Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Pavlov, Tengis S.; Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Pochynyuk, Oleh; Staruschenko, Alexander

doi:10.3791/53035

Cited by 10 publications

(6 citation statements)

References 50 publications

(54 reference statements)

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“…Patch-clamp experiments. ENaC activity was assessed in freshly isolated nondilated CDs and cysts as previously described (22,24). Briefly, kidneys from 16-wk-old rats were flushed and cut in longitudinal plane in 1-to 2-mm slices.…”

Section: Methodsmentioning

confidence: 99%

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

Arkhipov

Potter

Geurts

et al. 2019

American Journal of Physiology-Renal Physiology

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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.

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Section: Methodsmentioning

confidence: 99%

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

Arkhipov

Potter

Geurts

et al. 2019

American Journal of Physiology-Renal Physiology

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“…Blood was collected during the experiment with bleeds from the arterial catheters on conscious rats while resting, and plasma glucose level was then measured with a Contour strip glucometer (Bayer, Mishawaka, IN). At the end of the protocols, the kidneys of rats were perfused (6 ml/min) through the distal aorta with saline to clear them from blood, and then the renal tissues were collected for further analyses (Pavlov et al, 2015a ).…”

Section: Methodsmentioning

confidence: 99%

Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats

et al. 2017

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In the kidney, reabsorption via the epithelial sodium channel (ENaC) is involved in long-term blood pressure control. Previously we demonstrated that ENaC hyperactivity is associated with development of salt-sensitive (SS) hypertension in Dahl SS rats. AMP-activated kinase (AMPK), playing a role in cellular energy homeostasis, has been shown to decrease ENaC activity. Here, we tested whether metformin and AICAR, two drugs that activate AMPK, affect the development of salt-induced hypertension. High salt diet significantly increased mean arterial pressure (MAP) in Dahl SS rats. Blood pressure elevation was accompanied by a short-term decline of heart rate and increased circadian arterial pressure dipping. Metformin and AICAR were delivered intravenously at doses of 200 and 20 mg/kg/day, respectively. However, both control and drug-treated groups had similar development of high blood pressure within 3 weeks of 8% NaCl dietary salt intake. In the metformin-treated animals MAP reached 164.9 ± 9.1 mmHg, which was not significantly different from the control group (171.8 ± 5.6 mmHg). Patch clamp analysis revealed that the metformin-treated rats had no difference in the activity of ENaC. AICAR treatment also did not affect the development of hypertension and kidney injury. MAP reached 182.8 ± 4.8 and 178.0 ± 2.8 mmHg in AICAR and vehicle treated groups, respectively. Of note, we found that high-salt diet activated AMPK in the Dahl SS rats, and treatment with these AMPK activators had no significant further effect on AMPK activity. We conclude that AMPK activators, at least under these conditions, do not affect development of hypertension during high-salt diet in the Dahl SS rat model.

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“…Finally, it was shown that inhibition of P2X 7 receptors with antagonist A438079 or through morpholino-mediated knockdown of P2RX7 strongly regulates cyst development in the pkd2 knockdown zebrafish model of PKD [27]. We and others also recently reported the abnormal effects of ATP in cystic tubules isolated from PCK rats compared to normal tubules from Sprague-Dawley (SD) rats [28,29].…”

mentioning

confidence: 88%

“…At the age of 16 weeks, rats were euthanized and their kidneys were flushed, harvested and cut to 1-1.5-mm-thick slices. The internal monolayer of the cyst-lining cells was manually microdissected and attached to a cover glass as described earlier [29]. Samples were incubated with 3.5 μM Fluo-8 dye (no.…”

Section: Calcium Imaging In Isolated Cystsmentioning

confidence: 99%

Characterization of purinergic receptor expression in ARPKD cystic epithelia

Palygin

Ilatovskaya

Levchenko

et al. 2018

Purinergic Signalling

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Polycystic kidney diseases (PKD) are a group of inherited nephropathies marked with the formation of fluid‐filled cysts along the nephron. Growing evidence suggests that autocrine and paracrine effects of purinergic signaling via P2 receptors could detrimentally contribute to cyst expansion. However, little is known about purinergic signaling in renal cyst epithelium, which is characterized by loss of polarity, dedifferentiation and other abnormalities which can lead to purinergic signaling remodeling. We have proposed that ATP via associated intracellular signaling can contribute to cystogenesis by modulation of calcium influx. PCK/CrljCrl‐Pkhd1pck/CRL (PCK) rat, an established model of ARPKD, was used here to test this hypothesis. The cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley (SD) rat kidney cortical interstitium as assessed by highly sensitive for ATP enzymatic biosensors (211±55 vs 1082±147 nM for SD and PCK cortex correspondingly, and 2,078±391 nM for cystic fluid). 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 absence of ATP evoked transient, which pointed towards the extracellular (ionotropic) calcium entry in cyst‐lining cells rather than the metabotropic P2Y‐mediated internal depot. Application of iso‐PPADS (a non‐selective P2X antagonist) resulted in partial blockade of ATP response (calcium release after ATP was 30.1 ± 0.9, 22.2 ± 1.8 and 19.5 ± 3.2 a.u. in control, after incubation with iso‐PPADS, and after washout, respectively) indicating the contribution of P2X4 purinoreceptor in the cystic monolayer. Next, to specifically assess the role of P2X7 in the ATP‐mediated calcium influx, we employed AZ10606120, a potent P2X7 receptor antagonist. Application of AZ10606120 (5 μM) resulted in a bunted calcium response to ATP (32.4 ± 2.2, 13.5 ± 6.4, and 11.1 ± 3.1 a.u. of total calcium release in control, after incubation with AZ10606120, and after washout, respectively), which corroborates the commonly hypothesized role of P2X7 in cyst development. Further use of pharmacological agents (α,β‐methylene‐ATP, 5‐BDBD, and NF449) allowed to narrow down potential candidate receptors and suggested a significant involvement of the P2X4 and/or P2X7 signaling axis in the regulation of cytosolic calcium level in the cystic epithelia. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is altered in the ARPKD cystic epithelia towards the prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cited by 10 publications

References 50 publications

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

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

Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats

Characterization of purinergic receptor expression in ARPKD cystic epithelia

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