Characterization of purinergic receptor expression in ARPKD cystic epithelia

Abstract

Polycystic kidney diseases (PKDs) are a group of inherited nephropathies marked by formation of fluid-filled cysts along the nephron. Growing evidence suggests that in the kidney formation of cysts and alteration of cystic electrolyte transport are associated with purinergic signaling. PCK/CrljCrl-Pkhd1pck/CRL (PCK) rat, an established model of autosomal recessive polycystic kidney disease (ARPKD), was used here to test this hypothesis. Cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley rat kidney cortical interstitium as assessed by highly sensitive ATP enzymatic biosensors. Confocal calcium imaging of the freshly isolated cystic monolayers revealed a stronger response to ATP in a higher range of concentrations (above 100 μM). The removal of extracellular calcium results in the profound reduction of the ATP evoked transient, which suggests calcium entry into the cyst-lining cells is occurring via the extracellular (ionotropic) P2X channels. Further use of pharmacological agents (α,β-methylene-ATP, 5-BDBD, NF449, isoPPADS, AZ10606120) and immunofluorescent labeling of isolated cystic epithelia allowed us to narrow down potential candidate receptors. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is shifted in ARPKD cystic epithelia in an age-related manner towards prevalence of P2X₄ and/or P2X₇ receptors, which opens new avenues for the treatment of this disease.

Keywords: ARPKD; ATP; Intracellular calcium flux; Kidney; P2X receptors; P2X4; P2X7; P2rx4; P2rx7; PCK rat; Polycystic kidney disease; Purinergic receptor.

Fig. 1

ATP levels in PCK cystic fluid and cortical tissues of the isolated kidneys from PCK and Sprague-Dawley (SD) rats. a Enzymatic biosensor setup for detection of ATP in the isolated kidney tissues. ATP and Null (to subtract noise and non-selective interferences) biosensors were inserted into the kidney cortical layer and connected to a dual channel potentiostat for real-time amperometry recordings. Inset shows a close-up image with sensors’ tips inserted into a cortex of the freshly isolated kidney from PCK rat. b ATP concentrations were detected using enzymatic biosensors in the cortical tissues of the freshly extracted kidneys from PCK and SD rats and isolated cystic fluid from PCK rats (N = 4 rats for each group)

Fig. 2

Calcium level and response to ATP in the freshly isolated PCK rat cystic epithelium. a A representative confocal image showing a cystic monolayer isolated from a PCK rat kidney (bright field image, and the same cystic monolayer loaded with Fluo8 calcium dye before and after application of ATP). Scale bar is 100 μm. b Experimental protocol to determine intracellular calcium concentrations in the cells of the cystic epithelium. Average basal calcium concentration in the cystic cells is shown on the graph. To measure intracellular calcium concentration, cystic monolayers were loaded with Fluo-8, AM, and fluorescence intensity was recorded in the baseline and after addition of ionomycin and MnCl₂. The graph demonstrates the fluorescence signal changes in response to a calcium ionophore (ionomycin, producing the maximum Fluo-8 fluorescence, F_max) and MnCl₂, which quenches the dye and results in the lowest fluorescence intensity (F_min). Intensity of fluorescence (left axis) for each time point was recalculated into the actual calcium concentration in nanomoles (right axis). The transient shown on the graph reflects fluorescence intensity of a ROI (single cell) selected from a cystic monolayer; images were taken every 8 s. Calcium levels were assessed in 12 cystic monolayers isolated from 6 different rats, and 140 ROIs containing multiple cells were analyzed in this group. c Representative calcium transients recorded in an isolated cystic monolayer. The fluorescent responses to 200 μM ATP application in media containing 2 mM of Ca²⁺ (upper panel) and the same ATP concentration without extracellular Ca²⁺ (middle panel). Summary for the changes of the amplitude of intracellular calcium concentration in response to ATP with or without the presence of Ca²⁺ ions in extracellular solution (N ≥ 6 cyst monolayers for each group)

