Regulation of ENaC-Mediated Sodium Reabsorption by Peroxisome Proliferator-Activated Receptors

Peroxisome proliferator-activated receptors (PPARs) are members of a steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis. Peroxisomal proliferator-activated receptor subtype γ (PPARγ) has received much attention as the target for antidiabetic drugs, as well as its role in responding to endogenous compounds such as prostaglandin J2. However, thiazolidinediones (TZDs), the synthetic agonists of the PPARγ are tightly associated with fluid retention and edema, as potentially serious side effects. The epithelial sodium channel (ENaC) represents the rate limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. The role of PPARγ agonists on ENaC activity remains controversial. While PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via Serum- and Glucocorticoid-Regulated Kinase 1 (SGK1), other studies reported that PPARγ agonist-induced fluid retention is independent of ENaC activity. The current paper provides new insights into the control and function of ENaC and ENaC-mediated sodium transport as well as several other epithelial channels/transporters by PPARs and particularly PPARγ. The potential contribution of arachidonic acid (AA) metabolites in PPAR-dependent mechanisms is also discussed

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

ATP release into ADPKD cysts via pannexin-1/P2X7 channels decreases ENaC activity

Genetic predisposition is necessary for polycystic kidney disease (PKD) initiation, although there are other, incompletely identified downstream processes that are required for cyst growth. Their characterization may provide a unique opportunity for clinical interventions. One of the poorly studied phenomena in PKD is high ATP content in cysts. Unfortunately, neither origins of uncontrolled ATP release, nor consequences of abnormal purinergic signaling in relation to epithelial transport are well explored in the polycystic kidney. We tested the distribution of pannexin-1 (Panx1) and P2X7, two proteins potentially involved in ATP release, in the kidneys of the Pkd1(RC/RC) mice, a model of autosomal dominant PKD (ADPKD). Abundances of both proteins were abnormally increased in the cyst lining cells compared to non-dilated collecting ducts. To establish if pannexin-1 contributes to ATP release in the collecting ducts (CD), we measured luminal accumulation of ATP in M1 cell renal CD monolayers, and found that treatment with probenecid, a Panx1 blocker, prevents ATP release. Single channel patch clamp analysis of polarized M1 cells revealed that apical stimulation of P2X receptors with alphabeta-MeATP acutely reduces ENaC activity. We conclude that in ADPKD progression, an abnormal hyperexpression of both PANX1 and P2RX7 occurs in the cyst lining epithelial cells. High abundance of both proteins is not typical for non-dilated CDs but, when it happens in cysts, pannexin1/P2X7 cooperation elevates ATP release into the luminal space. High ATP level is a pathogenic factor facilitating cystogenesis by reducing ENaC-mediated reabsorption from the lumen

Involvement of ENaC in the development of salt-sensitive hypertension

Salt-sensitive hypertension is associated with renal and vascular dysfunctions, which lead to impaired fluid excretion, increased cardiac output, and total peripheral resistance. It is commonly accepted that increased renal sodium handling and plasma volume expansion are necessary factors for the development of salt-induced hypertension. The epithelial sodium channel (ENaC) is a trimeric ion channel expressed in the distal nephron that plays a critical role in the regulation of sodium reabsorption in both normal and pathological conditions. In this mini-review, we summarize recent studies investigating the role of ENaC in the development of salt-sensitive hypertension. On the basis of experimental data obtained from the Dahl salt-sensitive rats, we and others have demonstrated that abnormal ENaC activation in response to a dietary NaCl load contributes to the development of high blood pressure in this model. The role of different humoral factors, such as the components of the renin-angiotensin-aldosterone system, members of the epidermal growth factors family, arginine vasopressin, and oxidative stress mediating the effects of dietary salt on ENaC are discussed in this review to highlight future research directions and to determine potential molecular targets for drug development

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

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

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