis revealed that acute basolateral application of 100μM α,β-methylene-ATP to M1 collecting ducts cell culture grown on permeable membranes decreases activity of apical ENaC (NPo from 0.5 ±0.15 to 0.19±0.12, n=10). At the next step we investigated if the P2X7 knockout affect ENaC activity. Patch clamp technique was also applied on three types of tubular epithelia isolated from knockout PCK rats and their wild-type littermates: split-opened non-dilated collecting ducts, small developing cysts and large mature cysts. In these preparations we found that developing cysts exhibit increased ENaC activity whereas mature cysts had impaired channel activity (NPo= 0.79±0.12; 1.1±0.2 and 0.39±0.16). In all three tissue types P2X7 knockout increased ENaC activity (NPo=1.36±0.17; 2.3±0.36; 0.86±0.05), as expected. In conclusion, knockout of the P2rx7 gene in PCK rats increases cystogenesis and aggravates renal insufficiency. P2X7 deficiency also increases activity of ENaC in PCK rat collecting ducts and cysts.