Fig. 3

Calcium response in the cystic monolayer is triggered by high concentrations of ATP and consecutive ATP applications/washouts protocol do not cause P2 receptor desensitization. a Changes of the cystic cells’ intracellular calcium levels in response to various concentrations of ATP. Shown are representative transients from different cystic monolayers (12÷15 cells/ROIs were analyzed per each transient). b Representative calcium transients in response to three consecutive applications of ATP interleaved with 15-min long washout periods; 15 cells/ROIs were analyzed per each transient

Fig. 4

Calcium transient in the cystic monolayer in response to P2X receptor agonist αβmeATP. a Representative changes of the cystic cells’ intracellular calcium levels in response to various concentrations of α,β-methylene-ATP (αβmeATP). Shown are representative transients from different cysts (12÷15 cells/ROIs were analyzed per each transient). b Response to ATP in a cystic monolayer is sustained after application of αβmeATP with 15-min-long washouts between applications of the compounds. N = 4 rats per group, 5÷8 cystic monolayers were tested in each group. c Summary for the amplitude and total [Ca²⁺]_i production (AUC) for ATP and αβmeATP (200 μM each) (N ≥ 3 cyst monolayers for each group at selected concentration of agonist)

Fig. 5

The effects of P2X antagonists isoPPADS, NF449 and AZ10606120 on calcium transients evoked by ATP in freshly isolated cystic epithelium. ATP was applied to establish the control level of the response (left), then, during the washout period, isoPPADS (a), NF449 (b), or AZ10606120 (c) were added to the bath solution, and incubation was performed (15 min); ATP was applied to the cystic monolayer in presence of the antagonist (middle). Following a second washout of all applied drugs (next 15 min), ATP was applied again to test the reversibility of the response (right). Shown are the representative responses from single cystic monolayers (11÷15 cells/ROIs were analyzed per transient). d The summary of the changes in the [Ca²⁺]_i transient amplitude for the applied agonists/antagonists of purinergic signaling to the cystic epithelial cells (N = 3–8 cyst monolayers for each group)

Fig. 6

P2X₄ receptor inhibition results in a blunted calcium transient in response to ATP or αβmeATP. The specific P2X₄ receptor inhibitor 5-BDBD was applied to the cystic monolayers following an application of ATP (a) or αβmeATP (b) and a washout. After incubation with 5-BDBD was performed, ATP was applied to the cystic monolayer in presence of the antagonist; then following a second washout, ATP was applied to test the reversibility of the response. Representative responses from single cystic monolayers are shown. c Summary of the changes in the [Ca²⁺]_i transient amplitude for the applied agonists/5-BDBD of purinergic signaling to the cystic epithelial cells (N = 3–8 cyst monolayers for each group)

Fig. 7

Expression of P2X₇ and P2X₄ receptors in the cortex of PCK rats during the development of cytogenesis. Average relative density analysis for Western blotting (N ≥ 8) and mRNA levels (N ≥ 6) of P2X₇ (a) P2X₄ receptors (b), and corresponding for the P2X₇ receptor (normalized to loading controls) in the kidney cortex lysates of 5, 12, and 40 weeks old PCK rats. c Examples of the corresponding Western blotting (each line represents 1 rat)

Fig. 8

Intracellular localizations of P2X₇ and P2X₄ receptors in PCK cysts. Transmitted light images depict the epithelial cellular structure in cystic monolayers. Due to the thin, squamous nature of the epithelia, nuclear protrusions can occasionally be observed in the transmitted light. Freshly isolated cyst sections were fixed and probed for P2X₇ (green) and AQP2 (red) (a). Cells demonstrate distinctive membrane localization in classic Voronoi patterning for both P2X₇ and AQP2. b P2X₄ channels (red) are also targeted to the plasma membrane of cyst epithelia. c PCK cyst epithelia contain unique microvasculature. In this image, the intermingling microvessel can be observed by lack of P2X₄ staining and structural features in the transmitted light. Approximate outlines of the cystic and vessel regions are shown in the transmitted light image. d Polycystic kidneys were embedded in paraffin, sectioned, and probed for P2X₇ and P2Y₂. Similar to the isolated cyst epithelial staining, P2X₇ is observed in cystic cells, while P2Y₂ are observed in small, early cysts, but not in larger cysts