Intact Cytoskeleton Is Required for Small G Protein Dependent Activation of the Epithelial Na+ Channel

BACKGROUND: The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity. METHODS AND FINDINGS: First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity. CONCLUSIONS: We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity

Purinergic receptors profile in the ARPKD cystic epithelia

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

Modulation of blood pressure regulatory genes in the Agtrap‐Plod1 locus associated with a deletion in Clcn6

Abstract The AGTRAP‐PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt‐Sensitive (SS) rat background (SS‐Clcn6) resulted in lower diastolic blood pressure compared to SS‐WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS‐Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS‐WT. Nppb mRNA expression in SS‐Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors

Mechanosensitive cation channels in human leukaemia cells: calcium permeation and blocking effect

Cell-attached and inside-out patch-clamp methods were employed to identify and characterize mechanosensitive (MS) ionic channels in the plasma membrane of human myeloid leukaemia K562 cells. A reversible activation of gadolinium-blockable mechanogated currents in response to negative pressure application was found in 58 % of stable patches (n = 317). I-V relationships measured with a sodium-containing pipette solution showed slight inward rectification. Data analysis revealed the presence of two different populations of channels that were distinguishable by their conductance properties (17.2 ± 0.3 pS and 24.5 ± 0.5 pS), but were indistinguishable with regard to their selective and pharmacological properties. Ion-substitution experiments indicated that MS channels in leukaemia cells were permeable to cations but not to anions and do not discriminate between Na+ and K+. The channels were fully impermeable to large organic cations such as Tris+ and N-methyl-d-glucamine ions (NMDG+). Ca2+ permeation and blockade of MS channels were examined using pipettes containing different concentrations of Ca2+. In the presence of 2 mm CaCl2, when other cations were impermeant, both outward and inward single-channel currents were observed; the I-V relationship showed a unitary conductance of 7.7 ± 1.0 pS. The relative permeability value, PCa/PK, was equal to 0.75, as estimated at physiological Ca2+ concentrations. Partial or full inhibition of inward Ca2+ currents through MS channels was observed at higher concentrations of external Ca2+ (10 or 20 mm). No MS channels were activated when using a pipette containing 90 mm CaCl2. Monovalent mechanogated currents were not significantly affected by extracellular Ca2+ at concentrations within the physiological range (0-2 mm), and at some higher Ca2+ concentrations

Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli

A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis. Here we examined the effects of angiotensin II on intracellular calcium ion levels and endogenous channels in intact podocytes of freshly isolated decapsulated mouse glomeruli. An ion channel with distinct TRPC6 properties was identified in wild-type, but was absent in TRPC6 knockout mice. Single-channel electrophysiological analysis found that angiotensin II acutely activated native TRPC-like channels in both podocytes of freshly isolated glomeruli and TRPC6 channels transiently overexpressed in CHO cells; the effect was mediated by changes in the channel open probability. Angiotensin II evoked intracellular calcium transients in the wild-type podocytes, which was blunted in TRPC6 knockout glomeruli. Pan-TRPC inhibitors gadolinium and SKF 96365 reduced the response in wild-type glomerular epithelial cells, whereas the transient in TRPC6 knockout animals was not affected. Thus, angiotensin II-dependent activation of TRPC6 channels in podocytes may have a significant role in the development of kidney diseases.Fil: Ilatovskaya, Daria V.. Medical College Of Wisconsin; Estados Unidos. Russian Academy of Sciences; RusiaFil: Palygin, Oleg. Medical College Of Wisconsin; Estados UnidosFil: Chubinskiy Nadezhdin, Vladislav. Russian Academy of Sciences; RusiaFil: Negulyaev, Yuri A.. Russian Academy of Sciences; Rusia. St. Petersburg State Polytechnical University; RusiaFil: Ma, Rong. University of North Texas; Estados UnidosFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Staruschenko, Alexander. Medical College Of Wisconsin; Estados Unido