The actin cytoskeleton and small G protein RhoA are not involved in flow-dependent activation of ENaC

<p>Abstract</p> <p>Background</p> <p>Epithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na⁺channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity.</p> <p>Findings</p> <p>Pretreatment with Cytochalasin D and Latrunculin B for 20 min and 1-2 hrs to disassemble F-actin had no effect on flow-mediated increase of amiloride-sensitive current. Overexpression of ENaC with constitutively active (G14V) or dominant negative (T19N) RhoA similarly had no effect on flow-dependent activation of ENaC activity. In addition, we did not observe changes when we inhibited Rho-kinase with Y27632.</p> <p>Conclusions</p> <p>Our results suggest that the flow-dependent activation of ENaC is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton.</p

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

Probenecid slows disease progression in a murine model of autosomal dominant polycystic kidney disease

Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1(RC/RC) mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1(RC/RC) and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1(RC/RC) mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCD(cl4) cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCD(cl4) epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology

Adaptive Immunity-Driven Inflammation and Cardiovascular Disease

The adaptive immune response has recently emerged as an important factor in a wide variety of cardiovascular disorders including atherosclerosis, hypertension, cardiac remodeling, and heart failure; however, its role is not fully understood. Since an assortment of innate responsive cells, e.g., neutrophils and monocytes/macrophages, coordinate with adaptive immunity, e.g., T cells, dendritic cells, and B cells, the temporal response and descriptions pertinent to the cellular phenotype and inflammation processes, in general, need additional investigation, clarification, and consensus particularly in cardiovascular disease. This Perspectives article reviews the contributions of 15 articles (including 7 reviews) published in the American Journal of Physiology-Heart and Circulatory Physiology in response to the Call for Papers: Adaptive Immunity in Cardiovascular Disease. Here, we summarize the crucial reported findings at the cardiac, vascular, immune, and molecular levels and discuss the translational feasibility and benefits of future prospective research into the adaptive immune response. Readers are encouraged to evaluate the data and learn from this collection of novel studies

Effects of dietary sodium content on cysts formation in ARPKD

Polycystic kidney diseases (PKD) are a group of nephropathies marked with the formation of fluid-filled cysts along the nephron. Generally, patients with PKD are advised to restrict their dietary sodium intake to 100 mmol/day or less, as it is expected to reduce blood pressure and albuminuria. Here we hypothesize that general manipulation with sodium content in the diet can alter cyst formation. To test this, PCK rats were fed a normal (0.4%; NS), high salt (4%; HS), and sodiumdeficient (0.01%; SD) NaCl diets for 8 weeks (starting at 6 weeks of age). Immunohistochemistry, Western blotting, GFR measurements in conscious animals, and routine molecular biology approaches were applied to evaluate effects of various diets. Both HS and SD diets resulted in a dramatic increase in the cyst formation: 43.6% and 39.8% of whole kidney areas were cystic, compared to 28.5% in the NS group. However, the development of cysts was different between HS and SD diet fed groups. HS diet provoked cyst enlargement in a manner seen in NS group; in contrast, SD diet caused an extensive growth of small cysts in the cortical area, and increased hypertrophy of the renal tissue (2 kidney to body weight ratio was 15.9 ± 0.7 in animals fed a SD diet vs 11.5 ± 0.9 and 13.7 ± 0.8 mg/g in NS and HS groups). Additionally, SD diet-fed PCK rats exhibited reduced body weight (324 ± 11 compared to 517 ± 6 and 496 ± 5 g in NS and HS groups). Urinary output was significantly higher in the HS animals compared to both SD and NS groups; interestingly, we found no difference in food intake. There was no change in urinary creatinine over the course of diet challenge in either group; urinary chloride and sodium excretion were elevated in HS fed animals compared to NS and SD groups, as expected. GFR levels were assessed in conscious unrestrained animals after an injection of FITC-conjugated inulin and were found to be 0.40 ± 0.05, 0.65 ± 0.03, and 0.98 ± 0.11 uL/min/100g of body weight in SD, NS and HS fed rats, respectively. Plasma electrolytes (K+, Na+, Cl-, and Ca2+) were significantly lower in PCK rats fed SD diet, but not different between NS and HS groups. Consistent with other data, BUN was almost 130 mg/dL in the SD group compared to \u3c 20 mg/dL in NS and HS animals, indicative of renal failure in the SD fed rats. Western blotting and immunohistochemistry demonstrated a prominent decrease in the expression of the alpha and beta ENaC subunits in the SD fed group compared to the NS and HS groups. These data are consistent with our earlier studies where we reported that lower ENaC activity can contribute to cyst development in ARPKD; however the exact mechanism remains to be revealed in the future studies. In summary, both HS and SD diets significantly increased cystic area in PCK rats, although cyst formation and its effects on kidney function are different between these groups

Nitric-Oxide-Mediated Signaling in Podocyte Pathophysiology

Nitric oxide (NO) is a potent signaling molecule involved in many physiological and pathophysiological processes in the kidney. NO plays a complex role in glomerular ultrafiltration, vasodilation, and inflammation. Changes in NO bioavailability in pathophysiological conditions such as hypertension or diabetes may lead to podocyte damage, proteinuria, and rapid development of chronic kidney disease (CKD). Despite the extensive data highlighting essential functions of NO in health and pathology, related signaling in glomerular cells, particularly podocytes, is understudied. Several reports indicate that NO bioavailability in glomerular cells is decreased during the development of renal pathology, while restoring NO level can be beneficial for glomerular function. At the same time, the compromised activity of nitric oxide synthase (NOS) may provoke the formation of peroxynitrite and has been linked to autoimmune diseases such as systemic lupus erythematosus. It is known that the changes in the distribution of NO sources due to shifts in NOS subunits expression or modifications of NADPH oxidases activity may be linked to or promote the development of pathology. However, there is a lack of information about the detailed mechanisms describing the production and release of NO in the glomerular cells. The interaction of NO and other reactive oxygen species in podocytes and how NO-calcium crosstalk regulates glomerular cells’ function is still largely unknown. Here, we discuss recent reports describing signaling, synthesis, and known pathophysiological mechanisms mediated by the changes in NO homeostasis in the podocyte. The understanding and further investigation of these essential mechanisms in glomerular cells will facilitate the design of novel strategies to prevent or manage health conditions that cause glomerular and kidney damage

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