Knockout ofP2rx7purinergic receptor attenuates cyst growth in a rat model of ARPKD

Abstract

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK. P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK. P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

An Engineered Mouse to Identify Proliferating Cells and Their Derivatives

“…In contrast, Aurkb protein expression level in Aurkb ER Cre/+ heterozygous mice was quite similar to their wildtype controls (Figure 3D). This is consistent with other reports that heterozygous knockout mice can express similar or more than half of the proteins relative to wildtype control mice in the target genes (Gineste et al, 2013;Zhou et al, 2015;Arkhipov et al, 2019). It is reported that an Aurkb knockout mouse in which exons 2-6 were excised is embryonic lethal, older Aurkb heterozygous mice approximately 12-24 months of age show decreased survival due to susceptibility for tumorigenesis, and a fraction of Aurkb heterozygous males suffer from oligospermia by 12 months of age (Fernandez-Miranda et al, 2011).…”

Characterization of purinergic receptor 2 signaling in podocytes from diabetic kidneys

“…Our experiments in both T1D and T2D podocytes with specific inhibitors provide compelling evidence that P2X 4 and P2X 7 receptors play a major role in ATP-induced [Ca 2+ ] i levels in diabetic podocytes. Previous research has established that P2X 7 receptor expression increases are linked to PKD, glomerulonephritis, human lupus nephritis, and hypertension-associated renal injury ( Arkhipov et al., 2019 ; Hillman et al., 2005 ; Pereira et al., 2020 ; Vallon et al., 2020 ). In addition, the upregulation of glomerular P2X 7 receptor mRNA and protein expression has been reported in STZ-induced diabetic and ren-2 transgenic hypertensive rat models, and was associated with inflammatory cytokine release and cell death ( Vonend et al., 2004a ).…”

“…Clinical and experimental studies suggest that purinergic signaling is an important contributor to various physiological and pathophysiological processes in the kidney, including diabetes ( Vallon et al., 2020 ). For example, ionotropic P2X 7 receptors are implicated in cyst formation in polycystic kidney disease and also renal interstitial inflammation associated with different kidney pathologies ( Arkhipov et al., 2019 ; Chang et al., 2011 ; Goncalves et al., 2006 ; Taylor et al., 2009a ). Renal proximal tubular P2X 4 activation promotes tubular inflammation and exacerbates ischemic acute kidney injury ( Han et al., 2020 ), and deficient metabotropic P2Y 2 signaling has been shown to aggravate the progression of chronic kidney disease in mice ( Potthoff et al., 2013 ).…”

Pannexin‐1 mediates fluid shear stress‐sensitive purinergic signaling and cyst growth in polycystic kidney disease

“…Dysfunctional PC1 also critically affects the transcriptome of kidney cells . In addition, increased expression of pannexin‐1 in cyst lining cells of a mouse and rat PKD model has recently been reported . Under given stimuli (eg, mechanic stimulation, intracellular concentrations of certain ions, etc), higher pannexin‐1 expression may result in higher transport activity.…”

“…Thus, our findings unequivocally indicate the contribution of pannexin‐1 to cyst growth and suggest this channel as druggable target for PKD therapy. Interestingly, pannexin‐1 expression levels appear to be dramatically increased in pathological conditions compared to the healthy kidney making it an especially interesting therapeutic target . BB‐FCF, is considered safe to use.…”